Mark Reid Philips, M.D.

Regulating the regulator: post-translational modification of RAS
Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.
2012 JAN ;13(1):39-51, Nature reviews. Molecular cell biology
RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation
— id: 150788, year: 2012, vol: 13, page: 39, stat: Journal Article,

Regulating the regulator: post-translational modification of RAS
Ahearn, Ian M; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R
2011 ;13(1):39-51, Nature reviews. Molecular cell biology
RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation
— id: 147707, year: 2011, vol: 13, page: 39, stat: Journal Article,

FKBP12 binds to acylated h-ras and promotes depalmitoylation
Ahearn, Ian M; Tsai, Frederick D; Court, Helen; Zhou, Mo; Jennings, Benjamin C; Ahmed, Mahiuddin; Fehrenbacher, Nicole; Linder, Maurine E; Philips, Mark R
2011 Jan 21;41(2):173-185, Molecular cell
A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines
— id: 121308, year: 2011, vol: 41, page: 173, stat: Journal Article,

Cytosolic ras supports eye development in Drosophila
Sung, Pamela J; Rodrigues, Aloma B; Kleinberger, Andrew; Quatela, Steven; Bach, Erika A; Philips, Mark R
2010 Dec;30(24):5649-5657, Molecular & cellular biology
Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development
— id: 114824, year: 2010, vol: 30, page: 5649, stat: Journal Article,

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis
Botelho, Roberto J; Harrison, Rene E; Stone, James C; Hancock, John F; Philips, Mark R; Jongstra-Bilen, Jenny; Mason, David; Plumb, Jonathan; Gold, Michael R; Grinstein, Sergio
2009 Oct 16;284(42):28522-28532, Journal of biological chemistry
We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response
— id: 133732, year: 2009, vol: 284, page: 28522, stat: Journal Article,

Ras/MAPK signaling from endomembranes
Fehrenbacher, Nicole; Bar-Sagi, Dafna; Philips, Mark
2009 Aug;3(4):297-307, Molecular oncology
Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output
— id: 101955, year: 2009, vol: 3, page: 297, stat: Journal Article,

Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation
Madigan, James P; Bodemann, Brian O; Brady, Donita C; Dewar, Brian J; Keller, Patricia J; Leitges, Michael; Philips, Mark R; Ridley, Anne J; Der, Channing J; Cox, Adrienne D
2009 Nov 15;424(1):153-161, Biochemical journal
The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization
— id: 150609, year: 2009, vol: 424, page: 153, stat: Journal Article,

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane
Mor, Adam; Wynne, Joseph P; Ahearn, Ian M; Dustin, Michael L; Du, Guangwei; Philips, Mark R
2009 Jun;29(12):3297-3306, Molecular & cellular biology
Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane
— id: 99231, year: 2009, vol: 29, page: 3297, stat: Journal Article,

Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe
Wright, Latasha P; Court, Helen; Mor, Adam; Ahearn, Ian M; Casey, Patrick J; Philips, Mark R
2009 Apr;29(7):1826-1833, Molecular & cellular biology
Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the alpha carboxyl group of prenylated proteins including Ras and related GTPases. Methyl esterification neutralizes the negative charge of the prenylcysteine and thereby increases membrane affinity. Icmt is an integral membrane protein restricted to the endoplasmic reticulum (ER). The Saccharomyces cerevisiae ortholog, Ste14p, traverses the ER membrane six times. We used a novel fluorescent reporter to map the topology of human Icmt in living cells. Our results indicate that Icmt traverses the ER membrane eight times, with both N and C termini disposed toward the cytosol and with a helix-turn-helix structure comprising transmembrane (TM) segments 7 and 8. Several conserved amino acids that map to cytoplasmic portions of the enzyme are critical for full enzymatic activity. Mammalian Icmt has an N-terminal extension consisting of two TM segments not found in Ste14p and therefore likely to be regulatory. Icmt is a target for anticancer drug discovery, and these data may facilitate efforts to develop small-molecule inhibitors
— id: 97752, year: 2009, vol: 29, page: 1826, 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,

Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis
Quatela, Steven E; Sung, Pamela J; Ahearn, Ian M; Bivona, Trever G; Philips, Mark R
2008 ;439:87-102, Methods in enzymology
K-Ras is a member of a family of proteins that associate with the plasma membrane by virtue of a lipid modification that inserts into the membrane and a polybasic region that associates with the anionic head groups of inner leaflet phospholipids. In the case of K-Ras, the lipid is a C-terminal farnesyl isoprenoid adjacent to a polylysine sequence. The affinity of K-Ras for the plasma membrane can be modulated by diminishing the net charge of the polybasic region. Among the ways this can be accomplished is phosphorylation by protein kinase C (PKC) of serine 181 within the polybasic region. Phosphorylation at this site regulates a farnesyl-electrostatic switch that controls association of K-Ras with the plasma membrane. Surprisingly, engagement of the farnesyl-electrostatic switch promotes apoptosis. This chapter describes methods for directly analyzing the phosphorylation status of K-Ras using metabolic labeling with (32)P, for indirectly assessing the farnesyl-electrostatic switch by following GFP-tagged K-Ras in live cells, for artificially activating the farnesyl-electrostatic switch by directing the kinase domain of a PKC to activated K-Ras using a Ras-binding domain, and for assessing apoptosis of individual cells using a YFP-tagged caspase 3 biosensor
— id: 79382, year: 2008, vol: 439, page: 87, stat: Journal Article,

Activated Kras, but not Hras or Nras, may initiate tumors of endodermal origin via stem cell expansion
Quinlan, Margaret P; Quatela, Steven E; Philips, Mark R; Settleman, Jeffrey
2008 Apr;28(8):2659-2674, Molecular & cellular biology
The three closely related human Ras genes, Hras, Nras, and Kras, are all widely expressed, engage a common set of downstream effectors, and can each exhibit oncogenic activity. However, the vast majority of activating Ras mutations in human tumors involve Kras. Moreover, Kras mutations are most frequently seen in tumors of endodermally derived tissues (lung, pancreas, and colon), suggesting that activated Kras may affect an endodermal progenitor to initiate oncogenesis. Using a culture model of retinoic acid (RA)-induced stem cell differentiation to endoderm, we determined that while activated HrasV12 promotes differentiation and growth arrest in these endodermal progenitors, KrasV12 promotes their proliferation. Furthermore, KrasV12-expressing endodermal progenitors fail to differentiate upon RA treatment and continue to proliferate and maintain stem cell characteristics. NrasV12 neither promotes nor prevents differentiation. A structure-function analysis demonstrated that these distinct effects of the Ras isoforms involve their variable C-terminal domains, implicating compartmentalized signaling, and revealed a requirement for several established Ras effectors. These findings indicate that activated Ras isoforms exert profoundly different effects on endodermal progenitors and that mutant Kras may initiate tumorigenesis by expanding a susceptible stem/progenitor cell population. These results potentially explain the high frequency of Kras mutations in tumors of endodermal origin
— id: 133405, year: 2008, vol: 28, page: 2659, stat: Journal Article,

Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans
Yun, Chi; Stanhill, Ariel; Yang, Yun; Zhang, Yuhong; Haynes, Cole M; Xu, Chong-Feng; Neubert, Thomas A; Mor, Adam; Philips, Mark R; Ron, David
2008 May 13;105(19):7094-7099, Proceedings of the National Academy of Sciences of the United States of America
The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions
— id: 94504, year: 2008, vol: 105, page: 7094, stat: Journal Article,

The lymphocyte function-associated antigen-1 receptor costimulates plasma membrane Ras via phospholipase D2
Mor, Adam; Campi, Gabriele; Du, Guangwei; Zheng, Yang; Foster, David A; Dustin, Michael L; Philips, Mark R
2007 Jun;9(6):713-719, Nature cell biology
Ras activation as a consequence of antigen receptor (T-cell receptor; TCR) engagement on T lymphocytes is required for T-cell development, selection and function. Lymphocyte function-associated antigen-1 (LFA-1) mediates lymphocyte adhesion, stabilization of the immune synapse and bidirectional signalling. Using a fluorescent biosensor we found that TCR activation with or without costimulation of CD28 led to activation of Ras only on the Golgi apparatus, whereas costimulation with LFA-1 induced Ras activation on both the Golgi and the plasma membrane. Ras activation on both compartments required RasGRP1, an exchange factor regulated by calcium and diacylglycerol (DAG), but phospholipase C (PLC) activity was required only for activation on the Golgi. Engagement of LFA-1 increased DAG levels at the plasma membrane by stimulating phospholipase D (PLD). PLD2 and phosphatidic acid phosphatase (PAP) were required for Ras activation on the plasma membrane. Thus, LFA-1 acts through PLD2 to reshape the pattern of Ras activation downstream of the TCR
— id: 73108, year: 2007, vol: 9, page: 713, stat: Journal Article,

Small GTPases and LFA-1 reciprocally modulate adhesion and signaling
Mor, Adam; Dustin, Michael L; Philips, Mark R
2007 Aug;218:114-125, Immunological reviews
Leukocyte-function-associated antigen-1 (LFA-1) is an integrin that is critical for T-cell adhesion and immunologic responses. As a transmembrane receptor and adhesion molecule, LFA-1 signals bidirectionally, whereby information about extracellular ligands is passed outside-in while cellular activation is transmitted inside-out to the adhesive ectodomain. Here, we review the role of small guanosine triphosphatases (GTPases) in LFA-1 signaling. Rap1, a Ras-related GTPase, appears to be central to LFA-1 function. Rap1 is regulated by receptor signaling [e.g. T-cell receptor (TCR), CD28, and cytotoxic T-lymphocyte antigen-4 (CTLA-4)] and by adapter proteins [e.g. adhesion and degranulation-promoting adapter protein (ADAP) and Src kinase-associated phosphoprotein of 55 kDa (SKAP-55)]. Inside-out signaling flows through Rap1 to regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP interacting adapter molecule (RIAM) that act in conjunction with the cytoskeleton on the cytosolic domain of LFA-1 to increase adhesion of the ectodomain. Outside-in signaling also relies on small GTPases such as Rho proteins. Vav-1, a guanine nucleotide exchange factor for Rho proteins, is activated as a consequence of LFA-1 engagement. Jun-activating binding protein-1 (JAB-1) and cytohesin-1 have been implicated as possible outside-in signaling intermediates. We have recently shown that Ras is also downstream of LFA-1 engagement: LFA-1 signaling through phospholipase D (PLD) to RasGRP1 was required for Ras activation on the plasma membrane following stimulation of TCR
— id: 73951, year: 2007, vol: 218, page: 114, stat: Journal Article,

The role of Ras signaling in lupus T lymphocytes: biology and pathogenesis
Mor, Adam; Philips, Mark R; Pillinger, Michael H
2007 Dec;125(3):215-223, Clinical immunology
Ras is a GTP-binding protein that plays multiple important roles in cell activation, including proliferative and inflammatory responses. Ras regulation is complex and depends upon post-translational processing, organelle-specific localization and the activation/deactivation of Ras by a number of regulatory molecules. Ras activation in T lymphocytes demonstrates unique features, including its dependence on the T cell receptor and the ability of Ras to signal from both the plasma membrane and the Golgi. Abnormalities of Ras expression, activation and signaling pathways in T lymphocytes appear to play important roles in the development of autoimmunity in general, and systemic lupus erythematosus in particular. In this manuscript, we review the basic biology of Ras in T lymphocytes, and the ways in which T lymphocyte Ras abnormalities may contribute to the development of a lupus phenotype
— id: 75658, year: 2007, vol: 125, page: 215, stat: Journal Article,

Geranylgeranyltransferase I as a target for anti-cancer drugs
Philips, Mark R; Cox, Adrienne D
2007 May;117(5):1223-1225, Journal of clinical investigation
Posttranslational modification is critical for the function of the gene products of ras oncogenes, which are frequently mutated in cancer. Ras proteins are modified by farnesyltransferase (FTase), but many related small GTPases that also end in a CAAX motif (where C is cysteine, A is often an aliphatic amino acid, and X is any amino acid) are modified by a closely related enzyme known as geranylgeranyltransferase type I (GGTase-I). Accordingly, inhibitors for both of these enzymes have been developed, and those active against FTase are in clinical trials. In this issue of the JCI, Sjogren et al. report the development of a mouse strain homozygous for a conditional allele of the gene that encodes GGTase-I (see the related article beginning on page 1294). They found that ablation of the GGTase-I-encoding gene in cells destined to produce lung tumors driven by oncogenic K-Ras resulted in delayed onset and decreased severity of disease, validating in a genetic model the theory that GGTase-I is a good target for anti-cancer drug development.
— id: 72874, year: 2007, vol: 117, page: 1223, stat: Journal Article,

