Michele Pagano, M.D.

FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas
Duan, Shanshan; Cermak, Lukas; Pagan, Julia K; Rossi, Mario; Martinengo, Cinzia; di Celle, Paola Francia; Chapuy, Bjoern; Shipp, Margaret; Chiarle, Roberto; Pagano, Michele
2012 Jan 5;481(7379):90-93, Nature
BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene
— id: 149798, year: 2012, vol: 481, page: 90, stat: Journal Article,

APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage
Cotto-Rios, Xiomaris M; Jones, Mathew J K; Busino, Luca; Pagano, Michele; Huang, Tony T
2011 Jul 25;194(2):177-186, Journal of cell biology
Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C(Cdh1). USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C(Cdh1) inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C(Cdh1) in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry
— id: 135576, year: 2011, vol: 194, page: 177, stat: Journal Article,

Linking metabolism and cell cycle progression via the APC/CCdh1 and SCFbetaTrCP ubiquitin ligases
Duan, Shanshan; Pagano, Michele
2011 Dec 27;108(52):20857-20858, Proceedings of the National Academy of Sciences of the United States of America
— id: 149805, year: 2011, vol: 108, page: 20857, stat: Journal Article,

mTOR Generates an Auto-Amplification Loop by Triggering the betaTrCP- and CK1alpha-Dependent Degradation of DEPTOR
Duan, Shanshan; Skaar, Jeffrey R; Kuchay, Shafi; Toschi, Alfredo; Kanarek, Naama; Ben-Neriah, Yinon; Pagano, Michele
2011 Oct 21;44(2):317-324, Molecular cell
DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein betaTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the betaTrCP degron in DEPTOR is executed by CK1alpha after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the betaTrCP-dependent degradation of DEPTOR via betaTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind betaTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1alpha and betaTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways
— id: 139744, year: 2011, vol: 44, page: 317, stat: Journal Article,

MCL1 meets its end during mitotic arrest
Millman, Scott E; Pagano, Michele
2011 May 1;12(5):384-385, EMBO reports
— id: 131815, year: 2011, vol: 12, page: 384, stat: Journal Article,

Loss of p27KIP1 Expression in Fully-staged Node-negative Breast Cancer: Association with Lack of Hormone Receptors in T1a/b, but not T1c Infiltrative Ductal Carcinoma
Mirchandani, Deepu; Roses, Daniel F; Inghirami, Giorgio; Zeleniuch-Jacquotte, Anne; Cangiarella, Joan; Guth, Amber; Safyan, Rachael Ann; Formenti, Silvia C; Pagano, Michele; Muggia, Franco
2011 Dec;31(12):4401-4405, Anticancer research
Nuclear expression of the cell cycle inhibitor p27(KIP1) is reduced in a variety of human malignancies, including breast cancer. Loss of nuclear p27(KIP1) during tumor progression, documented by immunohistochemistry (IHC), has been studied for its potential prognostic implication. We examined by IHC the association between nuclear p27(KIP1) expression and hormone receptor status in T1N0M0 breast cancer. PATIENTS AND METHODS: The correlation between nuclear p27(KIP1) expression and estrogen (ER) and progesterone (PR) hormone receptor status was analyzed in 122 human T1N0M0 (68 T1a/b, 54 T1c) breast cancer specimens. All patients were staged as N0 by axillary node dissection. RESULTS: A statistically significant reduction in p27(KIP1) expression was observed as tumor size increased from T1a/b (7%) to T1c (22%). The proportion of tumors with low nuclear p27(KIP1) expression was higher in the ER-negative/PR-negative group compared to the ER-positive/PR-positive group, but this difference was only statistically significant in the T1a/b subgroup (p=0.0007). CONCLUSION: Further investigations into causes of p27(KIP1) deregulation and their relationship to hormone receptor expression in T1N0M0 breast ductal carcinomas are warranted. Such studies may help identify prognostic, as well as predictive, markers of therapy resistance
— id: 149934, year: 2011, vol: 31, page: 4401, stat: Journal Article,

FBXW5 controls centrosome number
Pagan, Julia; Pagano, Michele
2011 ;13(8):888-890, Nature cell biology
Regulatory mechanisms to prevent centriole overduplication during the cell cycle are not completely understood. In this issue, FBXW5 is shown to control the degradation of the centriole assembly factor HsSAS-6. Moreover, the study proposes that FBXW5 is a substrate of both PLK4 and APC/C, two established regulators of centriole duplication
— id: 135567, year: 2011, vol: 13, page: 888, stat: Journal Article,

Clinical relevance of SKP2 alterations in metastatic melanoma
Rose, Amy E; Wang, Guimin; Hanniford, Douglas; Monni, Stefano; Tu, Ting; Shapiro, Richard L; Berman, Russell S; Pavlick, Anna C; Pagano, Michele; Darvishian, Farbod; Mazumdar, Madhu; Hernando, Eva; Osman, Iman
2011 Feb;24(1):197-206, Pigment cell & melanoma research
In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2
— id: 138133, year: 2011, vol: 24, page: 197, stat: Journal Article,

SCF ubiquitin ligases in the maintenance of genome stability
Silverman, JS; Skaar, JR; Pagano, M
2011 Nov;:?-?, Trends Biochem Sci
In response to genotoxic stress, eukaryotic cells activate the DNA damage response (DDR), a series of pathways that coordinate cell cycle arrest and DNA repair to prevent deleterious mutations. In addition, cells possess checkpoint mechanisms that prevent aneuploidy by regulating the number of centrosomes and spindle assembly. Among these mechanisms, ubiquitin-mediated degradation of key proteins has an important role in the regulation of the DDR, centrosome duplication and chromosome segregation. This review discusses the functions of a group of ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) family, in the maintenance of genome stability. Given that general proteasome inhibitors are currently used as anticancer agents, a better understanding of the ubiquitylation of specific targets by specific ubiquitin ligases may result in improved cancer therapeutics.
— id: 155521, year: 2011, vol: , page: ?, stat: Journal Article,

Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization
Bashir, Tarig; Pagan, Julia K; Busino, Luca; Pagano, Michele
2010 Mar;9(5):971-974, Cell cycle
F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2
— id: 109207, year: 2010, vol: 9, page: 971, stat: Journal Article,

Dissecting the role of ubiquitylation in the DNA damage response checkpoint in G2
Bassermann, F; Pagano, M
2010 Jan;17(1):78-85, Cell death & differentiation
Maintenance of genomic integrity is one of the fundamental biological properties shared by all living organisms. To counterbalance deleterious and potentially mutagenic effects of omnipresent DNA damaging assaults, organisms have developed a network of genome surveillance and maintenance pathways known as the DNA damage response. In eukaryotes, the orchestration of cell-cycle checkpoints, DNA damage repair, and apoptosis in response to DNA damage relies on posttranslational modifications of key regulatory proteins. Although the role of phosphorylation in these pathways is relatively well established, the significance of ubiquitylation has only recently emerged. In this review, we survey current research on the ubiquitin-proteasome system, focusing on the DNA damage response in the G2 phase of the cell cycle and two prominent classes of ubiquitin ligases, the SCF- and APC/C complexes. These ubiquitin ligases are reviewed with regard to their function in activating, maintaining, and terminating the checkpoint and in light of increasing evidence that suggests a dynamic balance of substrate ubiquitylation and deubiquitylation. We further discuss the impact of defective G2 checkpoint signaling on genomic stability and cancer risk, highlighting strategies for targeted antitumor drug discovery
— id: 136505, year: 2010, vol: 17, page: 78, stat: Journal Article,

SCF(Cyclin F) controls centrosome homeostasis and mitotic fidelity through CP110 degradation
D'Angiolella, Vincenzo; Donato, Valerio; Vijayakumar, Sangeetha; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Dynlacht, Brian; Pagano, Michele
2010 Jul 1;466(7302):138-142, Nature
Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity
— id: 110690, year: 2010, vol: 466, page: 138, stat: Journal Article,

Tumor Suppressor Function of Androgen Receptor Coactivator ARA70{alpha} in Prostate Cancer
Ligr, Martin; Li, Yirong; Zou, Xuanyi; Daniels, Garrett; Melamed, Jonathan; Peng, Yi; Wang, Wei; Wang, Jinhua; Ostrer, Harry; Pagano, Michele; Wang, Zhengxin; Garabedian, Michael J; Lee, Peng
2010 Apr;176(4):1891-1900, American journal of pathology
Androgen receptor (AR), a member of the steroid receptor family, is a transcription factor that has an important role in the regulation of both prostate cell proliferation and growth suppression. AR coactivators may influence the transition between cell growth and growth suppression. We have shown previously that the internally spliced ARA70 isoform, ARA70beta, promotes prostate cancer cell growth and invasion. Here we report that the full length ARA70alpha, in contrast, represses prostate cancer cell proliferation and anchorage-independent growth in vitro and inhibits tumor growth in nude mice xenograft experiments in vivo. Further, the growth inhibition by ARA70alpha is AR-dependent and mediated through induction of apoptosis rather than cell cycle arrest. Interestingly, AR with T877A mutation in LNCaP cells decreased its physical and functional interaction with ARA70alpha, facilitating the growth of LNCaP cells. This is consistent with our previous findings that ARA70alpha expression is decreased in prostate cancer cells compared with benign prostate. ARA70alpha also reduced the invasion ability of LNCaP cells. Although growth inhibition by ARA70alpha is AR-dependent, the inhibition of cell invasion is an androgen-independent process. These results strongly suggest that ARA70alpha functions as a tumor suppressor gene
— id: 107298, year: 2010, vol: 176, page: 1891, stat: Journal Article,

The ubiquitin-specific protease USP47 is a novel beta-TRCP interactor regulating cell survival
Peschiaroli, A; Skaar, JR; Pagano, M; Melino, G
2010 MAR 4 ;29(9):1384-1393, Oncogene
Ubiquitin-specific proteases (USPs) are a subclass of cysteine proteases that catalyze the removal of ubiquitin (either monomeric or chains) from substrates, thus counteracting the activity of E3 ubiquitin ligases. Although the importance of USPs in a multitude of processes, from hereditary cancer to neurodegeneration, is well established, our knowledge on their mode of regulation, substrate specificity and biological function is quite limited. In this study we identify USP47 as a novel interactor of the E3 ubiquitin ligase, Skp1/Cul1/F-box protein beta-transducin repeat-containing protein (SCF beta-Trcp). We found that both beta-Trcp1 and beta-Trcp2 bind specifically to USP47, and point mutations in the beta-Trcp WD-repeat region completely abolished USP47 binding, indicating an E3-substrate-type interaction. However, unlike canonical beta-Trcp substrates, USP47 protein levels were neither affected by silencing of beta-Trcp nor modulated in a variety of processes, such as cell-cycle progression, DNA damage checkpoint responses or tumor necrosis factor (TNF) pathway activation. Notably, genetic or siRNA-mediated depletion of USP47 induced accumulation of Cdc25A, decreased cell survival and augmented the cytotoxic effects of anticancer drugs. In conclusion, we showed that USP47, a novel beta-Trcp interactor, regulates cell growth and survival, potentially providing a novel target for anticancer therapies. Oncogene (2010) 29, 1384-1393; doi:10.1038/onc.2009.430; published online 7 December 2009
— id: 108313, year: 2010, vol: 29, page: 1384, stat: Journal Article,

Cdc25 phosphatases: Differential regulation by ubiquitin-mediated proteolysis
Young, Lauren M.; Pagano, Michele
2010 DEC 1 ;9(23):4613-4614, Cell cycle
— id: 115372, year: 2010, vol: 9, page: 4613, stat: Journal Article,

betaTrCP- and Rsk1/2-mediated degradation of BimEL inhibits apoptosis
Dehan, Elinor; Bassermann, Florian; Guardavaccaro, Daniele; Vasiliver-Shamis, Gaia; Cohen, Michael; Lowes, Kym N; Dustin, Michael; Huang, David C S; Taunton, Jack; Pagano, Michele
2009 Jan 16;33(1):109-116, Molecular cell
The BimEL tumor suppressor is a potent proapoptotic BH3-only protein. We found that, in response to survival signals, BimEL was rapidly phosphorylated on three serine residues in a conserved degron, facilitating binding and degradation via the F box protein betaTrCP. Phosphorylation of the BimEL degron was executed by Rsk1/2 and promoted by the Erk1/2-mediated phosphorylation of BimEL on Ser69. Compared to wild-type BimEL, a BimEL phosphorylation mutant unable to bind betaTrCP was stabilized and consequently potent at inducing apoptosis by the intrinsic mitochondrial pathway. Moreover, although non-small cell lung cancer (NSCLC) cells often become resistant to gefitinib (a clinically relevant tyrosine kinase inhibitor that induces apoptosis through BimEL), silencing of either betaTrCP or Rsk1/2 resulted in BimEL-mediated apoptosis of both gefitinib-sensitive and gefitinib-insensitive NSCLC cells. Our findings reveal that betaTrCP promotes cell survival in cooperation with the ERK-RSK pathway by targeting BimEL for degradation
— id: 92189, year: 2009, vol: 33, page: 109, stat: Journal Article,

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation
Ge, Sheng; Skaar, Jeffrey R; Pagano, Michele
2009 Jan 1;8(1):167-171, Cell cycle
The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores. Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1. Here, we report that the SAC negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation. Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2. We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C(Cdc20) in response to the SAC
— id: 93223, year: 2009, vol: 8, page: 167, stat: Journal Article,

Thrombin induces tumor cell cycle activation and spontaneous growth by down-regulation of p27Kip1, in association with the up-regulation of Skp2 and MiR-222
Hu, Liang; Ibrahim, Sherif; Liu, Cynthia; Skaar, Jeffrey; Pagano, Michele; Karpatkin, Simon
2009 Apr 15;69(8):3374-3381, Cancer research
The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G(0) and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27(Kip1) and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27(Kip1) with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27(Kip1)-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27(Kip1), was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27(Kip1)
— id: 98895, year: 2009, vol: 69, page: 3374, stat: Journal Article,

Wnt Signaling in Mitosis
Kaldis, P; Pagano, M
2009 DEC 15 ;17(6):749-750, Developmental cell
Previously, the connection between cell proliferation and Wnt signaling focused on transcriptional activation of cyclin D1 and c-myc, which control the G1/S transition of the cell cycle. In this issue of Developmental Cell, the Niehrs group demonstrates mitotic activation of Wnt signaling by a novel Cdk/cyclin complex containing Cdk14 (PFTK1) and cyclin Y
— id: 106396, year: 2009, vol: 17, page: 749, stat: Journal Article,

Degradation of cyclin A is regulated by acetylation
Mateo, F; Vidal-Laliena, M; Canela, N; Busino, L; Martinez-Balbas, MA; Pagano, M; Agell, N; Bachs, O
2009 JUL ;28(29):2654-2666, Oncogene
Cyclin A accumulates at the onset of S phase, remains high during G(2) and early mitosis and is degraded at prometaphase. Here, we report that the acetyltransferase P/CAF directly interacts with cyclin A that as a consequence becomes acetylated at lysines 54,68, 95 and 112. Maximal acetylation occurs simultaneously to ubiquitylation at mitosis, indicating importance of acetylation on cyclin A stability. This was further confirmed by the observation that the pseudoacetylated cyclin A mutant can be ubiquitylated whereas the nonacetylatable mutant cannot. The nonacetylatable mutant is more stable than cyclin A WT (cycA WT) and arrests cell cycle at mitosis. Moreover, in cells treated with histone deacetylase inhibitors cyclin A acetylation increases and its stability decreases, thus supporting the function of acetylation on cyclin A degradation. Although the nonacetylatable mutant cannot be ubiquitylated, it interacts with the proteins needed for its degradation (cdks, Cks, Cdc 20, Cdh1 and APC/C). In fact, its association with cdks is increased and its complexes with these kinases display higher activity than control cycA WT-cdk complexes. All these results indicate that cyclin A acetylation at specific lysines is crucial for cyclin A stability and also has a function in the regulation of cycA-cdk activity. Oncogene (2009) 28, 2654-2666; doi: 10.1038/onc.2009.127; published online 1 June 2009
— id: 101237, year: 2009, vol: 28, page: 2654, stat: Journal Article,

Hypertension and diabetes mellitus are associated with cardiovascular events in the elderly without cardiovascular disease. Results of a 15-year follow-up in a Mediterranean population
Noto, D; Cefalu, A B; Barbagallo, C M; Sapienza, M; Cavera, G; Nardi, I; Pagano, M; Vivona, N; Notarbartolo, A; Averna, M R
2009 Jun;19(5):321-326, Nutrition, metabolism, & cardiovascular diseases : NMCD
BACKGROUND AND AIMS: Epidemiological prospective data on cardiovascular (CV) events in elderly subjects from Mediterranean populations are lacking. We aimed to investigate 15-year incidence of CV events and to evaluate the association with CV risk factors in an elderly Mediterranean population. METHODS AND RESULTS: The population of a small Sicilian village were enrolled, visited and a blood sample was drawn at baseline. CV events were recorded in the 15years of follow-up. From 1351 subjects (75% of the resident population); 315 were in the age range 65-85years; 266 subjects free from CV disease were analysed. Seventy-seven CV events were recorded in 73 out of 266 subjects, with a 19.7% rate (in 10years). Hypertension (HTN) (hazards ratio=2.1) and diabetes mellitus (DM) (hazards ratio=1.8) were independently associated with CV events. Subjects with both DM and HTN showed a lower survival free of CV events compared to those with DM or HTN. CONCLUSIONS: In a 15-year follow-up of an elderly Mediterranean population free from CV disease, diabetes mellitus and hypertension were related to CV events. The control of risk factors in the elderly needs to be reinforced to achieve better results in terms of CV prevention
— id: 79362, year: 2009, vol: 19, page: 321, stat: Journal Article,

The F-box protein FBXO45 promotes the proteasome-dependent degradation of p73
Peschiaroli, A; Scialpi, F; Bernassola, F; Pagano, M; Melino, G
2009 SEP ;28(35):3157-3166, Oncogene
The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modi. cation, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCFFBXO45 ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism. Oncogene (2009) 28, 3157-3166; doi: 10.1038/onc.2009.177; published online 6 July 2009
— id: 102139, year: 2009, vol: 28, page: 3157, stat: Journal Article,

SnapShot: F Box Proteins II
Skaar, Jeffrey R; D'Angiolella, Vincenzo; Pagan, Julia K; Pagano, Michele
2009 Jun 26;137(7):1358, 1358.e1-, Cell
— id: 100622, year: 2009, vol: 137, page: 1358, 1358.e1, stat: Journal Article,

SnapShot: F box proteins I
Skaar, Jeffrey R; Pagan, Julia K; Pagano, Michele
2009 Jun 12;137(6):1160-1160.e1, Cell
— id: 100193, year: 2009, vol: 137, page: 1160, stat: Journal Article,

Control of cell growth by the SCF and APC/C ubiquitin ligases
Skaar, Jeffrey R; Pagano, Michele
2009 Dec;21(6):816-824, Current opinion in cell biology
The ubiquitin-proteasome system plays key roles in the control of cell growth. The cell cycle, in particular, is highly regulated by the functions of the SCF and APC/C ubiquitin ligases, and perturbation of their function can result in tumorigenesis. Although the SCF and APC/C complexes are well established in growth control pathways, many aspects of their function remain unknown. Recent studies have shed light on the mechanism of SCF-mediated ubiquitination and new functions for the SCF complex and APC/C. Our expanding understanding of the roles of the SCF and APC/C complexes highlight the potential for targeted molecular therapies
— id: 105497, year: 2009, vol: 21, page: 816, stat: Journal Article,

INTS3 controls the hSSB1-mediated DNA damage response
Skaar, Jeffrey R; Richard, Derek J; Saraf, Anita; Toschi, Alfredo; Bolderson, Emma; Florens, Laurence; Washburn, Michael P; Khanna, Kum Kum; Pagano, Michele
2009 Oct 5;187(1):25-32, Journal of cell biology
Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3-MISE-hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function
— id: 103155, year: 2009, vol: 187, page: 25, stat: Journal Article,

PCNA-dependent regulation of p21 ubiquitylation and degradation via the CRL4Cdt2 ubiquitin ligase complex
Abbas, Tarek; Sivaprasad, Uma; Terai, Kenta; Amador, Virginia; Pagano, Michele; Dutta, Anindya
2008 Sep 15;22(18):2496-2506, Genes & development
The DNA polymerase delta processivity factor Proliferating Cell Nuclear Antigen (PCNA) promotes the DNA damage-induced degradation of the replication initiation factor Cdt1 via the CRL4(Cdt2) E3 ubiquitin ligase complex. Here we demonstrate that PCNA promotes the ubiquitylation and degradation of the CDK inhibitor p21 in cells irradiated with low dose of ultraviolet (UV) by a similar mechanism. Human cells that are depleted of Cul4, DDB1 (damage-specific DNA-binding protein-1), or the DCAF Cdt2, are deficient in the UV-induced ubiquitylation and degradation of p21. Depletion of mammalian cells of PCNA by siRNA, or mutations in p21 that abrogate PCNA binding, prevent UV-induced p21 ubiquitylation and degradation, indicating that physical binding with PCNA is necessary for the efficient ubiquitylation of p21 via the CRL4(Cdt2) ubiquitin ligase. Cdt2 functions as the substrate recruiting factor for p21 to the rest of the CRL4 ubiquitin ligase complex. The CRL4(Cdt2) E3 ubiquitin ligase ubiquitylates p21 both in vivo and in vitro, and its activity is dependent on the interaction of p21 with PCNA. Finally, we show that the CRL4(Cdt2) and the SCF(Skp2) ubiquitin ligases are redundant with each other in promoting the degradation of p21 during an unperturbed S phase of the cell cycle
— id: 96958, year: 2008, vol: 22, page: 2496, stat: Journal Article,

The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint
Bassermann, Florian; Frescas, David; Guardavaccaro, Daniele; Busino, Luca; Peschiaroli, Angelo; Pagano, Michele
2008 Jul 25;134(2):256-267, Cell
In response to DNA damage in G2, mammalian cells must avoid entry into mitosis and instead initiate DNA repair. Here, we show that, in response to genotoxic stress in G2, the phosphatase Cdc14B translocates from the nucleolus to the nucleoplasm and induces the activation of the ubiquitin ligase APC/C(Cdh1), with the consequent degradation of Plk1, a prominent mitotic kinase. This process induces the stabilization of Claspin, an activator of the DNA-damage checkpoint, and Wee1, an inhibitor of cell-cycle progression, and allows an efficient G2 checkpoint. As a by-product of APC/C(Cdh1) reactivation in DNA-damaged G2 cells, Claspin, which we show to be an APC/C(Cdh1) substrate in G1, is targeted for degradation. However, this process is counteracted by the deubiquitylating enzyme Usp28 to permit Claspin-mediated activation of Chk1 in response to DNA damage. These findings define a novel pathway that is crucial for the G2 DNA-damage-response checkpoint
— id: 80620, year: 2008, vol: 134, page: 256, stat: Journal Article,

KDM2A represses transcription of centromeric satellite repeats and maintains the heterochromatic state
Frescas, David; Guardavaccaro, Daniele; Kuchay, Shafi M; Kato, Hiroyuki; Poleshko, Andrey; Basrur, Venkatesha; Elenitoba-Johnson, Kojo S; Katz, Richard A; Pagano, Michele
2008 Nov 15;7(22):3539-3547, Cell cycle
Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes, and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated and HP1-interacting protein that promotes HP1 localization to chromatin. We show that KDM2A is required to maintain the heterochromatic state, as determined using a candidate-based approach coupled to an in vivo epigenetic reporter system. Remarkably, a parallel and independent siRNA screen also detected a role for KDM2A in epigenetic silencing. Moreover, we demonstrate that KDM2A associates with centromeres and represses transcription of small non-coding RNAs that are encoded by the clusters of satellite repeats at the centromere. Dissecting the relationship between heterochromatin and centromeric RNA transcription is the basis of ongoing studies. We demonstrate that forced expression of these satellite RNA transcripts compromise the heterochromatic state and HP1 localization to chromatin. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development
— id: 91978, year: 2008, vol: 7, page: 3539, stat: Journal Article,

Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer
Frescas, David; Pagano, Michele
2008 Jun;8(6):438-449, Nature reviews. Cancer
The maintenance and preservation of distinct phases during the cell cycle is a highly complex and coordinated process. It is regulated by phosphorylation--through the activity of cyclin-dependent kinases (CDKs)--and protein degradation, which occurs through ubiquitin ligases such as SCF (SKP1-CUL1-F-box protein) complexes and APC/C (anaphase-promoting complex/cyclosome). Here, we explore the functionality and biology of the F-box proteins, SKP2 (S-phase kinase-associated protein 2) and beta-TrCP (beta-transducin repeat-containing protein), which are emerging as important players in cancer biogenesis owing to the deregulated proteolysis of their substrates
— id: 79246, year: 2008, vol: 8, page: 438, stat: Journal Article,

Control of chromosome stability by the beta-TrCP-REST-Mad2 axis
Guardavaccaro, Daniele; Frescas, David; Dorrello, N Valerio; Peschiaroli, Angelo; Multani, Asha S; Cardozo, Timothy; Lasorella, Anna; Iavarone, Antonio; Chang, Sandy; Hernando, Eva; Pagano, Michele
2008 Mar 20;452(7185):365-369, Nature
REST/NRSF (repressor-element-1-silencing transcription factor/neuron-restrictive silencing factor) negatively regulates the transcription of genes containing RE1 sites. REST is expressed in non-neuronal cells and stem/progenitor neuronal cells, in which it inhibits the expression of neuron-specific genes. Overexpression of REST is frequently found in human medulloblastomas and neuroblastomas, in which it is thought to maintain the stem character of tumour cells. Neural stem cells forced to express REST and c-Myc fail to differentiate and give rise to tumours in the mouse cerebellum. Expression of a splice variant of REST that lacks the carboxy terminus has been associated with neuronal tumours and small-cell lung carcinomas, and a frameshift mutant (REST-FS), which is also truncated at the C terminus, has oncogenic properties. Here we show, by using an unbiased screen, that REST is an interactor of the F-box protein beta-TrCP. REST is degraded by means of the ubiquitin ligase SCF(beta-TrCP) during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2, an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TrCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2(+/-) cells. In particular, we observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and mis-segregation in anaphase, tetraploidy, and faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TrCP. Thus, SCF(beta-TrCP)-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis. The high levels of REST or its truncated variants found in certain human tumours may contribute to cellular transformation by promoting genomic instability
— id: 78365, year: 2008, vol: 452, page: 365, stat: Journal Article,

Thrombin Enhances Spontaneous Tumor Growth and Cell Cycle Activation by Downregulation of p27(Kipl) and Upregulation of Skp2 and MiR-222
Hu, L; Ibrahim, S; Liu, C; Skaar, J; Pagano, M; Karpatkin, S
2008 NOV 16 ;112(11):152-152, Blood
— id: 93282, year: 2008, vol: 112, page: 152, stat: Journal Article,

APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation
Merluzzi, Sonia; Gri, Giorgia; Gattei, Valter; Pagano, Michele; Pucillo, Carlo
2008 Aug;45(14):3731-3739, Molecular immunology
Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches
— id: 96959, year: 2008, vol: 45, page: 3731, stat: Journal Article,

Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division
Michaelson, David; Abidi, Wasif; Guardavaccaro, Daniele; Zhou, Mo; Ahearn, Ian; Pagano, Michele; Philips, Mark R
2008 May 5;181(3):485-496, Journal of cell biology
Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division
— id: 79148, year: 2008, vol: 181, page: 485, stat: Journal Article,

The metabolic syndrome predicts cardiovascular events in subjects with normal fasting glucose: results of a 15 years follow-up in a Mediterranean population
Noto, Davide; Barbagallo, Carlo Maria; Cefalu, Angelo Baldassare; Falletta, Angelo; Sapienza, Michelangelo; Cavera, Giovanni; Amato, Salvatore; Pagano, Michele; Maggiore, Maria; Carroccio, Antonio; Notarbartolo, Alberto; Averna, Maurizio R
2008 Mar;197(1):147-153, Atherosclerosis
The aim of this study was to evaluate the cardiovascular (CV) risk due to the metabolic syndrome in a 15-year prospective study of a Sicilian population. In the Mediterranean area obesity is highly prevalent, but epidemiological data on the metabolic syndrome are limited. METHODS AND RESULTS: Among the 1351 subjects enrolled in the 'Ventimiglia di Sicilia' epidemiological project, we selected 687 subjects between 35 and 75 years of age; baseline parameters were assessed and subjects have been followed for 15 years recording CV events, total and cardiovascular mortality. The metabolic syndrome was defined according to both the Adult Treatment Panel III and the International Diabetes Federation criteria. Metabolic syndrome (ATPIII criteria) was significantly (p<0.00001) more prevalent in women (31.5%) than in men (12.4%). The metabolic syndrome increased the risk of CV events with a hazard ratio of 1.9 (confidence interval CI; 1.46-2.46). Using a Cox proportional hazards estimation model, the survival curve of subjects with metabolic syndrome and normal fasting glucose did not significantly differ from the curve of subjects with metabolic syndrome and impaired fasting glucose (IFG). CONCLUSIONS: In a 15-year follow-up the metabolic syndrome is predictive of CV events regardless of the presence of IFG or diabetes mellitus
— id: 79364, year: 2008, vol: 197, page: 147, stat: Journal Article,

Stimulation of prostate cancer cellular proliferation and invasion by the androgen receptor co-activator ARA70
Peng, Yi; Li, Caihong X; Chen, Fei; Wang, Zhengxin; Ligr, Martin; Melamed, Jonathan; Wei, Jianjun; Gerald, William; Pagano, Michele; Garabedian, Michael J; Lee, Peng
2008 Jan;172(1):225-235, American journal of pathology
ARA70 was first identified as a gene fused to the ret oncogene in thyroid carcinoma and subsequently as a co-activator for androgen receptor (AR). Two isoforms of ARA70 have been identified: a 70-kDa version called ARA70 alpha and an internally spliced 35-kDa variant termed ARA70 beta. We have previously reported that ARA70 alpha expression is reduced in prostate cancer, and its overexpression inhibits proliferation of LNCaP prostate cancer cells. However, the function of the ARA70 beta isoform in prostate cancer is not understood. In this report we examined the effects of ARA70 beta on AR transcriptional regulation as well as prostate cancer cellular proliferation and invasion. Although both ARA70 alpha and ARA70 beta functioned as transcriptional co-activators of AR in cell-based reporter assays, ARA70 beta overexpression, in contrast to ARA70 alpha, promoted prostate cancer cellular proliferation and invasion through Matrigel. Interestingly, genome-wide expression profiling of cells expressing ARA70 beta revealed an increase in the expression of genes involved in the control of cell division and adhesion, compatible with a role for ARA70 beta in proliferation and invasion. Consistent with its function in promoting cell growth and invasion, ARA70 beta expression was increased in prostate cancer. Our findings implicate ARA70 beta as a regulator of tumor cell growth and metastasis by affecting gene expression
— id: 76451, year: 2008, vol: 172, page: 225, stat: Journal Article,