Agpat6--a novel lipid biosynthetic gene required for triacylglycerol production in mammary epithelium
Beigneux, Anne P; Vergnes, Laurent; Qiao, Xin; Quatela, Steven; Davis, Ryan; Watkins, Steven M; Coleman, Rosalind A; Walzem, Rosemary L; Philips, Mark; Reue, Karen; Young, Stephen G
2006 Apr;47(4):734-744, Journal of lipid research
In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, 1-acylglycerol-3-phosphate O-acyltransferase (Agpat6), with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6's closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum (ER) in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6-/-) mice. Agpat6-/- mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6-/- mice was a defect in lactation. Pups nursed by Agpat6-/- mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6-/- lactating mammary glands were underdeveloped, and there was a dramatic decrease in the size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6-/- mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the ER, AGPAT6, which is crucial for the production of milk fat by the mammary gland
— id: 150604, year: 2006, vol: 47, page: 734, stat: Journal Article,

Analysis of Ras activation in living cells with GFP-RBD
Bivona, TG; Quatela, S; Philips, MR
2006 MAY ;407(11):128-143, Methods in enzymology
Several genetically encoded fluorescent biosensors for Ras family GTPases have been developed that permit spatiotemporal analysis of the activation of these signaling molecules in living cells. We describe here the use of the simplest of these probes, the Ras binding domain (RBD) of selected effectors fused with green fluorescent protein (GFP) or one of its spectral mutants. When expressed in quiescent cells, these probes are distributed homogeneously through the cytosol and nucleoplasm. On activation of their cognate GTPases on membranes, they are recruited to these compartments, and activation can be scored by redistribution of the probe. The advantage of this system is its simplicity: the probes are genetically encoded and can easily be constructed with standard cloning techniques, and the readout of activation requires only standard epifluorescence or confocal microscopy. The disadvantage of the system is that only rarely are Ras-related GTPases expressed at high enough levels to permit detection of the activation of the endogenous proteins. In general, the method requires overexpressing untagged, wild-type versions of the GTPase of interest. However, we describe a FRET-based method called bystander FRET developed to detect endogenous proteins that can be used to validate the results obtained by overexpressing Ras proteins. By use of this technique, we and others have uncovered important new features of the spatiotemporal regulation of Ras and related GTPases
— id: 98072, year: 2006, vol: 407, page: 128, stat: Journal Article,

PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis
Bivona, Trever G; Quatela, Steven E; Bodemann, Brian O; Ahearn, Ian M; Soskis, Michael J; Mor, Adam; Miura, John; Wiener, Heidi H; Wright, Latasha; Saba, Shahryar G; Yim, Duke; Fein, Adam; Perez de Castro, Ignacio; Li, Chi; Thompson, Craig B; Cox, Adrienne D; Philips, Mark R
2006 Feb 17;21(4):481-493, Molecular cell
K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-XL. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181
— id: 64117, year: 2006, vol: 21, page: 481, stat: Journal Article,

Spatial segregation of Ras signaling: new evidence from fission yeast
Chang, Eric C; Philips, Mark R
2006 Sep;5(17):1936-1939, Cell cycle
The Ras GTPases act as binary switches for signal transduction pathways that are important for growth regulation and tumorigenesis. Despite the biochemical simplicity of this switch, Ras proteins control multiple pathways, and the functions of the four mammalian Ras proteins are not overlapping. This raises an important question--how does a Ras protein selectively regulate a particular activity? One recently emerging model suggests that a single Ras protein can control different functions by acting in distinct cellular compartments. A critical test of this model is to identify pathways that are selectively controlled by Ras when it is localized to a particular compartment. A recent study has examined Ras signaling in the fission yeast Schizosaccharomyces pombe, which expresses only one Ras protein that controls two separate evolutionarily conserved pathways. This study demonstrates that whereas Ras localized to the plasma membrane selectively regulates a MAP kinase pathway to mediate mating pheromone signaling, Ras localized to the endomembrane activates a Cdc42 pathway to mediate cell polarity and protein trafficking. This study has provided unambiguous evidence for compartmentalized signaling of Ras
— id: 150608, year: 2006, vol: 5, page: 1936, stat: Journal Article,

The use of GFP to localize Rho GTPases in living cells
Michaelson, David; Philips, Mark
2006 ;406:296-315, Methods in enzymology
The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems
— id: 150605, year: 2006, vol: 406, page: 296, stat: Journal Article,

Compartmentalized ras/mapk signaling
Mor, Adam; Philips, Mark R
2006 ;24:771-800, Annual review of immunology
Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCgamma and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thus, the Ras/MAPK pathway represents a clear example of compartmentalized signaling
— id: 64114, year: 2006, vol: 24, page: 771, stat: Journal Article,

Compartmentalized signaling of Ras in fission yeast
Onken, Brian; Wiener, Heidi; Philips, Mark R; Chang, Eric C
2006 Jun 13;103(24):9045-9050, Proceedings of the National Academy of Sciences of the United States of America
Compartment-specific Ras signaling is an emerging paradigm that may explain the multiplex outputs from a single GTPase. The fission yeast, Schizosaccharomyces pombe, affords a simple system in which to study Ras signaling because it has a single Ras protein, Ras1, that regulates two distinct pathways: one that controls mating through a Byr2-mitogen-activated protein kinase cascade and one that signals through Scd1-Cdc42 to maintain elongated cell morphology. We generated Ras1 mutants that are restricted to either the endomembrane or the plasma membrane. Protein binding studies showed that each could interact with the effectors of both pathways. However, when examined in ras1 null cells, endomembrane-restricted Ras1 supported morphology but not mating, and, conversely, plasma membrane-restricted Ras1 supported mating but did not signal to Scd1-Cdc42. These observations provide a striking demonstration of compartment-specific Ras signaling and indicate that spatial specificity in the Ras pathway is evolutionarily conserved
— id: 150606, year: 2006, vol: 103, page: 9045, stat: Journal Article,