Cdh1: a master G0/G1 regulator
Skaar, Jeffrey R; Pagano, Michele
2008 Jul;10(7):755-757, Nature cell biology
APC/C(Cdh1) controls the G0 and G1 phases of the cell cycle. Using a conditional knockout of the Cdh1 coding gene Fizzy-related (Fzr), a new study demonstrates that Cdh1 is essential for viability and that it functions as a tumour suppressor by preventing genomic instability
— id: 80618, year: 2008, vol: 10, page: 755, stat: Journal Article,

The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein
Zhao, Xudong; Heng, Julian Ik-Tsen; Guardavaccaro, Daniele; Jiang, Richeng; Pagano, Michele; Guillemot, Francois; Iavarone, Antonio; Lasorella, Anna
2008 Jun;10(6):643-653, Nature cell biology
Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue
— id: 79363, year: 2008, vol: 10, page: 643, stat: Journal Article,

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase
Amador, Virginia; Ge, Sheng; Santamaria, Patricia G; Guardavaccaro, Daniele; Pagano, Michele
2007 Aug 3;27(3):462-473, Molecular cell
During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation
— id: 73589, year: 2007, vol: 27, page: 462, stat: Journal Article,

Multisite phosphorylation of nipa at G2/M involves cyclin B1/CDK1
Bassermann, Florian; von Klitzing, Christine; Illert, Anna Lena; Munch, Silvia; Morris, Stephan W; Pagano, Michele; Peschel, Christian; Duyster, Justus
2007 Jun 1;282(22):15965-15972, Journal of biological chemistry
NIPA is an F-box containing protein that defines a nuclear SCF-type ubiquitin E3 ligase (SCFNIPA) implicated in the regulation of mitotic entry. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. Further to the recently described serine residue 354, we specify two new residues, Ser 359 and 395, implicated in the phosphorylation process at G2/M within this region. Moreover, we find cyclin B1/Cdk1 to phosphorylate NIPA at Ser 395 in mitosis. Mutation of both, Ser 359 and 395 impaired effective inactivation of the SCFNIPA complex resulting in reduced levels of mitotic cyclin B1. These data are compatible with a process of sequential NIPA phosphorylation where cyclin B1/Cdk1 amplifies phosphorylation of NIPA once an initial phosphorylation event has dissociated the SCFNIPA complex. Thus, cyclin B1/Cdk1 may contribute to the regulation of its own abundance in early mitosis
— id: 72422, year: 2007, vol: 282, page: 15965, stat: Journal Article,

SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins
Busino, Luca; Bassermann, Florian; Maiolica, Alessio; Lee, Choogon; Nolan, Patrick M; Godinho, Sofia I H; Draetta, Giulio F; Pagano, Michele
2007 May 11;316(5826):900-904, Science
One component of the circadian clock in mammals is the Clock-Bmal1 heterodimeric transcription factor. Among its downstream targets, two genes, Cry1 and Cry2, encode inhibitors of the Clock-Bmal1 complex that establish a negative-feedback loop. We found that both Cry1 and Cry2 proteins are ubiquitinated and degraded via the SCF(Fbxl3) ubiquitin ligase complex. This regulation by SCF(Fbxl3) is a prerequisite for the efficient and timely reactivation of Clock-Bmal1 and the consequent expression of Per1 and Per2, two regulators of the circadian clock that display tumor suppressor activity. Silencing of Fbxl3 produced no effect in Cry1-/-;Cry2-/- cells, which shows that Fbxl3 controls clock oscillations by mediating the degradation of CRY proteins
— id: 72420, year: 2007, vol: 316, page: 900, stat: Journal Article,

Wrenches in the works: drug discovery targeting the SCF ubiquitin ligase and APC/C complexes
Cardozo, Timothy; Pagano, Michele
2007 ;8 Suppl 1:S9-S9, BMC biochemistry
Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)
— id: 75675, year: 2007, vol: 8 Suppl 1, page: S9, stat: Journal Article,

DRE-1: an evolutionarily conserved F box protein that regulates C. elegans developmental age
Fielenbach, Nicole; Guardavaccaro, Daniele; Neubert, Kerstin; Chan, Tammy; Li, Dongling; Feng, Qin; Hutter, Harald; Pagano, Michele; Antebi, Adam
2007 Mar;12(3):443-455, Developmental cell
During metazoan development, cells acquire both positional and temporal identities. The Caenorhabditis elegans heterochronic loci are global regulators of larval temporal fates. Most encode conserved transcriptional and translational factors, which affect stage-appropriate programs in various tissues. Here, we describe dre-1, a heterochronic gene, whose mutant phenotypes include precocious terminal differentiation of epidermal stem cells and altered temporal patterning of gonadal outgrowth. Genetic interactions with other heterochronic loci place dre-1 in the larval-to-adult switch. dre-1 encodes a highly conserved F box protein, suggesting a role in an SCF ubiquitin ligase complex. Accordingly, RNAi knockdown of the C. elegans SKP1-like homolog SKR-1, the cullin CUL-1, and ring finger RBX homologs yielded similar heterochronic phenotypes. DRE-1 and SKR-1 form a complex, as do the human orthologs, hFBXO11 and SKP1, revealing a phyletically ancient interaction. The identification of core components involved in SCF-mediated modification and/or proteolysis suggests an important level of regulation in the heterochronic hierarchy
— id: 79365, year: 2007, vol: 12, page: 443, stat: Journal Article,

JHDM1B/FBXL10 is a nucleolar protein that represses transcription of ribosomal RNA genes
Frescas, David; Guardavaccaro, Daniele; Bassermann, Florian; Koyama-Nasu, Ryo; Pagano, Michele
2007 Nov 8;450(7167):309-313, Nature
JHDM1B is an evolutionarily conserved and ubiquitously expressed member of the JHDM (JmjC-domain-containing histone demethylase) family. Because it contains an F-box motif, this protein is also known as FBXL10 (ref. 4). With the use of a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identified as a gene that regulates growth. In the mouse, four independent screens have identified JHDM1B as a putative tumour suppressor by retroviral insertion analysis. Here we identify human JHDM1B as a nucleolar protein and show that JHDM1B preferentially binds the transcribed region of ribosomal DNA to repress the transcription of ribosomal RNA genes. We also show that repression of ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary for the specific demethylation of trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative effect of JHDM1B on cell size and cell proliferation. Because aberrant ribosome biogenesis and the disruption of epigenetic control mechanisms contribute to cellular transformation, these results, together with the low levels of JHDM1B expression found in aggressive brain tumours, suggest a role for JHDM1B in cancer development
— id: 75415, year: 2007, vol: 450, page: 309, stat: Journal Article,

The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period
Godinho, Sofia I H; Maywood, Elizabeth S; Shaw, Linda; Tucci, Valter; Barnard, Alun R; Busino, Luca; Pagano, Michele; Kendall, Rachel; Quwailid, Mohamed M; Romero, M Rosario; O'neill, John; Chesham, Johanna E; Brooker, Debra; Lalanne, Zuzanna; Hastings, Michael H; Nolan, Patrick M
2007 May 11;316(5826):897-900, Science
By screening N-ethyl-N-nitrosourea-mutagenized animals for alterations in rhythms of wheel-running activity, we identified a mouse mutation, after hours (Afh). The mutation, a Cys(358)Ser substitution in Fbxl3, an F-box protein with leucine-rich repeats, results in long free-running rhythms of about 27 hours in homozygotes. Circadian transcriptional and translational oscillations are attenuated in Afh mice. The Afh allele significantly affected Per2 expression and delayed the rate of Cry protein degradation in Per2::Luciferase tissue slices. Our in vivo and in vitro studies reveal a central role for Fbxl3 in mammalian circadian timekeeping
— id: 72419, year: 2007, vol: 316, page: 897, stat: Journal Article,

Constitutive phosphorylation of aurora-a on ser51 induces its stabilization and consequent overexpression in cancer
Kitajima, Shojiro; Kudo, Yasusei; Ogawa, Ikuko; Tatsuka, Masaaki; Kawai, Hidehiko; Pagano, Michele; Takata, Takashi
2007 ;2(9):e944-e944, PLoS ONE
BACKGROUND: The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer. PRINCIPAL FINDINGS: Aur-A protein was ubiquitinylated by APC(Cdh1) and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1)-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation. CONCLUSIONS/SIGNIFICANCE: Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy
— id: 79130, year: 2007, vol: 2, page: e944, stat: Journal Article,

The pRb-Cdh1-p27 autoamplifying network
Santamaria, Patricia G; Pagano, Michele
2007 Feb;9(2):137-138, Nature cell biology
— id: 72423, year: 2007, vol: 9, page: 137, stat: Journal Article,

Substrate Recognition and Ubiquitination of SCFSkp2/Cks1 Ubiquitin-Protein Isopeptide Ligase
Xu, Shuichan; Abbasian, Mahan; Patel, Palka; Jensen-Pergakes, Kristen; Lombardo, Christian R; Cathers, Brian E; Xie, Weilin; Mercurio, Frank; Pagano, Michele; Giegel, David; Cox, Sarah
2007 May 25;282(21):15462-15470, Journal of biological chemistry
p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2
— id: 72421, year: 2007, vol: 282, page: 15462, stat: Journal Article,

APC/CCDC20 controls the ubiquitin-mediated degradation of p21 during early mitosis
Amador, V; Gonzalez-Santamaria, P; Pagano, M
2006 JUN ;273(11):136-136, FEBS journal
— id: 69260, year: 2006, vol: 273, page: 136, stat: Journal Article,

Modification of Cul1 regulates its association with proteasomal subunits
Bloom, Joanna; Peschiaroli, Angelo; Demartino, George; Pagano, Michele
2006 ;1:5-5, Cell division
ABSTRACT : BACKGROUND : Ubiquitylation targets proteins for degradation by the 26S proteasome. Some yeast and plant ubiquitin ligases, including the highly conserved SCF (Skp1/Cul1/F-box protein) complex, have been shown to associate with proteasomes. We sought to characterize interactions between SCF complexes and proteasomes in mammalian cells. RESULTS : We found that the binding of SCF complexes to proteasomes is conserved in higher eukaryotes. The Cul1 subunit associated with both sub-complexes of the proteasome, and high molecular weight forms of Cul1 bound to the 19S proteasome. Cul1 is ubiquitylated in vivo. Ubiquitylation of Cul1 promotes its binding to the S5a subunit of the 19S sub-complex without affecting Cul1 stability. CONCLUSION : The association of ubiquitylating enzymes with proteasomes may be an additional means to target ubiquitylated substrates for degradation
— id: 72425, year: 2006, vol: 1, page: 5, stat: Journal Article,

S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth
Dorrello, N Valerio; Peschiaroli, Angelo; Guardavaccaro, Daniele; Colburn, Nancy H; Sherman, Nicholas E; Pagano, Michele
2006 Oct 20;314(5798):467-471, Science
The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth
— id: 69030, year: 2006, vol: 314, page: 467, stat: Journal Article,

Stabilizers and destabilizers controlling cell cycle oscillators
Guardavaccaro, Daniele; Pagano, Michele
2006 Apr 7;22(1):1-4, Molecular cell
Various destabilizing factors of the ubiquitin system contribute to the synchrony and unidirectionality of the cell cycle clock by finely tuning the activity of various CDKs. The recent findings of hierarchical and connected waves of cyclin stabilizers highlight the complexity of this network
— id: 64207, year: 2006, vol: 22, page: 1, stat: Journal Article,

Skp2 Contains a Novel Cyclin A Binding Domain That Directly Protects Cyclin A from Inhibition by p27Kip1
Ji, Peng; Goldin, Luba; Ren, Hao; Sun, Daqian; Guardavaccaro, Daniele; Pagano, Michele; Zhu, Liang
2006 Aug 18;281(33):24058-24069, Journal of biological chemistry
Skp2 is well known as the F-box protein of the SCF(Skp2).Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interaction. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 compromises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mechanism of Skp2 and a new regulatory mechanism of cyclin A
— id: 66921, year: 2006, vol: 281, page: 24058, stat: Journal Article,

Cell Division, a new open access online forum for and from the cell cycle community
Kaldis, Philipp; Pagano, Michele
2006 ;1(1):1-1, Cell division
ABSTRACT : Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases
— id: 66922, year: 2006, vol: 1, page: 1, stat: Journal Article,

Degradation of Id2 by the anaphase-promoting complex couples cell cycle exit and axonal growth
Lasorella, Anna; Stegmuller, Judith; Guardavaccaro, Daniele; Liu, Guangchao; Carro, Maria S; Rothschild, Gerson; de la Torre-Ubieta, Luis; Pagano, Michele; Bonni, Azad; Iavarone, Antonio
2006 Jul 27;442(7101):471-474, Nature
In the developing nervous system, Id2 (inhibitor of DNA binding 2, also known as inhibitor of differentiation 2) enhances cell proliferation, promotes tumour progression and inhibits the activity of neurogenic basic helix-loop-helix (bHLH) transcription factors. The anaphase promoting complex/cyclosome and its activator Cdh1 (APC/C(Cdh1)) restrains axonal growth but the targets of APC/C(Cdh1) in neurons are unknown. Id2 and other members of the Id family are very unstable proteins that are eliminated as cells enter the quiescent state, but how they are targeted for degradation has remained elusive. Here we show that Id2 interacts with the core subunits of APC/C and Cdh1 in primary neurons. APC/C(Cdh1) targets Id2 for degradation through a destruction box motif (D box) that is conserved in Id1 and Id4. Depletion of Cdh1 stabilizes Id proteins in neurons, whereas Id2 D-box mutants are impaired for Cdh1 binding and remain stable in cells that exit from the cell cycle and contain active APC/C(Cdh1). Mutants of the Id2 D box enhance axonal growth in cerebellar granule neurons in vitro and in the context of the cerebellar cortex, and overcome the myelin inhibitory signals for growth. Conversely, activation of bHLH transcription factors induces a cluster of genes with potent axonal inhibitory functions including the gene coding for the Nogo receptor, a key transducer of myelin inhibition. Degradation of Id2 in neurons permits the accumulation of the Nogo receptor, thereby linking APC/C(Cdh1) activity with bHLH target genes for the inhibition of axonal growth. These findings indicate that deregulated Id activity might be useful to reprogramme quiescent neurons into the axonal growth mode
— id: 66920, year: 2006, vol: 442, page: 471, stat: Journal Article,

Response: More money, and less time!
Pagano, M
2006 NOV 17 ;127(4):664-665, Cell
— id: 69456, year: 2006, vol: 127, page: 664, stat: Journal Article,

American Idol and NIH Grant Review
Pagano, Michele
2006 Aug 25;126(4):637-638, Cell
— id: 72424, year: 2006, vol: 126, page: 637, stat: Journal Article,

SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response
Peschiaroli, Angelo; Dorrello, N Valerio; Guardavaccaro, Daniele; Venere, Monica; Halazonetis, Thanos; Sherman, Nicholas E; Pagano, Michele
2006 Aug 4;23(3):319-329, Molecular cell
During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint
— id: 66918, year: 2006, vol: 23, page: 319, stat: Journal Article,

A peptidomimetic siRNA transfection reagent for highly effective gene silencing
Utku, Yeliz; Dehan, Elinor; Ouerfelli, Ouathek; Piano, Fabio; Zuckermann, Ronald N; Pagano, Michele; Kirshenbaum, Kent
2006 Jun;2(6-7):312-317, Molecular bioSystems
RNA interference (RNAi) techniques hold forth great promise for therapeutic silencing of deleterious genes. However, clinical applications of RNAi require the development of safe and efficient methods for intracellular delivery of small interfering RNA (siRNA) oligonucleotides specific to targeted genes. We describe the use of a lipitoid, a cationic oligopeptoid-phospholipid conjugate, for non-viral transfection of synthetic siRNA oligos in cell culture. This peptidomimetic delivery vehicle allows for efficient siRNA transfection in a variety of human cell lines with negligible toxicity and promotes extensive downregulation of the targeted genes at both the protein and the mRNA level. We compare the lipitoid reagent to a standard commercial transfection reagent. The lipitoid is highly efficient even in primary IMR-90 human lung fibroblasts in which other commercial reagents are typically ineffective
— id: 66919, year: 2006, vol: 2, page: 312, stat: Journal Article,

Cdk1: the dominant sibling of Cdk2
Bashir, Tarig; Pagano, Michele
2005 Aug;7(8):779-781, Nature cell biology
— id: 64218, year: 2005, vol: 7, page: 779, stat: Journal Article,

Involvement of the SCF complex in the control of Cdh1 degradation in S-phase
Benmaamar, Ramla; Pagano, Michele
2005 Sep;4(9):1230-1232, Cell cycle
The anaphase promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that acts as a key regulator in the progression through mitosis (when mostly in complex with Cdc20) and as a stabilizer of the G1 phase (when in complex with Cdh1). Cdh1 is an activator of APC/C, and it has previously been reported that it is capable of mediating its own degradation during Go and G1. Herein, we show that the SCF complex (Skp1/Cul1/F-box protein/Roc1) intervenes in the surveillance of Cdh1 cellular abundance in S-phase
— id: 64217, year: 2005, vol: 4, page: 1230, stat: Journal Article,

The acidic tail domain of human Cdc34 is required for p27Kip1 ubiquitination and complementation of a cdc34 temperature sensitive yeast strain
Block, Karen; Appikonda, Srikanth; Lin, Horng-Ru; Bloom, Joanna; Pagano, Michele; Yew, P Renee
2005 Oct;4(10):1421-1427, Cell cycle
Human Cdc34 is an ubiquitin conjugating enzyme or E2 that ubiquitinates substrates including p27(Kip1), IkappaBalpha, Wee1, and MyoD. Cdc34 possesses a core catalytic domain encoding the active site cysteine and an acidic tail domain within the carboxyl terminal 36 amino acids. Studies suggest that Cdc34 is phosphorylated in mammalian cells at 5 potential residues within the tail domain. In order to study the biological significance of the Cdc34 acidic tail domain and the possible significance of phosphorylation within this region, we tested the ability of human Cdc34 mutants to complement the cdc34-2 temperature sensitive (ts) strain of Saccharomyces cerevisiae. Our studies indicated that complementation of the cdc34-2 ts strain was critically dependent upon the carboxyl-terminal 36 amino acids of human Cdc34, but did not require phosphorylation of human Cdc34 residues S203, S222, S231, T233, and S236. Further studies demonstrated that although a Cdc34 mutant bearing a deletion of the C-terminal 36 amino acids (Cdc34 1-200) was efficiently charged with ubiquitin by E1, it was severely reduced for the ability to ubiquitinate p27(Kip1) in vitro compared to wildtype Cdc34. Both in vivo and in vitro binding studies indicated that Cdc34 1-200 bound to the E3-SCF components, Cul1 and Roc1, at levels comparable to the wildtype Cdc34. These studies suggest that the 36 amino acid acidic tail domain of human Cdc34 is critical for its ability to transfer ubiquitin to a substrate and is dispensable for the association of Cdc34 with Cul1 and Roc1. We postulate that the tail domain of Cdc34 may be important for its efficient dissociation from Cul1 and Roc1, an essential requirement for ubiquitination by the budding yeast Cdc34p, or it may be required more directly for ubiquitin transfer to the substrate
— id: 64216, year: 2005, vol: 4, page: 1421, stat: Journal Article,

Experimental tests to definitively determine ubiquitylation of a substrate
Bloom, Joanna; Pagano, Michele
2005 ;399:249-266, Methods in enzymology
Ubiquitin-mediated proteolysis is a major pathway of protein degradation that regulates numerous cellular processes. An understanding of the circumstances that contribute to the ubiquitylation of a specific protein can yield vast insight into its regulation. This article examines multiple procedures that explain whether a protein is ubiquitylated and suggests methods to investigate the factors that specifically target the substrate for ubiquitylation, as well as the site of ubiquitin conjugation
— id: 64215, year: 2005, vol: 399, page: 249, stat: Journal Article,

Skp2, the FoxO1 hunter
Dehan, Elinor; Pagano, Michele
2005 Mar;7(3):209-210, Cancer cell
Skp2 is an oncoprotein that mediates the degradation of several negative regulators of the cell cycle to promote cell proliferation. A recent report by Huang and colleagues reveals that Skp2 directs the ubiquitylation and subsequent degradation of FoxO1, a member of the FoxO family of transcription factors. Since FoxO proteins possess tumor suppressor functions, this new finding suggests a new mechanism by which Skp2 may favor tumorigenesis
— id: 51787, year: 2005, vol: 7, page: 209, stat: Journal Article,

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase
Hao, Bing; Zheng, Ning; Schulman, Brenda A; Wu, Geng; Miller, Julie J; Pagano, Michele; Pavletich, Nikola P
2005 Oct 7;20(1):9-19, Molecular cell
The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex
— id: 64214, year: 2005, vol: 20, page: 9, stat: Journal Article,

Previous preterm cesarean delivery: identification of a new risk factor for uterine rupture in VBAC candidates
Rochelson, Burton; Pagano, Michelle; Conetta, Laurie; Goldman, Benjamin; Vohra, Nidhi; Frey, Michael; Day, Catherine
2005 Nov;18(5):339-342, Journal of maternal-fetal & neonatal medicine
OBJECTIVE: A major risk of trials of labor in patients with prior cesarean delivery is uterine rupture. We evaluated the question of whether a previous cesarean delivery at an early gestational age predisposes the patient to subsequent uterine rupture. METHODS: This was a retrospective chart review of patients delivering at North Shore University Hospital with a trial of labor after previous cesarean delivery to ascertain all cases of uterine rupture. Patients who had had a previous cesarean delivery at our institution who did not suffer uterine rupture during a trial of labor served as controls. RESULTS: Twenty-five patients suffered a uterine rupture. The incidence of prior preterm cesarean delivery (PPCD) in this group was 40%, compared to 10.9% of 691 laboring vaginal birth after cesarean (VBAC) patients without rupture (p < 0.001). Patients in the rupture group with a PPCD were less likely to have experienced labor in the index pregnancy and more likely to have had an interdelivery interval of less than two years. CONCLUSIONS: An undeveloped lower segment in the preterm uterus represents a risk for later rupture, even if the incision is transverse
— id: 62636, year: 2005, vol: 18, page: 339, stat: Journal Article,

Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase
Bashir, Tarig; Dorrello, N Valerio; Amador, Virginia; Guardavaccaro, Daniele; Pagano, Michele
2004 Mar 11;428(6979):190-193, Nature
Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state
— id: 42119, year: 2004, vol: 428, page: 190, stat: Journal Article,

Don't skip the G(1) phase: How APC/C(Cdh1) Keeps SCF(Skp2) in Check
Bashir, Tarig; Pagano, Michele
2004 Jul 14;3(7):1556-1560, Cell cycle
By keeping the levels of Skp2 and Cks1 low during G(1) progression, APC/C(Cdh1) prevents unscheduled degradation of SCF(Skp2) substrates and premature entry into S phase. Thus, APC/C(Cdh1), a ubiquitin ligase involved in mitotic exit and maintenance of G(0)/G(1) phase, directly controls SCF(Skp2), a ubiquitin ligase involved in the regulation of S phase entry
— id: 44902, year: 2004, vol: 3, page: 1556, stat: Journal Article,

Varshavsky's contributions
Baumeister, Wolfgang; Bachmair, Andreas; Chau, Vincent; Cohen, Robert; Coffino, Phil; Demartino, George; Deshaies, Raymond; Dohmen, Juergen; Emr, Scott; Finley, Daniel; Hampton, Randy; Hill, Christopher; Hochstrasser, Mark; Huber, Robert; Jackson, Peter; Jentsch, Stefan; Johnson, Erica; Kwon, Yong Tae; Pagano, Michele; Pickart, Cecile; Rechsteiner, Martin; Scheffner, Martin; Sommer, Thomas; Tansey, William; Tyers, Mike; Vierstra, Richard; Weissman, Allan; Wilkinson, Keith D; Wolf, Dieter
2004 Nov 19;306(5700):1290-1292, Science
— id: 64220, year: 2004, vol: 306, page: 1290, stat: Journal Article,

Ubiquitin-dependent degradation of p73 is inhibited by PML
Bernassola, Francesca; Salomoni, Paolo; Oberst, Andrew; Di Como, Charles J; Pagano, Michele; Melino, Gerry; Pandolfi, Pier Paolo
2004 Jun 7;199(11):1545-1557, Journal of experimental medicine
p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression
— id: 45027, year: 2004, vol: 199, page: 1545, stat: Journal Article,

To be or not to be ubiquitinated?
Bloom, Joanna; Pagano, Michele
2004 Mar-Apr;3(2):138-140, Cell cycle
Levels of p21, a cyclin-dependent kinase (CDK) inhibitor, are controlled in part at the post-translational level by protein degradation. Although the signaling pathways leading to p21 degradation have not yet been fully elucidated, it is evident that p21 ubiquitination is an essential factor in its degradation. We discuss that, with the only notable exception of ornithine decarboxylase, ubiquitination appears to be a prerequisite for proteasomal degradation rather than an unnecessary byproduct of such proteolysis
— id: 42120, year: 2004, vol: 3, page: 138, stat: Journal Article,

The SCF ubiquitin ligase: insights into a molecular machine
Cardozo, Timothy; Pagano, Michele
2004 Sep;5(9):739-751, Nature reviews. Molecular cell biology
Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies
— id: 45024, year: 2004, vol: 5, page: 739, stat: Journal Article,

Oncogenic aberrations of cullin-dependent ubiquitin ligases
Guardavaccaro, Daniele; Pagano, Michele
2004 Mar 15;23(11):2037-2049, Oncogene
Accumulating evidence points to a key role of the ubiquitin-proteasome pathway in oncogenesis. Aberrant proteolysis of substrates involved in cellular processes such as the cell division cycle, gene transcription, the DNA damage response and apoptosis has been reported to contribute significantly to neoplastic transformation. Cullin-dependent ubiquitin ligases (CDLs) form a class of structurally related multisubunit enzymes central to the ubiquitin-mediated proteolysis of many important biological substrates. In this review, we describe the role of CDLs in the ubiquitinylation of cancer-related substrates and discuss how altered ubiquitinylation by CDLs may contribute to tumor development
— id: 42579, year: 2004, vol: 23, page: 2037, stat: Journal Article,

An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a partial-penetrance Rb mutant
Ji, Peng; Jiang, Hong; Rekhtman, Katya; Bloom, Joanna; Ichetovkin, Marina; Pagano, Michele; Zhu, Liang
2004 Oct 8;16(1):47-58, Molecular cell
It is believed that Rb blocks G1-S transition by inhibiting expression of E2F regulated genes. Here, we report that the effects of E2F repression lag behind the onset of G1 cell cycle arrest in timed Rb reexpression experiments. In comparison, kinase inhibitor p27Kip1 protein accumulates with a faster kinetics. Conversely, Rb knockout leads to faster p27 degradation. Rb interacts with the N terminus of Skp2, interferes with Skp2-p27 interaction, and inhibits ubiquitination of p27. Disruption of p27 function or expression of the Skp2 N terminus prevents Rb from causing G1 arrest. A full-penetrance, inactive Rb mutant fails to interfere with Skp2-p27 interaction but, interestingly, a partial-penetrance Rb mutant that is defective for E2F binding retains full activity in inhibiting Skp2-p27 interaction and can induce G1 cell cycle arrest with wild-type kinetics. These results identify an Rb-Skp2-p27 pathway in Rb function, which may be involved in inhibition of tumor progression
— id: 64223, year: 2004, vol: 16, page: 47, stat: Journal Article,

Systematic analysis and nomenclature of mammalian F-box proteins
Jin, Jianping; Cardozo, Timothy; Lovering, Ruth C; Elledge, Stephen J; Pagano, Michele; Harper, J Wade
2004 Nov 1;18(21):2573-2580, Genes & development
— id: 64222, year: 2004, vol: 18, page: 2573, stat: Journal Article,

Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation
Kitajima, Shojiro; Kudo, Yasusei; Ogawa, Ikuko; Bashir, Tarig; Kitagawa, Masae; Miyauchi, Mutsumi; Pagano, Michele; Takata, Takashi
2004 Dec;165(6):2147-2155, American journal of pathology
Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy
— id: 64219, year: 2004, vol: 165, page: 2147, stat: Journal Article,

Role of F-Box Protein betaTrcp1 in mammary gland development and tumorigenesis
Kudo, Yasusei; Guardavaccaro, Daniele; Santamaria, Patricia G; Koyama-Nasu, Ryo; Latres, Esther; Bronson, Roderick; Yamasaki, Lili; Pagano, Michele
2004 Sep;24(18):8184-8194, Molecular & cellular biology
The F-box protein betaTrcp1 controls the stability of several crucial regulators of proliferation and apoptosis, including certain inhibitors of the NF-kappaB family of transcription factors. Here we show that mammary glands of betaTrcp1(-/-) female mice display a hypoplastic phenotype, whereas no effects on cell proliferation are observed in other somatic cells. To investigate further the role of betaTrcp1 in mammary gland development, we generated transgenic mice expressing human betaTrcp1 targeted to epithelial cells under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. Compared to controls, MMTV betaTrcp1 mammary glands display an increase in lateral ductal branching and extensive arrays of alveolus-like protuberances. The mammary epithelia of MMTV betaTrcp1 mice proliferate more and show increased NF-kappaB DNA binding activity and higher levels of nuclear NF-kappaB p65/RelA. In addition, 38% of transgenic mice develop tumors, including mammary, ovarian, and uterine carcinomas. The targeting of betaTrcp1 to lymphoid organs produces no effects on these tissues. In summary, our results support the notion that betaTrcp1 positively controls the proliferation of breast epithelium and indicate that alteration of betaTrcp1 function and expression may contribute to malignant behavior of breast tumors, at least in part through NF-kappaB transactivation
— id: 45025, year: 2004, vol: 24, page: 8184, stat: Journal Article,

Fenton's pre-treatment of mature landfill leachate
Lopez, Antonio; Pagano, Michele; Volpe, Angela; Di Pinto, Appio Claudio
2004 Feb;54(7):1005-1010, Chemosphere
The aim of this study was to check the effectiveness of the Fenton's reagent (Fe2+ + H2O2 + H+) for the pre-treatment of a municipal landfill leachate with the objective of improving its overall biodegradability, evaluated in terms of BOD5/COD ratio, up to a value compatible with biological treatment. The leachate came from a municipal sanitary landfill located in southern Italy and the average values of its main parameters were: pH=8.2; COD=10,540 mgl(-1); BOD5=2,300 mgl(-1); TOC=3,900 mgl(-1); NH4-N=5210 mgl(-1); conductivity=45,350 microScm(-1); alkalinity=21,470 mgl(-1) CaCO3. The effect of initial pH value on the pre-treatment effectiveness was evaluated by titrating the amount of acidic by-products formed. The extent of leachate oxidation was monitored and controlled by both pH and redox potential measurements. The best operational conditions for achieving the desired goal (i.e., BOD5/COD> or =0.5) resulted: Fe2+=275 mgl(-1); H2O2=3,300 mgl(-1); initial pH=3; reaction time=2 h. At the end of the Fenton's pre-treatment, in order to permit a subsequent biological treatment, residual ferric ions were removed increasing the pH up to 8.5 by adding 3 gl(-1) of Ca(OH)2 and 3 mgl(-1) of a cationic polyelectrolyte, the latter as an aid to coagulation. This final step also resulted in a further modest removal of residual COD due to co-precipitation phenomena
— id: 64226, year: 2004, vol: 54, page: 1005, stat: Journal Article,

Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome
Moshe, Yakir; Boulaire, Jerome; Pagano, Michele; Hershko, Avram
2004 May 25;101(21):7937-7942, Proceedings of the National Academy of Sciences of the United States of America
Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis
— id: 64224, year: 2004, vol: 101, page: 7937, stat: Journal Article,

Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/2 axis
Pagano, Michele
2004 May 21;14(4):414-416, Molecular cell
A new study reveals a novel role for p27 in inhibiting Cdk1 activity at G2/M and shows that p27 deficiency almost completely rescues the aberrations observed in Skp2(-/-) mice, demonstrating that p27 is the principal downstream effector of the SCF(Skp2) ubiquitin ligase
— id: 45028, year: 2004, vol: 14, page: 414, stat: Journal Article,

Wagging the dogma; tissue-specific cell cycle control in the mouse embryo
Pagano, Michele; Jackson, Peter K
2004 Sep 3;118(5):535-538, Cell
The family of cyclin-dependent kinases (Cdks) lies at the core of the machinery that drives the cell division cycle. Studies in cultured mammalian cells have provided insight into the cellular functions of many Cdks. Recent Cdk and cyclin knockouts in the mouse show that the functions of G1 cell cycle regulatory genes are often essential only in specific cell types, pointing to our limited understanding of tissue-specific expression, redundancy, and compensating mechanisms in the Cdk network
— id: 45026, year: 2004, vol: 118, page: 535, stat: Journal Article,

Alterations in the expression of the cell cycle regulatory protein cyclin kinase subunit 1 in colorectal carcinoma
Shapira, Ma'anit; Ben-Izhak, Ofer; Bishara, Bishara; Futerman, Boris; Minkov, Ira; Krausz, Michael M; Pagano, Michele; Hershko, Dan D
2004 Apr 15;100(8):1615-1621, Cancer
BACKGROUND: Low levels of p27(Kip1) are associated with high aggressiveness and poor prognosis in various malignancies, including colorectal carcinoma. The authors showed that S phase kinase protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27(Kip1) for degradation, was overexpressed and was inversely related to p27(Kip1) levels in patients with colorectal carcinoma. The essential role of cyclin kinase subunit 1 (Cks1) in Skp2-dependent p27 degradation was recently discovered, but its role in human malignancies is unknown. METHODS: Quick-frozen colorectal tumor samples from 30 patients were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Cks1, Skp2, and p27(Kip1). The expression of Cks1 was also examined by immunohistochemistry using formalin-fixed, paraffin-embedded tissue sections from the same patients. RESULTS: A strong correlation was found between Cks1 levels and Skp2 expression and loss of tumor differentiation. A significant inverse relation was also observed between levels of Cks1 and p27(Kip1) and overall survival. CONCLUSIONS: The results of the current study suggest that increased expression of Cks1 may have an important causative role in decreasing levels of p27 in patients with aggressive colorectal carcinoma
— id: 64225, year: 2004, vol: 100, page: 1615, stat: Journal Article,

Cell cycle, proteolysis and cancer
Yamasaki, Lili; Pagano, Michele
2004 Dec;16(6):623-628, Current opinion in cell biology
Research in the past 15 years has shown that the mammalian cell cycle is controlled by the action of cyclin-dependent kinases (CDKs). A crucial substrate of the CDKs in G1-phase is the retinoblastoma tumor suppressor (pRB), which restrains proliferation largely by repressing the activity of the E2F transcription factors. More recent work has shown that the cell cycle is also a tale of two classes of ubiquitin ligases, referred to as SCF and APC/C ligases. CDKs, E2F and ubiquitin ligases reciprocally regulate each other, resulting in complex feedback loops. Perturbation of this network of molecular machines is associated with proliferative diseases, including cancer
— id: 64221, year: 2004, vol: 16, page: 623, stat: Journal Article,

Aberrant ubiquitin-mediated proteolysis of cell cycle regulatory proteins and oncogenesis
Bashir, Tarig; Pagano, Michele
2003 ;88(Pt 1):101-144, Advances in cancer research
The ubiquitin pathway plays a central role in the regulation of cell growth and cell proliferation by controlling the abundance of key cell cycle proteins. Increasing evidence indicates that unscheduled proteolysis of many cell cycle regulators contributes significantly to tumorigenesis and is indeed found in many types of human cancers. Aberrant proteolysis with oncogenic potential is elicited by two major mechanisms: defective degradation of positive cell cycle regulators (i.e., proto-oncoproteins) and enhanced degradation of negative cell cycle regulators (i.e., tumor suppressor proteins). In many cases, increased protein stability is a result of mutations in the substrate that prevent the recognition of the protein by the ubiquitin-mediated degradation machinery. Alternatively, the specific recognition proteins mediating ubiquitination (ubiquitin ligases) are not expressed or harbor mutations rendering them inactive. In contrast, the overexpression of a ubiquitin ligase may result in the enhanced degradation of a negative cell cycle regulator. This chapter aims to review the involvement of the ubiquitin pathway in the scheduled destruction of some important cell cycle regulators and to discuss the implications of their aberrant degradation for the development of cancer
— id: 39260, year: 2003, vol: 88, page: 101, stat: Journal Article,

Proteasome-Mediated Degradation of p21 via N-Terminal Ubiquitinylation
Bloom, Joanna; Amador, Virginia; Bartolini, Francesca; DeMartino, George; Pagano, Michele
2003 Oct 3;115(1):71-82, Cell
We examined the mechanism responsible for the degradation of p21, a negative regulator of the cell division cycle. We found that p21 proteolysis requires functional ubiquitin and Nedd8 systems. Ubiquitinylated forms of p21 and p21(K0), a p21 mutant missing all lysines, are detected in vivo and in vitro, showing that the presence of lysines is dispensable for p21 ubiquitinylation. Instead, the free amino group of the N-terminal methionine of p21 is a site for ubiquitinylation in vivo. Although wild-type p21 is more abundantly ubiquitinylated than p21(K0) mutant due to the presence of internal lysine residues, their rates of proteolysis are indistinguishable. These results demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome
— id: 38122, year: 2003, vol: 115, page: 71, stat: Journal Article,

Deregulated degradation of the cdk inhibitor p27 and malignant transformation
Bloom, Joanna; Pagano, Michele
2003 Feb;13(1):41-47, Seminars in cancer biology
p27 acts as a critical negative regulator of the cell cycle by inhibiting the activity of cyclin/cdk complexes during G0 and G1. Degradation of p27 is a critical event for the G1/S transition and occurs through ubiquitination by SCF(Skp2) and subsequent degradation by the 26S-proteasome. A tumor suppressing function of p27 has been demonstrated in mouse models and studies of human tumors. More recent evidence suggests that Skp2, the specific recognition factor for p27 ubiquitination, has oncogenic properties. This review will focus on the regulation of p27 proteolysis and its consequences for tumorigenesis
— id: 39340, year: 2003, vol: 13, page: 41, stat: Journal Article,

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase
Bornstein, Gil; Bloom, Joanna; Sitry-Shevah, Danielle; Nakayama, Keiko; Pagano, Michele; Hershko, Avram
2003 Jul 11;278(28):25752-25757, Journal of biological chemistry
The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase
— id: 64228, year: 2003, vol: 278, page: 25752, stat: Journal Article,

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage
Busino, Luca; Donzelli, Maddalena; Chiesa, Massimo; Guardavaccaro, Daniele; Ganoth, Dvora; Dorrello, N Valerio; Hershko, Avram; Pagano, Michele; Draetta, Giulio F
2003 Nov 6;426(6962):87-91, Nature
The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation
— id: 42121, year: 2003, vol: 426, page: 87, stat: Journal Article,

Altered expression of p27 and Skp2 proteins in prostate cancer of African-American patients
Drobnjak, Marija; Melamed, Jonathan; Taneja, Samir; Melzer, Kate; Wieczorek, Rosemary; Levinson, Benjamin; Zeleniuch-Jacquotte, Anne; Polsky, David; Ferrara, Jay; Perez-Soler, Roman; Cordon-Cardo, Carlos; Pagano, Michele; Osman, Iman
2003 Jul;9(7):2613-2619, Clinical cancer research
PURPOSE: The purpose is to investigate the clinical relevance of altered patterns of p27 and Skp2 expression in African-American patients with localized prostate cancer. The abundance of p27, an inhibitor of cell proliferation, is controlled by Skp2-dependent proteolysis. EXPERIMENTAL DESIGN: A well-characterized cohort of 162 African-Americans who underwent radical prostatectomy at the Veterans Affairs Medical Center of New York between 1990 and 2000 was studied. We analyzed p27 and Skp2 expression by immunohistochemistry. Altered expression of p27 (defined as <40% tumor cells expressing the protein) and Skp2 (defined as > or ==' BORDER='0'>20% tumor cells expressing the protein) were correlated with clinicopathological parameters and time to prostate-specific antigen (PSA) recurrence. RESULTS: Altered expression of p27 and Skp2 was observed in 112 of 162 (69.1%) and 93 of 162 (57.4%) cases, respectively. Inverse patterns of Skp2 and p27 protein expression were seen in 87 of 162 (53.7%) cases. A marginally significant association was found between Skp2 overexpression and extracapsular extension (P = 0.065). Moreover, patients with Skp2 overexpression had a 2.77 years decreased median time to PSA recurrence compared with patients with low Skp2 expression; however, the difference was not statistically significant. In multivariate analysis, only tumor grade and stage independently predicted PSA recurrence in this cohort. CONCLUSIONS: Our data suggest a role for Skp2 overexpression in prostate cancer pathogenesis that might not be exclusively related to p27 degradation. More studies are needed to determine the mechanistic role of Skp2 in prostate cancer
— id: 38153, year: 2003, vol: 9, page: 2613, stat: Journal Article,

Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo
Guardavaccaro, Daniele; Kudo, Yasusei; Boulaire, Jerome; Barchi, Marco; Busino, Luca; Donzelli, Maddalena; Margottin-Goguet, Florence; Jackson, Peter K; Yamasaki, Lili; Pagano, Michele
2003 Jun;4(6):799-812, Developmental cell
SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events
— id: 39205, year: 2003, vol: 4, page: 799, stat: Journal Article,

Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration independent of cell cycle arrest functions
McAllister, Sandra S; Becker-Hapak, Michelle; Pintucci, Giuseppe; Pagano, Michele; Dowdy, Steven F
2003 Jan;23(1):216-228, Molecular & cellular biology
Hepatocyte growth factor (HGF) signaling via its receptor, the proto-oncogene Met, alters cell proliferation and motility and has been associated with tumor metastasis. HGF treatment of HepG2 human hepatocellular carcinoma cells induces cell migration concomitant with increased levels of the p27(kip1) cyclin-cdk inhibitor. HGF signaling resulted in nuclear export of endogenous p27 to the cytoplasm, via Ser-10 phosphorylation, where it colocalized with F-actin. Introduction of transducible p27 protein (TATp27) was sufficient for actin cytoskeletal rearrangement and migration of HepG2 cells. TATp27 mutational analysis identified a novel p27 C-terminal domain required for cell migration, distinct from the N-terminal cyclin-cyclin-dependent kinase (cdk) binding domain. Loss or disruption of the p27 C-terminal domain abolished both actin rearrangement and cell migration. The cell-scattering activity of p27 occurred independently of its cell cycle arrest functions and required cytoplasmic localization of p27 via Ser-10 phosphorylation. Furthermore, Rac GTPase was necessary for p27-dependent migration but alone was insufficient for HepG2 cell migration. These results predicted a migration defect in p27-deficient cells. Indeed, p27-deficient primary fibroblasts failed to migrate, and reconstitution with TATp27 rescued the motility defect. These observations define a novel role for p27 in cell motility that is independent of its function in cell cycle inhibition
— id: 64229, year: 2003, vol: 23, page: 216, stat: Journal Article,

When protein destruction runs amok, malignancy is on the loose
Pagano, Michele; Benmaamar, Ramla
2003 Oct;4(4):251-256, Cancer cell
Ubiquitin-dependent proteolysis ensures that specific protein functions are turned off at the right time, in the right place, and in a unidirectional fashion. The high substrate specificity of the system is determined by a large family of ubiquitin ligases, which competes with the protein kinases to be the largest family of enzymes in mammals. Given the crucial function of the proteolytic machinery, altered degradation of cellular regulators contributes to the unchecked proliferation typical of cancer cells. Here we review the aberrant activity of a variety of ubiquitin ligases in human cancer, hence the prospect of targeting them in cancer therapy
— id: 38583, year: 2003, vol: 4, page: 251, stat: Journal Article,

Low prepregnancy ideal weight:height ratio in women with hyperemesis gravidarum
Rochelson, Burton; Vohra, Nidhi; Darvishzadeh, J; Pagano, Michelle
2003 Jun;48(6):422-424, Journal of reproductive medicine
OBJECTIVE: To determine if a low prepregnancy weight:height ratio is a risk factor for hyperemesis. STUDY DESIGN: A retrospective chart review was conducted on 38 patients admitted to North Shore University Hospital for hyperemesis over a 5-year period. Prepregnancy weight, body mass index and percent ideal weight:height ratio in this group were compared to findings on 79 randomly selected control patients not admitted for hyperemesis. RESULTS: There was a significantly higher incidence of prepregnancy underweight women (low percent ideal weight for height) in patients admitted for hyperemesis as compared to controls (P < .01). Thyroid-stimulating hormone was low in 19% of patients tested. CONCLUSION: A low prepregnancy percent ideal weight:height ratio may predispose women to the development of hyperemesis
— id: 94035, year: 2003, vol: 48, page: 422, stat: Journal Article,

Olive husk: an alternative sorbent for removing heavy metals from aqueous streams
Volpe, Angela; Lopez, Antonio; Pagano, Michele
2003 Sep;110(3):137-149, Applied biochemistry & biotechnology
Sorption properties of olive husk were investigated under equilibrium (batch tests) and dynamic (column tests) conditions in order to assess the possibility of using such a waste material for removing heavy metals from aqueous streams. Husk samples were contacted, at 25 degrees C, with aqueous solutions of nitric salts of Pb, Cd, Cu, and Zn. Sorption isotherms obtained from equilibrium data were fitted and interpreted by the Freundlich model. Metals-saturated husk samples resulting from column tests were air-dried and incinerated to simulate combustion in order to assess the fate of sorbed metals. The results demonstrated that, under both equilibrium and dynamic conditions, metal sorption capacity of the husk was in the sequence Pb>Cd>Cu>Zn. For all the metals, calculated Freundlich constants decreased by increasing initial metal concentration or decreasing solution pH. In dynamic tests, a significant reduction of sorption capacity was recorded (except for copper) when a metal was fed simultaneously to the others: Pb (77%); Cd (93%); Zn (68%). Combustion tests carried out on metals-saturated husk samples showed that the average losses of lead and cadmium, as volatile species, were always three to four times greater than the losses of copper and zinc, in both single-metal- and multimetal-saturated samples
— id: 64227, year: 2003, vol: 110, page: 137, stat: Journal Article,