Ras signaling on the Golgi
Quatela, Steven E; Philips, Mark R
2006 Apr;18(2):162-167, Current opinion in cell biology
The discovery that Ras proteins are modified by enzymes restricted to the endoplasmic reticulum and Golgi apparatus and that, at steady state, a significant pool of Ras is localized on the Golgi has led to the hypothesis that Ras can become activated on and signal from intracellular membranes. Fluorescent probes capable of showing when and where in living cells Ras becomes activated together with studies of Ras proteins stringently tethered to intracellular membranes have confirmed this hypothesis. Thus, recent studies of Ras have contributed to the rapidly expanding field of compartmentalized signaling
— id: 64116, year: 2006, vol: 18, page: 162, stat: Journal Article,

Thematic review series: Lipid Posttranslational Modifications. CAAX modification and membrane targeting of Ras
Wright, Latasha P; Philips, Mark R
2006 May;47(5):883-891, Journal of lipid research
Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking
— id: 64115, year: 2006, vol: 47, page: 883, stat: Journal Article,

Receptor activation alters inner surface potential during phagocytosis
Yeung, Tony; Terebiznik, Mauricio; Yu, Liming; Silvius, John; Abidi, Wasif M; Philips, Mark; Levine, Tim; Kapus, Andras; Grinstein, Sergio
2006 Jul 21;313(5785):347-351, Science
The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis
— id: 150607, year: 2006, vol: 313, page: 347, stat: Journal Article,

Analysis of Ras and Rap activation in living cells using fluorescent Ras binding domains
Bivona, Trever G; Philips, Mark R
2005 Oct;37(2):138-145, Methods
Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1
— id: 61854, year: 2005, vol: 37, page: 138, stat: Journal Article,

Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion
Chan, Amanda Y; Coniglio, Salvatore J; Chuang, Ya-yu; Michaelson, David; Knaus, Ulla G; Philips, Mark R; Symons, Marc
2005 Nov 24;24(53):7821-7829, Oncogene
Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells
— id: 64120, year: 2005, vol: 24, page: 7821, stat: Journal Article,

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway
Goodwin, J Shawn; Drake, Kimberly R; Rogers, Carl; Wright, Latasha; Lippincott-Schwartz, Jennifer; Philips, Mark R; Kenworthy, Anne K
2005 Jul 18;170(2):261-272, Journal of cell biology
Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes
— id: 64121, year: 2005, vol: 170, page: 261, stat: Journal Article,

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII
Jordan, J Dedrick; He, John Cijiang; Eungdamrong, Narat J; Gomes, Ivone; Ali, Wasif; Nguyen, Tracy; Bivona, Trever G; Philips, Mark R; Devi, Lakshmi A; Iyengar, Ravi
2005 Mar 25;280(12):11413-11421, Journal of biological chemistry
The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth
— id: 64122, year: 2005, vol: 280, page: 11413, stat: Journal Article,

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases
Michaelson, David; Ali, Wasif; Chiu, Vi K; Bergo, Martin; Silletti, Joseph; Wright, Latasha; Young, Stephen G; Philips, Mark
2005 Apr;16(4):1606-1616, Molecular biology of the cell
The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins
— id: 57676, year: 2005, vol: 16, page: 1606, stat: Journal Article,

Compartmentalized signalling of Ras
Philips, M R
2005 Aug;33(Pt 4):657-661, Transactions (Biochemical Society (Great Britain))
Ras proteins associate with cellular membranes by virtue of a series of post-translational modifications of their C-terminal CAAX sequences. The discovery that two of the three enzymes that modify CAAX proteins are restricted to the endoplasmic reticulum led to the recognition that all nascent Ras proteins transit endomembranes en route to the PM (plasma membrane) and that at steady-state N-Ras and H-Ras are highly expressed on the Golgi apparatus. To test the hypothesis that Ras proteins on internal membranes can signal, we developed a fluorescent probe that reports when and where in living cells Ras becomes active. We found that growth factors stimulated rapid and transient activation of Ras on the PM followed by delayed and sustained activation on the Golgi. We mapped one pathway responsible for this activity as involving PLCgamma (phospholipase Cgamma)/DAG (diacylglycerol)+Ca2+/RasGRP1. Using mammalian cells and fission yeast, we have shown that differential localization of activated Ras preferentially activates distinct signalling pathways. In very recent work, we have found that (i) the subcellular localization of K-Ras can be acutely modulated by phosphorylation of its C-terminal hypervariable region by PKC, (ii) among the membranes upon which phosphorylated K-Ras accumulates is the outer mitochondrial membrane and (iii) phosphorylated, internalized K-Ras promotes apoptosis. Thus the signalling output of Ras depends on its subcellular localization
— id: 64119, year: 2005, vol: 33, page: 657, stat: Journal Article,

Analysis of monomeric GTPases of the Ras and Rho families
Philips, Mark R
2005 Oct;37(2):129-130, Methods
— id: 64118, year: 2005, vol: 37, page: 129, stat: Journal Article,

Teaching resources. Imaging signal transduction in living cells with fluorescent proteins
Philips, Mark R
2005 Dec 13;2005(314):tr28-tr28, Science's STKE
Until recently, studies in this field of signal transduction have involved the 'what' and 'when' of signaling. Who talks to whom and for how long? With the advent of genetically encoded fluorescent proteins, it has become possible to monitor signaling events in living cells in real time. This has added the dimension of 'where' to the study of cellular signaling. This lecture, which is a part of 'Cell Signaling Systems: A Course for Graduate Students,' provides a survey of how green fluorescent protein (GFP)-tagged probes for signaling events have been used to elucidate new pathways, to describe the kinetics of signaling events at the single-cell level, and to reveal upon which subcellular compartments these events take place. Some of the findings confirm previous ones using biochemical techniques, and others have been surprising. Examples include those utilizing protein localization, relocalization, fluorescence recovery after photobleaching (FRAP), and fluorescence resonance energy transfer (FRET). The design of FRET probes is described. The detection of small guanosine triphosphatase (GTPase) signaling in living cells is used as an example to explore the creative and diverse ways investigators have developed to look at this system
— id: 62640, year: 2005, vol: 2005, page: tr28, stat: Journal Article,

Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion
Bivona, Trever G; Wiener, Heidi H; Ahearn, Ian M; Silletti, Joseph; Chiu, Vi K; Philips, Mark R
2004 Mar 2;164(3):461-470, Journal of cell biology
Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases
— id: 46166, year: 2004, vol: 164, page: 461, stat: Journal Article,