Nutritional characteristics of a rural Southern Italy population: the Ventimiglia di Sicilia Project
Barbagallo, Carlo M; Cavera, Giovanni; Sapienza, Michelangelo; Noto, Davide; Cefalu, Angelo B; Polizzi, Francesco; Onorato, Francesco; Rini, GiovanBattista; Di Fede, Gaetana; Pagano, Michele; Montalto, Giuseppe; Rizzo, Manfredi; Descovich, GianCarlo; Notarbartolo, Alberto; Averna, Maurizio R
2002 Dec;21(6):523-529, Journal of the American College of Nutrition
OBJECTIVE: Knowledge of alimentary habits among populations permits a better definition of appropriate public health interventions. We designed the epidemiological project 'Ventimiglia di Sicilia' to characterize the risk profile in a rural village with low total cholesterol levels and low early cardiovascular mortality but with a large prevalence of overweight and obesity, which previously have been significantly associated with total mortality. METHODS: 488 individuals of age 20 to 69 years were included in the dietary survey conducted by a seven-day food record. RESULTS: Alimentary habits were characterized by high consumption of total and complex carbohydrates (respectively 52.5 +/- 7.6% and 46.6 +/- 8.2% of daily energy) and by a low cholesterol intake (92.5 +/- 35.0 mg/1000 kcal/day). Fat intake was 34.7 +/- 7.7% of daily energy due to a higher consumption of monounsaturated fats in respect to saturated fats (respectively 20.5 +/- 5.1% and 10.2 +/- 2.9% of daily energy). In particular, in this population there was a large consumption of bread, pasta, fresh vegetables, olive oil and fruits. We also observed an excess of total calories (about 2900 kcal/day in men and 2100 kcal/day in women) not balanced by a high degree of physical activity levels. Furthermore we found a significant higher total and saturated fat consumption in the youngest individuals and in people with higher educational levels. CONCLUSIONS: Dietary habits of Ventimiglia di Sicilia still follow the nutritional characteristics typical of the Mediterranean diet. The high total calorie intake indicates a quantitative more than qualitative problem, which may account the large prevalence of overweight and obesity and may represent a public health issue that needs to be corrected in such a rural population
— id: 64230, year: 2002, vol: 21, page: 523, stat: Journal Article,

Butyrolactone: more than a kinase inhibitor?
Bloom, Joanna; Pagano, Michele
2002 Mar-Apr;1(2):117-118, Cell cycle
— id: 39371, year: 2002, vol: 1, page: 117, stat: Journal Article,

S-phase kinase-associated protein 2 expression in non-Hodgkin's lymphoma inversely correlates with p27 expression and defines cells in S phase
Chiarle, Roberto; Fan, Yan; Piva, Roberto; Boggino, Hugo; Skolnik, Jeffrey; Novero, Domenico; Palestro, Giorgio; De Wolf-Peeters, Chris; Chilosi, Marco; Pagano, Michele; Inghirami, Giorgio
2002 Apr;160(4):1457-1466, American journal of pathology
The protein expression of the cyclin-dependent kinase inhibitor p27 is often deregulated in human tumors. In lymphomas the inactivation of p27 is achieved through either increased degradation(1) or sequestration via D cyclins,(2) and p27 protein levels have been shown to have a prognostic significance.(1,3) Recently, S-phase kinase-associated protein 2 (Skp2) has been proved to mediate p27 degradation in normal cells(4-7) and to have oncogenetic properties.(8,9) In this study, B-, T-, and myeloid hematopoietic cell lines and a well-characterized panel of human lymphomas (n = 244) were studied for the expression of Skp2. In human lymphomas, the expression of Skp2 strongly related to the grade of malignancy, being low in indolent tumors and very high in aggressive lymphomas. Moreover, the percentages of Skp2- and S-phase-positive cells, as measured by DNA content or BrdU labeling, strictly matched and closely parallel that of Ki-67 and cyclin A. An inverse correlation between Skp2 and p27 was found in the majority of lymphoma subtypes. Nonetheless, most mantle cell lymphomas and a subset of diffuse large cell lymphomas failed to show this correlation, suggesting that alternative pathway(s) for the regulation of p27 might exist. The detection of Skp2 protein either by flow cytometry or by immunohistochemistry represents a simple method to precisely assess the S phase of lymphomas. The potential diagnostic and prognostic value of Skp2 is discussed
— id: 39682, year: 2002, vol: 160, page: 1457, stat: Journal Article,

Dual mode of degradation of Cdc25 A phosphatase
Donzelli, Maddalena; Squatrito, Massimo; Ganoth, Dvora; Hershko, Avram; Pagano, Michele; Draetta, Giulio F
2002 Sep 16;21(18):4875-4884, EMBO journal
The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment
— id: 64231, year: 2002, vol: 21, page: 4875, stat: Journal Article,

CDC25 phosphatases and checkpoint controls
Draetta, G; Donzelli, M; Squatrito, M; Ganoth, D; Hershko, A; Pagano, M
2002 NOV abstract #386;38(5):S116-S116, European journal of cancer
— id: 36591, year: 2002, vol: 38, page: S116, stat: Journal Article,

In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation
Piva, Roberto; Liu, Jian; Chiarle, Roberto; Podda, Antonello; Pagano, Michele; Inghirami, Giorgio
2002 Dec;22(23):8375-8387, Molecular & cellular biology
Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation together with Cul1 of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, we generated transgenic (Tg) mice expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. We show that the in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. Our findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation
— id: 39375, year: 2002, vol: 22, page: 8375, stat: Journal Article,

Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer
Signoretti, Sabina; Di Marcotullio, Lucia; Richardson, Andrea; Ramaswamy, Sridhar; Isaac, Beth; Rue, Montserrat; Monti, Franco; Loda, Massimo; Pagano, Michele
2002 Sep;110(5):633-641, Journal of clinical investigation
Estrogen receptor (ER) expression and Her-2 amplification define specific subsets of breast tumors for which specific therapies exist. The S-phase kinase-associated protein Skp2 is required for the ubiquitin-mediated degradation of the cdk-inhibitor p27 and is a bona fide proto-oncoprotein. Using microarray analysis and immunohistochemistry, we determined that higher levels of Skp2 are present more frequently in ER-negative tumors than in ER-positive cases. Interestingly, the subset of ER-negative breast carcinomas overexpressing Skp2 are also characterized by high tumor grade, negativity for Her-2, basal-like phenotype, high expression of certain cell cycle regulatory genes, and low levels of p27 protein. We also found that Skp2 expression is cell adhesion-dependent in normal human mammary epithelial cells but not in breast cancer cells and that an inhibition of Skp2 induces a decrease of adhesion-independent growth in both ER-positive and ER-negative cancer cells. Finally, forced expression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. We conclude that Skp2 has oncogenic potential in breast epithelial cells and is overexpressed in a subset of breast carcinomas (ER- and Her-2 negative) for which Skp2 inhibitors may represent a valid therapeutic option
— id: 64232, year: 2002, vol: 110, page: 633, stat: Journal Article,

Three different binding sites of Cks1 are required for p27-ubiquitin ligation
Sitry, Danielle; Seeliger, Markus A; Ko, Tun K; Ganoth, Dvora; Breward, Sadie E; Itzhaki, Laura S; Pagano, Michele; Hershko, Avram
2002 Nov 1;277(44):42233-42240, Journal of biological chemistry
Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound
— id: 64234, year: 2002, vol: 277, page: 42233, stat: Journal Article,

Lead removal and recovery from battery industry wastewaters by soluble starch xanthate
Tiravanti, Giovanni; Marani, Dario; Pagano, Michele; Presicce, Dominique Sara; Passino, Roberto
2002 Jul-Aug;92(7-8):677-688, Annali di chimica
Treatment, removal and recovery of lead (3 mg/L) from battery industry wastewaters have been investigated utilising a chemical precipitation process with soluble starch xanthate (SX) at pH 5-6. A reactant ratio, i.e., SX/Pb(II) = 6 mol/mol, a reaction time of 15 min., the addition of 15 mg/L of a cationic polyelectrolyte and a final filtration gave residual lead concentrations in the liquid phase less than 0.2 mg/L, well below the maximum limit established by the EU Directive. Lead was extracted from the obtained sludge by oxidation with sodium hypochlorite or hydrogen peroxide solutions. The amounts of oxidant needed were quantified as 13.5 mol NaClO/mol Pb and one order of magnitude larger, for H2O2, the latter due to the competitive disproportion reaction of the oxidant. The metal extraction was quantitative using sodium hypochlorite; when hydrogen peroxide was used, the formation of insoluble PbSO4 (Anglesite) gave a 80% metal extraction. In both cases molar ratios between sulphate and lead ions in the extracted solutions were in the range 2.1-2.2, in agreement with the stoichiometries of the reactions. Lead can be quantitatively recovered from the extracted (NaClO) solutions, for reuse, after a chemical precipitation process with 1M NaOH at pH 9-9.5, in the form of hydrocerussite [Pb3(CO3)2(OH)2]
— id: 64233, year: 2002, vol: 92, page: 677, stat: Journal Article,

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex
Zheng, Ning; Schulman, Brenda A; Song, Langzhou; Miller, Julie J; Jeffrey, Philip D; Wang, Ping; Chu, Claire; Koepp, Deanna M; Elledge, Stephen J; Pagano, Michele; Conaway, Ronald C; Conaway, Joan W; Harper, J Wade; Pavletich, Nikola P
2002 Apr 18;416(6882):703-709, Nature
SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold
— id: 64235, year: 2002, vol: 416, page: 703, stat: Journal Article,

Role of the F-box protein Skp2 in adhesion-dependent cell cycle progression
Carrano AC; Pagano M
2001 Jun 25;153(7):1381-1390, Journal of cell biology
Cell adhesion to the extracellular matrix (ECM) is a requirement for proliferation that is typically lost in malignant cells. In the absence of adhesion, nontransformed cells arrest in G1 with increased levels of the cyclin-dependent kinase inhibitor p27. We have reported previously that the degradation of p27 requires its phosphorylation on Thr-187 and is mediated by Skp2, an F-box protein that associates with Skp1, Cul1, and Roc1/Rbx1 to form the SCF(Skp2) ubiquitin ligase complex. Here, we show that the accumulation of Skp2 protein is dependent on both cell adhesion and growth factors but that the induction of Skp2 mRNA is exclusively dependent on cell adhesion to the ECM. Conversely, the expression of the other three subunits of the SCF(Skp2) complex is independent of cell anchorage. Phosphorylation of p27 on Thr-187 is also not affected significantly by the loss of cell adhesion, demonstrating that increased p27 stability is not dependent on p27 dephosphorylation. Significantly, ectopic expression of Skp2 in nonadherent G1 cells resulted in p27 downregulation, entry into S phase, and cell division. The ability to induce adhesion-independent cell cycle progression was potentiated by coexpressing Skp2 with cyclin D1 but not with cyclin E, indicating that Skp2 and cyclin D1 cooperate to rescue proliferation in suspension cells. Our study shows that Skp2 is a key target of ECM signaling that controls cell proliferation
— id: 21089, year: 2001, vol: 153, page: 1381, stat: Journal Article,

Human T cell leukemia virus type 1 Tax associates with a molecular chaperone complex containing hTid-1 and Hsp70
Cheng H; Cenciarelli C; Shao Z; Vidal M; Parks WP; Pagano M; Cheng-Mayer C
2001 Nov 13;11(22):1771-1775, Current biology. CB
Tax, an oncogenic viral protein encoded by human T cell leukemia virus type 1 (HTLV-1), induces cellular transformation of T lymphocytes by modulating a variety of cellular gene expressions [1]. Identifying cellular partners that interact with Tax constitutes the first step toward elucidating the molecular basis of Tax-induced transformation. Here, we report a novel Tax-interacting protein, hTid-1. hTid-1, a human homolog of the Drosophila tumor suppressor protein Tid56, was initially characterized based on its interaction with the HPV-16 E7 oncoprotein [2]. hTid-1 and Tid56 are members of the DnaJ family [2,3], which contains a highly conserved signature J domain that regulates the activities of heat shock protein 70 (Hsp70) by serving as cochaperone [4-6]. In this context, the molecular chaperone complex is involved in cellular signaling pathways linked to apoptosis, protein folding, and membrane translocation and in modulation of the activities of tumor suppressor proteins, including retinoblastoma, p53, and WT1[7-12]. We find that expression of hTid-1 inhibits the transformation phenotype of two human lung adenocarcinoma cell lines. We show that Tax interacts with hTid-1 via a central cysteine-rich domain of hTid-1 while a signature J domain of hTid-1 mediates its binding to Hsp70 in HEK cells. Importantly, Tax associates with the molecular chaperone complex containing both hTid-1 and Hsp70 and alters the cellular localization of hTid-1 and Hsp70. In the absence of Tax, expression of the hTid-1/Hsp70 molecular complex is targeted to perinuclear mitochondrial clusters. In the presence of Tax, hTid-1 and its associated Hsp70 are sequestered within a cytoplasmic 'hot spot' structure, a subcellular distribution that is characteristic of Tax in HEK cells
— id: 39468, year: 2001, vol: 11, page: 1771, stat: Journal Article,

The cyclin dependent kinase inhibitor p27 and its prognostic role in breast cancer
Chiarle R; Pagano M; Inghirami G
2001 ;3(2):91-94, Breast cancer research
p27 is an inhibitor of cyclin dependent kinase involved in the regulation of the cell cycle. In this commentary we discuss the current knowledge on p27 in breast cancer and its significance in predicting the outcome. p27 protein levels are high in most cases of breast carcinomas, are correlated with the levels of cyclin D1 and estrogen receptor, and could be a useful predictor of survival, because they are low in aggressive carcinomas. Immunodetection of p27 in breast tumors could be useful in the assessment of prognosis, especially in those cases in which the commonly used parameters are insufficient, and might ultimately influence the therapy of this disease
— id: 21234, year: 2001, vol: 3, page: 91, stat: Journal Article,

The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein
DeSalle LM; Latres E; Lin D; Graner E; Montagnoli A; Baker RT; Pagano M; Loda M
2001 Sep 6;20(39):5538-5542, Oncogene
The ubiquitin pathway is involved in the proteolytic turnover of many short-lived cellular regulatory proteins. Since selective degradation of substrates of this system requires the covalent attachment of a polyubiquitin chain to the substrates, degradation could be counteracted by de-ubiquitinating enzymes (or isopeptidases) which selectively remove the polyubiquitin chain. Unp is a human isopeptidase with still poorly understood biological functions. Here, we show that cellular Unp specifically interacts with the retinoblastoma gene product (pRb)
— id: 26666, year: 2001, vol: 20, page: 5538, stat: Journal Article,

Regulation of the G1 to S transition by the ubiquitin pathway
DeSalle LM; Pagano M
2001 Feb 16;490(3):179-189, FEBS letters
This year the most prestigious prize in medical sciences, the Lasker Award, has been presented to the three scientists who discovered the ubiquitin pathway: Aaron Ciechanover, Avram Hershko, and Alexander Varshavsky [Nature Med. 6 (2000) 1073-1081]. During a time when the scientific community was focused on understanding how proteins were synthesized, they intently pursued the novel idea that cells were programmed to selectively destroy proteins. Their work led to the identification of an elaborate system of protein degradation targeting a myriad of cellular substrates. A small protein called ubiquitin is at the center of this process. Although the ubiquitin pathway was first described in the early 1980s, it has only more recently advanced to the forefront of basic research as a significant regulatory network within the cell. The field continues to grow as new ubiquitination enzymes and novel functions of this system are identified. Scientists are focused on elucidating the mechanisms by which cells deploy the ubiquitin pathway to control levels of selected proteins, such as cell cycle regulatory proteins, transcription factors and signaling molecules. Accelerated or decelerated rates of degradation of particular substrates participate in the genesis of many human diseases. Thus, understanding the mechanisms that confer specificity to the ubiquitin system will allow the development of novel therapeutic approaches to target aberrations in this pathway underlying tumorigenesis and other human pathologies
— id: 21094, year: 2001, vol: 490, page: 179, stat: Journal Article,

The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27
Ganoth D; Bornstein G; Ko TK; Larsen B; Tyers M; Pagano M; Hershko A
2001 Mar;3(3):321-324, Nature cell biology
The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate
— id: 21092, year: 2001, vol: 3, page: 321, stat: Journal Article,