Carboxyl methylation of Ras regulates membrane targeting and effector engagement
Chiu, Vi K; Silletti, Joseph; Dinsell, Victoria; Wiener, Heidi; Loukeris, Kristina; Ou, Guoming; Philips, Mark R; Pillinger, Michael H
2004 Mar 20;279(8):7346-7352, Journal of biological chemistry
Post-translational modification of Ras proteins includes prenylcysteine-directed carboxyl methylation. Because Ras participates in Erk activation by epidermal growth factor (EGF), we tested whether Ras methylation regulates Erk activation. EGF stimulation of Erk was inhibited by AFC (N-acetyl-S-farnesyl-L-cysteine), an inhibitor of methylation, but not AGC (N-acetyl-S-geranyl-L-cysteine), an inactive analog of AFC. AFC inhibited Ras methylation as well as the activation of pathway enzymes between Ras and Erk but did not inhibit EGF receptor phosphorylation, confirming action at the level of Ras. Transient transfection of human prenylcysteine-directed carboxyl methyltransferase increased EGF-stimulated Erk activation. AFC but not AGC inhibited movement of transiently transfected green fluorescent protein-Ras from the cytosol to the plasma membrane of COS-1 cells and depleted green fluorescent protein-Ras from the plasma membrane in stably transfected Madin-Darby canine kidney cells, suggesting that methylation regulates Erk by ensuring proper membrane localization of Ras. However, when COS-1 cells were transfected with Ras complexed to CD8, plasma membrane localization of Ras was unaffected by AFC, yet EGF-stimulated Erk activation was inhibited by AFC. Thus, Ras methylation appears to regulate Erk activation both through the localization of Ras as well as the propagation of Ras-dependent signals
— id: 46235, year: 2004, vol: 279, page: 7346, stat: Journal Article,

Membranes as messengers in T cell adhesion signaling
Dustin, Michael L; Bivona, Trever G; Philips, Mark R
2004 Apr;5(4):363-372, Nature immunology
Talin and RapL are components of molecular pathways that regulate the avidity of the integrin lymphocyte function-associated antigen 1 (LFA-1) for its ligand, intercellular adhesion molecule 1. In this review, we discuss recent advances in our understanding of LFA-1 affinity regulation and signaling and discuss a scenario for how Talin and Rap1 might act in synergy to achieve regulation of LFA-1 that is tailored to the specific functional requirements of different situations. Speedy delivery of signals may be crucial, and membrane trafficking from endosomes and the Golgi apparatus seem to be essential in delivering the messages from spatially segregated surface receptors
— id: 44917, year: 2004, vol: 5, page: 363, stat: Journal Article,

Human mitochondrial peptide deformylase, a new anticancer target of actinonin-based antibiotics
Lee, Mona D; She, Yuhong; Soskis, Michael J; Borella, Christopher P; Gardner, Jeffrey R; Hayes, Paula A; Dy, Benzon M; Heaney, Mark L; Philips, Mark R; Bornmann, William G; Sirotnak, Francis M; Scheinberg, David A
2004 Oct;114(8):1107-1116, Journal of clinical investigation
Peptide deformylase activity was thought to be limited to ribosomal protein synthesis in prokaryotes, where new peptides are initiated with an N-formylated methionine. We describe here a new human peptide deformylase (Homo sapiens PDF, or HsPDF) that is localized to the mitochondria. HsPDF is capable of removing formyl groups from N-terminal methionines of newly synthesized mitochondrial proteins, an activity previously not thought to be necessary in mammalian cells. We show that actinonin, a peptidomimetic antibiotic that inhibits HsPDF, also inhibits the proliferation of 16 human cancer cell lines. We designed and synthesized 33 chemical analogs of actinonin; all of the molecules with potent activity against HsPDF also inhibited tumor cell growth, and vice versa, confirming target specificity. Small interfering RNA inhibition of HsPDF protein expression was also antiproliferative. Actinonin treatment of cells led to a tumor-specific mitochondrial membrane depolarization and ATP depletion in a time- and dose-dependent manner; removal of actinonin led to a recovery of the membrane potential consistent with indirect effects on the electron transport chain. In animal models, oral or parenteral actinonin was well tolerated and inhibited human prostate cancer and lung cancer growth. We conclude that HsPDF is a new human mitochondrial enzyme that may provide a novel selective target for anticancer therapy by use of actinonin-based antibiotics
— id: 64123, year: 2004, vol: 114, page: 1107, stat: Journal Article,

Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus
Perez de Castro, Ignacio; Bivona, Trever G; Philips, Mark R; Pellicer, Angel
2004 May;24(8):3485-3496, Molecular & cellular biology
Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells
— id: 46001, year: 2004, vol: 24, page: 3485, stat: Journal Article,

Differential spatio-temporal activation of Ras and Rap1 in living cells
Philips, M; Bivona, T
2004 MAY 14 ;18(8):C1-C1, FASEB journal
— id: 46553, year: 2004, vol: 18, page: C1, stat: Journal Article,

Methotrexate and Ras methylation: a new trick for an old drug?
Philips, Mark R
2004 Apr 23;2004(225):pe13-pe13, Science's STKE
Ras plays a central role in the development and progression of human cancer. Ras function depends on its ability to associate with cellular membranes. Nascent Ras is targeted to membranes by virtue of a series of posttranslational modifications of a C-terminal 'CAAX' motif that include farnesylation, proteolysis, and carboxyl methylation. This pathway is an attractive target for anti-Ras drug development. Farnesyltransferase inhibitors have been developed and are in clinical trials. Their success has prompted interest in developing pharmacologically useful inhibitors of the other two enzymes in the Ras processing pathway. Ironically, it now appears that methotrexate, one of the oldest chemotherapeutic drugs, may work, in part, by inhibiting carboxyl methylation of Ras
— id: 46205, year: 2004, vol: 2004, page: pe13, stat: Journal Article,

Sef: a MEK/ERK catcher on the Golgi
Philips, Mark R
2004 Aug 23;15(2):168-169, Molecular cell
Subcellular compartmentalization is an emerging paradigm in signaling pathways including the Ras/MAPK pathway. In a recent issue of Developmental Cell, Torii et al. (2004) characterize a new MAPK scaffold, Sef, that resides on the Golgi apparatus, binds active MEK/ERK complexes, and permits signaling to cytosolic substrates but not nuclear targets
— id: 45970, year: 2004, vol: 15, page: 168, stat: Journal Article,

Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations
Walker, Simon A; Kupzig, Sabine; Bouyoucef, Dalila; Davies, Louise C; Tsuboi, Takashi; Bivona, Trever G; Cozier, Gyles E; Lockyer, Peter J; Buckler, Alan; Rutter, Guy A; Allen, Maxine J; Philips, Mark R; Cullen, Peter J
2004 Apr 21;23(8):1749-1760, EMBO journal
Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras
— id: 64124, year: 2004, vol: 23, page: 1749, stat: Journal Article,

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment
Zhang, Si Qing; Yang, Wentian; Kontaridis, Maria I; Bivona, Trever G; Wen, Gengyun; Araki, Toshiyuki; Luo, Jincai; Thompson, Julie A; Schraven, Burkhart L; Philips, Mark R; Neel, Benjamin G
2004 Feb 13;13(3):341-355, Molecular cell
The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility
— id: 64125, year: 2004, vol: 13, page: 341, stat: Journal Article,

Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1
Bivona, Trever G; Perez De Castro, Ignacio; Ahearn, Ian M; Grana, Theresa M; Chiu, Vi K; Lockyer, Peter J; Cullen, Peter J; Pellicer, Angel; Cox, Adrienne D; Philips, Mark R
2003 Aug 7;424(6949):694-698, Nature
Ras proteins regulate cellular growth and differentiation, and are mutated in 30% of cancers. We have shown recently that Ras is activated on and transmits signals from the Golgi apparatus as well as the plasma membrane but the mechanism of compartmentalized signalling was not determined. Here we show that, in response to Src-dependent activation of phospholipase Cgamma1, the Ras guanine nucleotide exchange factor RasGRP1 translocated to the Golgi where it activated Ras. Whereas Ca(2+) positively regulated Ras on the Golgi apparatus through RasGRP1, the same second messenger negatively regulated Ras on the plasma membrane by means of the Ras GTPase-activating protein CAPRI. Ras activation after T-cell receptor stimulation in Jurkat cells, rich in RasGRP1, was limited to the Golgi apparatus through the action of CAPRI, demonstrating unambiguously a physiological role for Ras on Golgi. Activation of Ras on Golgi also induced differentiation of PC12 cells, transformed fibroblasts and mediated radioresistance. Thus, activation of Ras on Golgi has important biological consequences and proceeds through a pathway distinct from the one that activates Ras on the plasma membrane
— id: 39161, year: 2003, vol: 424, page: 694, stat: Journal Article,

Ras pathway signaling on endomembranes
Bivona, Trever G; Philips, Mark R
2003 Apr;15(2):136-142, Current opinion in cell biology
Until recently, the plasma membrane has been considered to be a unique platform from which emanate the signaling events regulating or regulated by Ras and its close relatives. For the past few years, the role of endosomes derived from the plasma membrane as platforms for Ras/mitogen-activated protein kinase signaling has been appreciated. More recently, the cytoplasmic face of the Golgi apparatus and endoplasmic reticulum have been shown to host Ras signaling. The biological implications of compartmentalized signaling are only beginning to emerge
— id: 39266, year: 2003, vol: 15, page: 136, stat: Journal Article,

Tie-2-dependent activation of RhoA and Rac1 participates in endothelial cell motility triggered by angiopoietin-1
Cascone, Ilaria; Audero, Enrica; Giraudo, Enrico; Napione, Lucia; Maniero, Fabrizio; Philips, Mark R; Collard, John G; Serini, Guido; Bussolino, Federico
2003 Oct 1;102(7):2482-2490, Blood
Angiopoietin-1 is implicated in the maturation and remodeling of the vascular network during embryo development and in adult life. Through its tyrosine kinase receptor Tie-2 it stimulates endothelial cells to migrate and change shape. Here we show that angiopoietin-1 elicits chemokinesis of endothelial cells by a phosphoinositide 3-OH kinase/son of sevenless-dependent modulation of Rac1 and RhoA. The resulting temporal events are associated with cytoskeletal rearrangements and occur in discrete zones of the cell. Endothelial cells carrying dominant-negative mutants of RhoA and Rac1 or treated with LY294002, an inhibitor of phosphoinositide 3-OH kinase, dramatically decrease their chemokinetic velocity. Taken together, these results further expand our understanding of angiopoietin-1-mediated endothelial cell motility during vascular network assembly and angiogenesis
— id: 64127, year: 2003, vol: 102, page: 2482, stat: Journal Article,

Immunotoxin resistance in multidrug resistant cells
McGrath, Melissa S; Rosenblum, Michael G; Philips, Mark R; Scheinberg, David A
2003 Jan 1;63(1):72-79, Cancer research
Multidrug resistance (MDR) can be mediated, in part, by overexpression of P-glycoprotein (P-gp) and is characterized by broad resistance to several structurally, chemically, and pharmacologically distinct chemotherapeutic compounds. It has been hypothesized that immunological approaches to cytolysis may be used to overcome drug resistance. RV+ is a P-gp-expressing variant of the human myeloid leukemic cell line HL60 that displays a typical MDR phenotype. MDR RV+ cells displayed relative resistance to the immunotoxin (IT) HuM195-gelonin and to free rGelonin. K562 leukemia cells retrovirally infected to overexpress P-gp are also resistant to HuM195-gelonin. In addition, a monoclonal antibody capable of inhibiting the function of P-gp was able to partially reverse resistance to the IT. These data indicated that the expression of P-gp may contribute to IT resistance in RV+. Resistance to the IT was not mediated through decreased binding to cells, nor reduced internalization into the cell because the IT displayed similar kinetics of binding and internalization for both the parental HL60 and MDR RV+ cell lines. Comparison of the cytotoxicity of other ribosome-inactivating toxins indicated that RV+ cells were not universally resistant to toxins: RV+ cells were sensitive to the actions of ricin A chain, which acts on precisely the same RNase target as gelonin. Sensitivity of the MDR RV+ cells to the protein synthesis inhibitor cycloheximide, saponin, and Pseudomonas exotoxin A additionally confirmed that the resistance was not mediated through the ribosome and that pathways downstream from the inactivation of protein synthesis leading to cell death were not substantially perturbed in the MDR cells. Resistance could be partially abrogated by bafilomycin A, which inhibits lysosomal function. Moreover, direct visualization by confocal microscopy of the intracellular trafficking route of the IT showed that the IT accumulated preferentially in the lysosome in MDR RV+ cells but not in sensitive cells. These observations implicated the process of increased lysosomal degradation as the most likely basis for resistance. Such pathways of resistance may be important in the therapeutic applications of ITs, now becoming available for human use
— id: 64128, year: 2003, vol: 63, page: 72, stat: Journal Article,

Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK
Mohney, Robert P; Das, Margaret; Bivona, Trever G; Hanes, Richard; Adams, Anthony G; Philips, Mark R; O'Bryan, John P
2003 Nov 21;278(47):47038-47045, Journal of biological chemistry
Intersectin (ITSN) is a molecular scaffold involved in regulating endocytosis and mitogenic signaling. We previously demonstrated that ITSN transformed rodent fibroblasts, accelerated hormone-induced maturation of Xenopus oocytes, and activated the Elk-1 transcription factor through an MEK- and Erk-independent mechanism. We now demonstrate that ITSN complexes with the Ras guanine nucleotide exchange factor Sos1 leading to increased RasGTP levels. Using fluorescence resonant energy transfer analysis, we demonstrate that ITSN complexes with Ras in living cells leading to Ras activation on intracellular vesicles. These vesicles contain epidermal growth factor receptor but are distinct from transferrin-positive vesicles. However, Ras is not required for ITSN stimulation of transcription. Rather, we demonstrate that ITSN signals through JNK to activate Elk-1. Although ITSN activation of Elk-1 was Ras-independent, ITSN cooperates with Ras to synergistically activate JNK. These findings indicate that ITSN activates multiple intracellular signaling pathways and suggest that this adaptor protein may coordinately regulate the activity of these pathways in vivo
— id: 64126, year: 2003, vol: 278, page: 47038, stat: Journal Article,

Localized diacylglycerol-dependent activation of Ras during Fc gamma receptor-mediated phagocytosis
Botelho, RJ; Mojdami, D; Chiu, VK; Bivona, T; Philips, MR; Grinstein, S
2002 NOV ;13(4):225A-225A, Molecular biology of the cell
— id: 37187, year: 2002, vol: 13, page: 225A, stat: Journal Article,

Ras signalling on the endoplasmic reticulum and the Golgi
Chiu, Vi K; Bivona, Trever; Hach, Angela; Sajous, J Bernard; Silletti, Joseph; Wiener, Heidi; Johnson, Ronald L 2nd; Cox, Adrienne D; Philips, Mark R
2002 May;4(5):343-350, Nature cell biology
Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane
— id: 32483, year: 2002, vol: 4, page: 343, stat: Journal Article,

Membrane trafficking of heterotrimeric G proteins via the endoplasmic reticulum and Golgi
Michaelson, David; Ahearn, Ian; Bergo, Martin; Young, Stephen; Philips, Mark
2002 Sep;13(9):3294-3302, Molecular biology of the cell
Membrane targeting of G-protein alphabetagamma heterotrimers was investigated in live cells by use of Galpha and Ggamma subunits tagged with spectral mutants of green fluorescent protein. Unlike Ras proteins, Gbetagamma contains a single targeting signal, the CAAX motif, which directed the dimer to the endoplasmic reticulum. Endomembrane localization of farnesylated Ggamma(1), but not geranylgeranylated Ggamma(2), required carboxyl methylation. Targeting of the heterotrimer to the plasma membrane (PM) required coexpression of all three subunits, combining the CAAX motif of Ggamma with the fatty acyl modifications of Galpha. Galpha associated with Gbetagamma on the Golgi and palmitoylation of Galpha was required for translocation of the heterotrimer to the PM. Thus, two separate signals, analogous to the dual-signal targeting mechanism of Ras proteins, cooperate to target heterotrimeric G proteins to the PM via the endomembrane
— id: 47809, year: 2002, vol: 13, page: 3294, stat: Journal Article,

Activity of G gamma prenylcysteine carboxyl methyltransferase
Michaelson, David; Philips, Mark R
2002 ;344(10):234-245, Methods in enzymology
— id: 27263, year: 2002, vol: 344, page: 234, stat: Journal Article,

Green fluorescent protein-tagged Ras proteins for intracellular localization
Choy E; Philips M
2001 ;332(4):50-64, Methods in enzymology
— id: 21199, year: 2001, vol: 332, page: 50, stat: Journal Article,

Activity of G[gamma] prenylcysteine carboxyl methyltransferase
Michaelson D; Philips MR
2001 ;344:234-245, Methods in enzymology
— id: 26863, year: 2001, vol: 344, page: 234, stat: Journal Article,

Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding
Michaelson D; Silletti J; Murphy G; D'Eustachio P; Rush M; Philips MR
2001 Jan 8;152(1):111-126, Journal of cell biology
Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI alpha. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI alpha and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic
— id: 17229, year: 2001, vol: 152, page: 111, stat: Journal Article,

Signaling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways
Murphy GA; Jillian SA; Michaelson D; Philips MR; D'Eustachio P; Rush MG
2001 Mar;12(3):157-167, Cell growth & differentiation
The mammalian Rho family GTPases TC10 and Cdc42 share many properties. Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation. In contrast, previously reported differences between TC10 and Cdc42 are not striking. We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate. These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins
— id: 26749, year: 2001, vol: 12, page: 157, stat: Journal Article,

Expression and activity of human prenylcysteine-directed carboxyl methyltransferase
Choy, E; Philips, M
2000 ;325:101-114, Methods in enzymology
— id: 150603, year: 2000, vol: 325, page: 101, stat: Journal Article,

Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi [see comments]
Choy E; Chiu VK; Silletti J; Feoktistov M; Morimoto T; Michaelson D; Ivanov IE; Philips MR
1999 Jul 9;98(1):69-80, Cell
We show that Nras is transiently localized in the Golgi prior to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminated motile, peri-Golgi vesicles, and prolonged BFA treatment blocked PM expression. GFP-Hras colocalized with GFP-Nras, but GFP-Kras4B revealed less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal was required for PM expression, the CAAX motif alone was necessary and sufficient to target proteins to the endomembrane where they were methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif
— id: 8499, year: 1999, vol: 98, page: 69, stat: Journal Article,