Inverse relation between levels of p27(Kip1) and of its ubiquitin ligase subunit Skp2 in colorectal carcinomas
Hershko D; Bornstein G; Ben-Izhak O; Carrano A; Pagano M; Krausz MM; Hershko A
2001 May 1;91(9):1745-1751, Cancer
BACKGROUND: Previous studies have shown that low levels of p27(Kip1), an inhibitor of G1 cyclin-dependent kinases, are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27 are caused, at least in part, by acceleration of the rate of its ubiquitin-mediated degradation. In cultured cells and cell-free biochemical systems, it has been shown that p27 is targeted for degradation by a ubiquitin ligase complex that contains Skp2 (S-phase kinase-associated protein 2) as the specific substrate-recognizing and rate-limiting subunit. This investigation was undertaken to examine the possible relation between levels of p27 and of its specific ubiquitin ligase subunit Skp2 in human cancers. METHODS: Quick-frozen colorectal tumor samples from 20 patients were homogenized at 0 degrees C in buffer containing a mixture of protease inhibitors. Samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Skp2 and p27. The expression of Skp2 also was examined by immunohistochemistry using formalin fixed, paraffin embedded tissue sections from the same cases. RESULTS: A strongly significant inverse correlation was found between levels of Skp2 and p27 (r = -0.812; P < 0.0001). Thus, decreased levels of p27 were associated with strongly increased levels of Skp2, whereas high levels of p27 coincided with low levels of Skp2. Immunohistochemical examination of Skp2 expression agreed with immunoblot analysis in 89% of cases. CONCLUSIONS: The results are compatible with the notion that increased expression of Skp2 may have a causative role in decreasing the levels of p27 in aggressive colorectal carcinomas.
— id: 21091, year: 2001, vol: 91, page: 1745, stat: Journal Article,

Role of the F-box protein Skp2 in lymphomagenesis
Latres E; Chiarle R; Schulman BA; Pavletich NP; Pellicer A; Inghirami G; Pagano M
2001 Feb 27;98(5):2515-2520, Proceedings of the National Academy of Sciences of the United States of America
The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G(1)-S transition by controlling the stability of several G(1) regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas
— id: 21093, year: 2001, vol: 98, page: 2515, stat: Journal Article,

Cul1 and Skp1 complexes inactivation leads to defective chromosome segregation, genetic instability and neoplastic transformation
Liu, J; Piva, R; Chiarle, R; Podda, A; Pagano, M; Inghirami, G
2001 OCT ;69(4):338-338, American journal of human genetics
— id: 54822, year: 2001, vol: 69, page: 338, stat: Journal Article,

Beware the baited hook of publicity - Headlines are tempting but lead to disillusionment-and a sour taste with your spaghetti
Pagano, M
2001 DEC 20 ;414(6866):843-843, Nature
— id: 54784, year: 2001, vol: 414, page: 843, stat: Journal Article,

p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis
Rodier, G; Montagnoli, A; Di Marcotullio, L; Coulombe, P; Draetta, GF; Pagano, M; Meloche, S
2001 Dec 3;20(23):6672-6682, EMBO journal
The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G(1) nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half- life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes
— id: 28201, year: 2001, vol: 20, page: 6672, stat: Journal Article,

Induction of beta -Transducin Repeat-containing Protein by JNK Signaling and Its Role in the Activation of NF-kappa B
Spiegelman VS; Stavropoulos P; Latres E; Pagano M; Ronai Z; Slaga TJ; Fuchs SY
2001 Jul 20;276(29):27152-27158, Journal of biological chemistry
Activation of Jun N-kinase (JNK) and NF-kappaB transcription factor are the hallmarks of cellular response to stress. Phosphorylation of NF-kappaB inhibitor (IkappaB) by respective stress-inducible kinases (IKK) is a key event in NF-kappaB activation. beta-TrCP F-box protein mediates ubiquitination of phosphorylated IkappaB via recruitment of SCF(beta-TrCP)-Roc1 E3 ubiquitin ligase complex. Subsequent proteasome-dependent degradation of IkappaB results in activation of the NF-kappaB pathway. We found that a variety of cellular stress stimuli induce an increase in the steady state levels of beta-TrCP mRNA and protein levels in human cells. Activation of stress-activated protein kinases JNK (and, to a lesser extent, p38) by forced expression of constitutively active mutants of JNKK2 and MKK6 (but not MEK1 or IKKbeta) also leads to accumulation of beta-TrCP. Transcription of the beta-TrCP gene is not required for JNK-mediated induction of beta-TrCP. A synergistic effect of stimulation of IKK and JNK on the transcriptional activity of NF-kappaB was observed. The mechanisms of beta-TrCP induction via stress and its role in NF-kappaB activation are discussed
— id: 21090, year: 2001, vol: 276, page: 27152, stat: Journal Article,

Role of the Cdc25A phosphatase in human breast cancer
Cangi MG; Cukor B; Soung P; Signoretti S; Moreira G Jr; Ranashinge M; Cady B; Pagano M; Loda M
2000 Sep;106(6):753-761, Journal of clinical investigation
The phosphatase Cdc25A plays an important role in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases, and it has been shown to transform diploid murine fibroblasts in cooperation with activated Ras. Here we show that Cdc25A is overexpressed in primary breast tumors and that such overexpression is correlated with higher levels of cyclin-dependent kinase 2 (Cdk2) enzymatic activity in vivo. Furthermore, in the breast cancer cell line MCF-7, Cdc25A activity is necessary for both the activation of Cdk2 and the subsequent induction of S-phase entry. Finally, in a series of small (< 1 cm) breast carcinomas, overexpression of Cdc25A was found in 47% of patients and was associated with poor survival. These data suggest that overexpression of Cdc25A contributes to the biological behavior of primary breast tumors and that both Cdc25A and Cdk2 are suitable therapeutic targets in early-stage breast cancer
— id: 21097, year: 2000, vol: 106, page: 753, stat: Journal Article,

Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma
Chiarle R; Budel LM; Skolnik J; Frizzera G; Chilosi M; Corato A; Pizzolo G; Magidson J; Montagnoli A; Pagano M; Maes B; De Wolf-Peeters C; Inghirami G
2000 Jan 15;95(2):619-626, Blood
Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription-polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P =.001), the loss of p27 (P =. 002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P =.002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626) (Blood. 2000;95:619-626)
— id: 11873, year: 2000, vol: 95, page: 619, stat: Journal Article,

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors
Chiaur DS; Murthy S; Cenciarelli C; Parks W; Loda M; Inghirami G; Demetrick D; Pagano M
2000 ;88(3-4):255-258, Cytogenetics & cell genetics
Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.
— id: 11681, year: 2000, vol: 88, page: 255, stat: Journal Article,

Low expression of p27 and low proliferation index do not correlate in hairy cell leukaemia [In Process Citation]
Chilosi M; Chiarle R; Lestani M; Menestrina F; Montagna L; Ambrosetti A; Prolla G; Pizzolo G; Doglioni C; Piva R; Pagano M; Inghirami G
2000 Oct;111(1):263-271, British journal of haematology
The molecular basis accounting for the peculiar clinical and biological features of hairy cell leukaemia (HCL) is currently unknown. Deregulation of cell cycle genes plays a significant role in oncogenesis and there is considerable evidence suggesting that Cdk inhibitors (Ckis) function as tumour suppressors. We and others have recently demonstrated low expression of Cki p27 in very aggressive neoplasms and high-grade lymphomas. To investigate whether HCL cases express normal p27 protein, as in other low-grade lymphomas with a low proliferation index, 58 cases of HCL were characterized using a sensitive biotin-streptavidin-immunoperoxidase technique and specific antibodies against p27. All HCL cases showed either no or very weak reactivity, in contrast to other types of low-grade B-cell lymphoma [22 cases of chronic lymphocytic leukaemia (CLL), 12 cases of gastric marginal B-cell lymphoma (MALT), 16 cases of follicular lymphomas and two cases of splenic marginal zone lymphomas]. To investigate the possible mechanism(s) accounting for the low p27 expression observed in hairy cells, multiple approaches were used. According to these molecular studies, low levels of p2 7 are not as a result of (1) increased ubiquitin-mediated degradation, (2) decreased levels of p27 transcription or (3) p27 somatic mutations and/or allelic loss. These findings suggest that low p27 protein expression in HCL may be achieved through post-transcriptional regulation. Finally, our data demonstrate that p27 expression in HCL does not correlate with either cell cycle progression or proliferation index, suggesting that low levels of p27 in hairy cells may be associated with their unique stage of B-cell differentiation and/or the activation of as yet unknown pathways
— id: 14849, year: 2000, vol: 111, page: 263, stat: Journal Article,

The F-box protein family
Kipreos ET; Pagano M
2000 ;1(5):3002-3002, Genome biology
SUMMARY: The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction. F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes (named after their main components, Skp I, Cullin, and an F-box protein), in which they bind substrates for ubiquitin-mediated proteolysis. The F-box motif links the F-box protein to other components of the SCF complex by binding the core SCF component Skp I. F-box proteins have more recently been discovered to function in non-SCF protein complexes in a variety of cellular functions. There are 11 F-box proteins in budding yeast, 326 predicted in Caenorhabditis elegans, 22 in Drosophila, and at least 38 in humans. F-box proteins often include additional carboxy-terminal motifs capable of protein-protein interaction; the most common secondary motifs in yeast and human F-box proteins are WD repeats and leucine-rich repeats, both of which have been found to bind phosphorylated substrates to the SCF complex. The majority of F-box proteins have other associated motifs, and the functions of most of these proteins have not yet been defined
— id: 21095, year: 2000, vol: 1, page: 3002, stat: Journal Article,

Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex
Schulman BA; Carrano AC; Jeffrey PD; Bowen Z; Kinnucan ER; Finnin MS; Elledge SJ; Harper JW; Pagano M; Pavletich NP
2000 Nov 16;408(6810):381-386, Nature
F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination (reviewed in refs 1-3). F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases. They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains. The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates. Most organisms have several Skp1 family members, but the function of these Skp1 homologues and the rules of recognition between different F-box and Skp1 proteins remain unknown. Here we describe the crystal structure of the human F-box protein Skp2 bound to Skp1. Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain. The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction
— id: 21098, year: 2000, vol: 408, page: 381, stat: Journal Article,

Regulation of the cdk inhibitor p27 and its deregulation in cancer
Slingerland J; Pagano M
2000 Apr;183(1):10-17, Journal of cellular physiology
p27 is a cell cycle inhibitor whose cellular abundance increases in response to many antimitogenic stimuli. In this review, we summarize the current knowledge on p27 function and its regulation by synthesis and by ubiquitin-mediated degradation. Importantly, p27 degradation is enhanced in many aggressive human tumors. The frequency with which this is observed suggests that loss of p27 may confer a growth advantage to these cancers. From a practical point of view, immunodetection of p27 in tumors may prove to be useful in the assessment of prognosis and may ultimately influence the therapy of this disease.
— id: 8530, year: 2000, vol: 183, page: 10, stat: Journal Article,

Wnt/beta-catenin signaling induces the expression and activity of betaTrCP ubiquitin ligase receptor
Spiegelman VS; Slaga TJ; Pagano M; Minamoto T; Ronai Z; Fuchs SY
2000 May;5(5):877-882, Molecular cell
Beta-transducing repeat-containing protein (betaTrCP) targets the ubiquitination and subsequent degradation of both beta-catenin and IkappaB, thereby playing an important role in beta-catenin/Tcf and NF-kappaB-dependent signaling. Here evidence is presented that beta-catenin/Tcf signaling elevates the expression of betaTrCP mRNA and protein in a Tcf-dependent manner, which does not require betaTrCP transcription. Induction of betaTrCP expression by the beta-catenin/Tcf pathway results in an accelerated degradation of the wild-type beta-catenin, suggesting that the negative feedback loop regulation may control the beta-catenin/Tcf pathway. This signaling also upregulated NF-kappaB transactivation without affecting the activity of IkappaB kinase, thereby establishing that the maintenance of the betaTrCP level is important for coordination between beta-catenin/Tcf and NF-kappaB signaling
— id: 21096, year: 2000, vol: 5, page: 877, stat: Journal Article,

SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27
Carrano AC; Eytan E; Hershko A; Pagano M
1999 Aug;1(4):193-199, Nature cell biology
Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation
— id: 6242, year: 1999, vol: 1, page: 193, stat: Journal Article,

Identification of a family of human F-box proteins
Cenciarelli C; Chiaur DS; Guardavaccaro D; Parks W; Vidal M; Pagano M
1999 Oct 21;9(20):1177-1179, Current biology. CB
F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins
— id: 6225, year: 1999, vol: 9, page: 1177, stat: Journal Article,

Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of I kappa B alpha
Gonen, H; Bercovich, B; Orian, A; Carrano, A; Takizawa, C; Yamanaka, K; Pagano, M; Iwai, K; Ciechanover, A
1999 MAY 21 ;274(21):14823-14830, Journal of biological chemistry
The last step in the activation of the transcription factor NF-kappa B is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor I kappa B alpha. Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive. Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process. The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of I kappa B alpha that cannot be phosphorylated and conjugated following an extracellular signal. Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor. Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor a-induced degradation of the inhibitor in vivo. Not surprisingly, they have a similar effect in a cell-free system as well. Although it is clear that the E2 enzymes are not entirely specific to I kappa B alpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases. The mechanisms that underlie the involvement of two different E2 species in I kappa B alpha conjugation are not clear at present. It is possible that different conjugating machineries operate under different physiological conditions or in different cells
— id: 53977, year: 1999, vol: 274, page: 14823, stat: Journal Article,

The human F box protein beta-Trcp associates with the Cul1/Skp1 complex and regulates the stability of beta-catenin
Latres E; Chiaur DS; Pagano M
1999 Jan 28;18(4):849-854, Oncogene
Ubiquitin-conjugation targets numerous cellular regulators for proteasome-mediated degradation. Thus, the identification of ubiquitin ligases and their physiological substrates is crucially important, especially for those cases in which aberrant levels of regulatory proteins (e.g., beta-catenin, p27) result from a deregulated ubiquitination pathway. In yeast, the proteolysis of several G1 regulators is controlled by ubiquitin ligases (or SCFs) formed by three subunits: Skp1, Cul A (Cdc53), and one of many F-box proteins. Specific F-box proteins (Fbps) recruit different substrates to the SCF. Although many Fbps have been identified in mammals, their specific substrates and the existence of multiple SCFs have not yet been reported. We have found that one human Fbp, beta-Trcp (beta-Transducin repeat containing protein), does indeed form a novel SCF with human Skp1 and Cul1. Consistent with recent reports indicating that Xenopus and Drosophila beta-Trcp homologs act as negative regulators of the Wnt/beta-catenin signaling pathway, we report here that human beta-Trcp interacts with beta-catenin in vivo. Furthermore, beta-catenin is specifically stabilized in vivo by the expression of a dominant negative beta-Trcp. These results indicate that the Cul1/Skp1/beta-Trcp complex forms a ubiquitin ligase that mediates the degradation of beta-catenin
— id: 7381, year: 1999, vol: 18, page: 849, stat: Journal Article,

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation
Montagnoli A; Fiore F; Eytan E; Carrano AC; Draetta GF; Hershko A; Pagano M
1999 May 1;13(9):1181-1189, Genes & development
The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor
— id: 6113, year: 1999, vol: 13, page: 1181, stat: Journal Article,

The cell cycle inhibitor p27 as a prognostic marker in human tumors and a novel target for therapeutic intervention
Pagano, Michele; Ravid, Katya
Signaling networks and cell cycles control: The molecual basis of cancer and other diseases Totowa, NJ : Humana Press,
— id: 2554, year: 1999, vol: , page: 545, stat: Chapter,

Activation of protein kinase C triggers its ubiquitination and degradation
Lu Z; Liu D; Hornia A; Devonish W; Pagano M; Foster DA
1998 Feb;18(2):839-845, Molecular & cellular biology
Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms alpha, delta, and epsilon in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC delta. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC alpha, delta, and epsilon were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC alpha could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway
— id: 30817, year: 1998, vol: 18, page: 839, stat: Journal Article,

Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma
Singh SP; Lipman J; Goldman H; Ellis FH Jr; Aizenman L; Cangi MG; Signoretti S; Chiaur DS; Pagano M; Loda M
1998 Apr 15;58(8):1730-1735, Cancer research
The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell division cycle. It is expressed at the highest levels during the quiescent (G0) and prereplicative (G1) phases, and its degradation is required for entry into the S phase. Because lack of p27 is associated with aggressive behavior in a variety of tumors of epithelial and lymphoid origin, we used immunohistochemistry and in situ hybridization to evaluate the expression of p27 in metaplastic and dysplastic Barrett's epithelium and to assess its prognostic significance in Barrett's associated adenocarcinoma (BAA) of the esophagus. In metaplastic Barrett's epithelium, p27 protein and mRNA were restricted to the superficial third of glands in all cases and extended to the lower third in 4 cases. In contrast, expression of p27 message and protein was both increased and full-thickness, in the 23 cases with high-grade dysplasia adjacent to BAA and in carcinoma in situ. Although all invasive carcinomas had elevated levels of p27 mRNA, 45 (83%) of 54 invasive carcinomas had low p27 protein levels (<50% positive tumor cells). Low p27 protein correlated with higher histological grade (P < 0.0001), depth of invasion (P = 0.0120), presence of lymph node metastasis (P = 0.05), and survival (P = 0.0197). In addition to the nuclear staining, cytoplasmic staining of p27 was noted in 11 of 23 (48%) of cases of dysplasia and in 14 of 54 (26%) adenocarcinomas and confirmed, in a subset of cases, by subcellular fractionation of protein lysates obtained from fresh tumor tissues. Cytoplasmic localization of p27 was also associated with decreased survival (P = 0.0239). Loss of p27 conferred poor prognosis independently of proliferative index, as assessed by Ki-67 (MIB-1) immunostaining, which was not significantly different in survivors versus nonsurvivors. These results show that: (a) distribution of p27 message and protein parallel one another in metaplastic and dysplastic Barrett's epithelium, suggesting transcriptional regulation of the gene in the nonneoplastic setting; (b) p27 is inactivated in the majority of BAA as a result of either post-transcriptional modification or altered subcellular localization; and (c) loss of the cell cycle inhibitor p27 is associated with parameters of aggressive behavior and unfavorable outcome in BAA
— id: 7795, year: 1998, vol: 58, page: 1730, stat: Journal Article,

Regulation of the cell cycle by the ubiquitin pathway
Slingerland J; Pagano M
1998 ;22:133-147, Results & problems in cell differentation
— id: 12092, year: 1998, vol: 22, page: 133, stat: Journal Article,

Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastases
Thomas GV; Szigeti K; Murphy M; Draetta G; Pagano M; Loda M
1998 Sep;153(3):681-687, American journal of pathology
The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell cycle and a potential tumor suppressor gene. Because we had previously demonstrated that loss of p27 protein is associated with aggressive behavior in colorectal adenocarcinomas, we used immunohistochemistry and in situ hybridization to evaluate the potential role of alterations in p27 expression in primary and metastatic colorectal adenocarcinomas. Parallel immunostaining was performed for Ki-67 and p53. We evaluated 13 cases of metachronous and 23 cases of synchronous primary and metastatic colorectal tumor pairs. In the synchronous subgroup (Stage IV tumors), 57% of the primary tumor and metastases pairs did not express p27 protein and the remainder were low expressors. In the metachronous subgroup, 54% of the primary tumors were low expressors and the remainder high expressors of p27 protein. There was a significant reduction in the expression of p27 in the metachronous metastases (mean positive cells: 14.5%) when compared to the corresponding primary tumors (mean positive cells: 41.8%), P = 0.0023. All the primary and metastatic tumors in the metachronous subgroup showed high levels of p27 mRNA expression. There was no association between loss of p27 and either Ki-67 count or p53 expression. Because p27 is known to be up-regulated when epithelial cells are grown in suspension, the down-regulation of p27 in circulating tumor cells may confer the ability to grow in an environment of altered extracellular matrix or intercellular adhesion properties, two situations which may facilitate metastases
— id: 7821, year: 1998, vol: 153, page: 681, stat: Journal Article,

Ubiquitin-dependent degradation of cyclin B is accelerated in polyploid megakaryocytes
Zhang Y; Wang Z; Liu DX; Pagano M; Ravid K
1998 Jan 16;273(3):1387-1392, Journal of biological chemistry
During the endomitotic cell cycle of megakaryocytic cell lines, the levels of cyclin B1 and the activity of cyclin B1-dependent Cdc2 kinase, although detectable, are reduced as compared with megakaryocytes undergoing a mitotic cell cycle. The levels of cyclin A, however, are comparable during both cell cycles. The expression of cyclin B1 mRNA is also equivalent in proliferating and polyploidizing cells. In the current study, we found that the rate of cyclin B1 protein degradation is enhanced in polyploidizing megakaryocytes. This finding has led us to further investigate whether the ubiquitin-proteosome pathway responsible for cyclin B degradation is accelerated in these cells. Our data indicate that polyploidizing megakaryocytic cell lines nad primary bone marrow cells treated with the megakaryocyte proliferation- and ploidy-promoting factor, the c-Mpl ligand, display increased activities of the ubiquitin-proteosome pathway, which degrades cyclin B, as compared with proliferating megakaryocytic cell lines or diploid bone marrow cells, respectively. This degradation has all the hallmarks of a ubiquitin pathway, including the dependence on ATP, the appearance of high molecular weight conjugated forms of cyclin B, and inhibition of the proteolytic process by a mutated form of the ubiquitin-conjugating enzyme Ubc4. Our studies also indicate that the ability to degrade cyclin A is equivalent in both the mitotic and endomitotic cell cycles. The increased potential of polyploid megakaryocytes to degrade cyclin B may be part of the cellular programming that leads to aborted mitosis
— id: 7867, year: 1998, vol: 273, page: 1387, stat: Journal Article,

Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation
Alessandrini, A; Chiaur, DS; Pagano, M
1997 MAR ;11(3):342-345, Leukemia
The cell cycle has been the object of extensive studies for the past years. A complex network of molecular interactions has been identified. In particular, a class of cell cycle inhibitory proteins has been cloned and characterized but details of the molecular mechanism of their action have yet to be resolved. These inhibitors regulate the progression through G1 and the G1/S transition via the inhibition of the cyclin-dependent kinase (Cdk) activity. The potential function of these negative regulators as tumor suppressors provides new insights into the link between the cell cycle and oncogenesis. p27 is a potent inhibitor of Cdks. In quiescent cells p27 accumulates without an increase in mRNA or protein synthesis. Cell cycle regulation of p27 levels, both in normal and transformed human cells, occurs via the ubiquitin-proteasome pathway and, compared to proliferating cells, quiescent cells contain a far lower amount of p27 ubiquitinating activity. The specific proteolysis of p27 is probably involved in the pathway of activation of Cdks. p27 is a phosphoprotein and its phosphorylation is cell cycle regulated. Often phosphorylation is a signal for ubiquitination. p27 is phosphorylated exclusively on serine by Erk1 and almost exclusively on threonine by Cdk1 in in vitro experiments. This finding raises the question of whether and how phosphorylation by these kinases is involved in the process of p27 proteolysis
— id: 53255, year: 1997, vol: 11, page: 342, stat: Journal Article,

Mechanism of p53 degradation
Brown JP; Pagano M
1997 Apr 18;1332(2):O1-O6, Biochimica & biophysica acta
— id: 12325, year: 1997, vol: 1332, page: O1, stat: Journal Article,

Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas [see comments]
Loda M; Cukor B; Tam SW; Lavin P; Fiorentino M; Draetta GF; Jessup JM; Pagano M
1997 Feb;3(2):231-234, Nature medicine
The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation
— id: 8310, year: 1997, vol: 3, page: 231, stat: Journal Article,

Enhanced ribosomal association of p27(Kip1) mRNA is a mechanism contributing to accumulation during growth arrest
Millard, SS; Yan, JS; Nguyen, HA; Pagano, M; Kiyokawa, H; Koff, A
1997 MAR 14 ;272(11):7093-7098, Journal of biological chemistry
p27(Kip1) regulates the decision to enter into S-phase or withdraw from the cell cycle by establishing an inhibitory threshold above which G(1) cyclin-dependent kinases accumulate before activation. We have used the HL-60 cell line to study regulation of p27 as cells withdraw from the cell cycle following treatment with 12-O-tetra-decanoylphorbol-13-acetate (TPA). We found that the amount of p27 is maximal in G(0) cells, lower in G(1) cells, and undetectable in S-phase cells, In contrast to the protein, the amount of p27 mRNA was the same in these populations, suggesting tliat accumulation of p27 during the cell cycle and as cells withdraw hom the cell cycle is controlled by post-transcriptional mechanisms, In S-phase cells, the degradation of p27 appears to predominate as a regulatory mechanism, In G(0) cells, there was an increase in the synthesis rate of p27, Our data demonstrate that, in G(0) cells, accumulation of p27 is due to an increase in the amount of p27 mRNA in polyribosomes
— id: 53249, year: 1997, vol: 272, page: 7093, stat: Journal Article,

Cell cycle regulation by the ubiquitin pathway
Pagano M
1997 Nov;11(13):1067-1075, FASEB journal
In the past 2 years, two ubiquitin-dependent proteolytic pathways have been established as important players in the regulation of the cell division cycle. In S. cerevisiae, the entry into S phase requires ubiquitin-mediated degradation of a cdk inhibitor, p40Sic1, in a pathway that involves the E2 enzyme Cdc34. Recent studies reviewed herein show that the Cdc34 pathway targets phosphorylated substrates. A second pathway that regulates chromosome segregation and mitotic exit by degrading anaphase inhibitors and mitotic cyclins involves a different E2 and a large molecular weight E3 complex, called the anaphase-promoting complex or cyclosome. This pathway targets substrates containing one or more destruction box motif
— id: 12224, year: 1997, vol: 11, page: 1067, stat: Journal Article,

The cell cycle inhibitor p27 is an independent prognostic marker in small (T-1a,T-b) invasive breast carcinomas
Tan, P; Cady, B; Wanner, M; Worland, P; Cukor, B; MagiGalluzzi, C; Lavin, P; Draetta, G; Pagano, M; Loda, M
1997 APR 1 ;57(7):1259-1263, Cancer research
Breast carcinomas less than or equal to 1 cm in size (T-1a,T-b) are being detected more frequently as a result of screening, Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy, A retrospective series of 202 patients with stage T-1a,T-b invasive breast carcinomas was evaluated, The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases, Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in nodenegative patients (median survival, 168 months; P = 0.0055), Patients with low p27 expression had a median survival of 139 months (17% mortality) versus 174 months (9% mortality) in the group with high p27 expression (P = 0.0233), Lack of p27 was associated with poor prognosis when node-positive patients were excluded (P = 0.0252), Nodal status and low p27 were found to be the only independent prognostic parameters by both univariate and multivariate analysis, with relative risks of dying of disease of 4.9 (P = 0.001) and 3.4 (P = 0.0306), respectively, Assessment of p27, which yields prognostic information in node-negative patients, could be useful to identify patients with small, invasive breast carcinomas who might benefit from adjuvant therapy
— id: 53198, year: 1997, vol: 57, page: 1259, stat: Journal Article,

Ubiquitin-dependent degradation of cyclin B is accelerated during an endomitotic cell cycle
Zhang, Y; Wang, Z; Pagano, M; Ravid, K
1997 JUL 31 ;11(9):A1217-A1217, FASEB journal
— id: 53457, year: 1997, vol: 11, page: A1217, stat: Journal Article,

Cell cycle control and cancer
Draetta, G; Pagano, M
1996 DEC ;31(4):241-248, Annual reports in medicinal chemistry
— id: 98376, year: 1996, vol: 31, page: 241, stat: Journal Article,

Human cyclin E, a nuclear protein essential for the G1-to-S phase transition
Ohtsubo M; Theodoras AM; Schumacher J; Roberts JM; Pagano M
1995 May;15(5):2612-2624, Molecular & cellular biology
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle
— id: 21100, year: 1995, vol: 15, page: 2612, stat: Journal Article,

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27
Pagano M; Tam SW; Theodoras AM; Beer-Romero P; Del Sal G; Chau V; Yew PR; Draetta GF; Rolfe M
1995 Aug 4;269(5224):682-685, Science
The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases
— id: 21099, year: 1995, vol: 269, page: 682, stat: Journal Article,

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts
Pagano M; Theodoras AM; Tam SW; Draetta GF
1994 Jul 15;8(14):1627-1639, Genes & development
Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint
— id: 21103, year: 1994, vol: 8, page: 1627, stat: Journal Article,

Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16Ink4
Tam SW; Shay JW; Pagano M
1994 Nov 15;54(22):5816-5820, Cancer research
p16Ink4 (inhibitor of cyclin-dependent kinase 4) is a cell cycle regulator that specifically binds to and inhibits Cdk4. Recently, the human mts1 (multiple tumor suppressor 1) gene, deleted or mutated in various primary tumors and in a large number of transformed cell lines, was found to be identical to ink4. In this study we have surveyed by immunoblotting the protein levels of p16Ink4 in normal and transformed human cells. We determined that p16Ink4 was differentially expressed in diploid cells derived from different tissues, in contrast to another cell cycle inhibitor, p21Waf1, which is ubiquitously expressed. In some tumor cell lines p16Ink4 protein was not detected, presumably because of a homozygous deletion of its gene. By contrast, it was found to be overexpressed in other cell lines when compared to levels in their normal counterparts. Interestingly, high levels of p16Ink4 protein correlated with functional inactivation of the retinoblastoma gene product. We also found that p16Ink4 protein expression varies during the cell cycle peaking during S phase. These results show a functional relationship between p16Ink4 and the retinoblastoma gene product and indicate that p16Ink4 is required for Cdk4 inhibition only at the G1-S transition at the time when Cdk4 kinase activity is no longer necessary
— id: 21101, year: 1994, vol: 54, page: 5816, stat: Journal Article,

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression
Tam SW; Theodoras AM; Shay JW; Draetta GF; Pagano M
1994 Sep;9(9):2663-2674, Oncogene
In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression
— id: 21102, year: 1994, vol: 9, page: 2663, stat: Journal Article,

Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts
Pagano M; Pepperkok R; Lukas J; Baldin V; Ansorge W; Bartek J; Draetta G
1993 Apr;121(1):101-111, Journal of cell biology
In mammalian cells inhibition of the cdc2 function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of affinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis
— id: 21104, year: 1993, vol: 121, page: 101, stat: Journal Article,

Association of cdk2 kinase with the transcription factor E2F during S phase
Pagano M; Draetta G; Jansen-Durr P
1992 Feb 28;255(5048):1144-1147, Science
The transcription factor E2F controls the expression of several proliferation-related genes and is a target of the adenovirus E1A oncogene. In human cells, both cyclin A and the cdk2 protein kinase were found in complexes with E2F. Although the total amounts of cdk2 were constant in the cell cycle, binding to E2F was detected only when cells entered S phase, a time when the cdk2 kinase is activated. These data suggest that the interaction between cdk2 and E2F requires an active kinase that has cyclin A as a targeting component
— id: 21105, year: 1992, vol: 255, page: 1144, stat: Journal Article,