Integrin-dependent homotypic adhesion of neutrophils. Arachidonic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase- dependent pathway
Capodici C; Pillinger MH; Han G; Philips MR; Weissmann G
1998 Jul 1;102(1):165-175, Journal of clinical investigation
AA stimulates integrin-dependent neutrophil adhesion, a critical early step in acute inflammation. However, neither the signaling pathway(s) of AA-stimulated adhesion, nor whether AA acts directly or through the generation of active metabolites, has been elucidated. Previously, we have observed a tight association between neutrophil Erk activation and homotypic adhesion in response to chemoattractants acting through G protein-linked receptors. We now report a similar association between homotypic adhesion and Erk activation in response to AA. Erk activation was cyclooxygenase independent and required AA metabolism to 5(S)- hydroperoxyeicosatetraenoic acid (5-HpETE) via 5-lipoxygenase, but not the further lipoxygenase-dependent metabolism of 5-HpETE to leukotrienes. AA stimulation of Erk was accompanied by Raf-1 activation and was sensitive to inhibitors of Raf-1 and Mek. Whereas activation of Erk by AA was pertussis toxin sensitive, [3H]-AA binding to neutrophils was not saturable, suggesting that an AA metabolite activates a G protein. Consistent with this hypothesis, Erk activation by 5(S)-hydroxyeicosatetraenoic acid (5-HETE; lipoxygenase-independent metabolite of 5-HpETE) was also pertussis toxin sensitive. These data suggest that a 5-lipoxygenase metabolite of AA, e.g., 5-HETE, is released from AA-treated cells to engage a plasma membrane-associated, pertussis toxin-sensitive, G protein-linked receptor, leading to activation of Erk and adhesion via the Raf-1/Mek signal transduction pathway
— id: 7521, year: 1998, vol: 102, page: 165, stat: Journal Article,

Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum
Dai Q; Choy E; Chiu V; Romano J; Slivka SR; Steitz SA; Michaelis S; Philips MR
1998 Jun 12;273(24):15030-15034, Journal of biological chemistry
Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane
— id: 7542, year: 1998, vol: 273, page: 15030, stat: Journal Article,

Brainstem origins of the n18 component of the somatosensory evoked response
Philips, M; Kotapka, M; Patterson, T; Bigelow, D C; Zager, E; Flamm, E S; Stecker, M
1998 ;8(3):133-140, Skull base surgery
Proposed generator sites for the N18 component of the somatosensory evoked potential (SEP) range in location from the medulla to the thalamus. Additional knowledge regarding the generators of the N18 will be important in interpreting the results of intra-operative monitoring during skull base surgery and providing the surgeon more specific information. The goal of this study was to use both intracranial electrical recording and the effects of acute brainstem ischemia in humans to further define the generators of N18. Monopolar electrodes were used to record SEP (after median nerve stimulation) from the brainstem surface in eight patients undergoing posterior fossa surgical procedures. Recordings were made from various locations, from the cervico-medullary junction to the level of the aqueduct of Sylvius. As the electrode moved rostrally on the brainstem surface, the difference in latencies between the scalp N18 potential and the electrode potential approached zero, suggesting an upper pontine-lower midbrain origin of the N18 potential. These findings were supported by the lack of change in the N18 potentials of ten patients with basilar tip aneurysms who experienced marked changes of their N20/P22 potentials during temporary occlusion of the distal basilar artery
— id: 123822, year: 1998, vol: 8, page: 133, stat: Journal Article,

Modes of action of aspirin-like drugs: salicylates inhibit erk activation and integrin-dependent neutrophil adhesion
Pillinger MH; Capodici C; Rosenthal P; Kheterpal N; Hanft S; Philips MR; Weissmann G
1998 Nov 24;95(24):14540-14545, Proceedings of the National Academy of Sciences of the United States of America
The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1-8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses
— id: 7745, year: 1998, vol: 95, page: 14540, stat: Journal Article,

Differential phosphorylation of the beta2 integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils
Buyon JP; Philips MR; Merrill JT; Slade SG; Leszczynska-Piziak J; Abramson SB
1997 Mar;61(3):313-321, Journal of leukocyte biology
Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation
— id: 9735, year: 1997, vol: 61, page: 313, stat: Journal Article,

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex
Clancy RM; Rediske J; Tang X; Nijher N; Frenkel S; Philips M; Abramson SB
1997 Oct 1;100(7):1789-1796, Journal of clinical investigation
Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions
— id: 9733, year: 1997, vol: 100, page: 1789, stat: Journal Article,

Mitogen-activated protein kinase in neutrophils and enucleate neutrophil cytoplasts: evidence for regulation of cell-cell adhesion
Pillinger MH; Feoktistov AS; Capodici C; Solitar B; Levy J; Oei TT; Philips MR
1996 May 17;271(20):12049-12056, Journal of biological chemistry
We employed neutrophils and enucleate neutrophil cytoplasts to study the activation of the mitogen-activated protein kinases (MAPKs) p44erk1 and p42erk2 in neutrophils by inflammatory agonists that engage G protein-linked receptors. Formyl-methionyl-leucyl-phenylalanine (FMLP) rapidly and transiently activated MAPK in neutrophils and cytoplasts, consistent with a role in signaling for neutrophil functions. FMLP stimulated p2lras activation in neutrophils and Raf-1 translocation from cytosol to plasma membrane in cytoplasts, with kinetics consistent with events upstream of MAPK activation. Insulin, a protein tyrosine kinase receptor (PTKR) agonist, stimulated neutrophil MAPK activation, demonstrating an intact system of PTKR signaling in these post-mitotic cells. FMLP- and insulin-stimulated MAPK activation in cytoplasts were inhibited by Bt2cAMP, consistent with signaling through Raf-1 and suggesting a mechanism for cAMP inhibition of neutrophil function. However, Bt2cAMP had no effect on FMLP-stimulated MAPK activation in neutrophils. The extent of MAPK activation by various chemoattractants correlated with their capacity to stimulate neutrophil and cytoplast homotypic aggregation. Consistent with its effects on MAPK, Bt2cAMP inhibited FMLP-stimulated aggregation in cytoplasts but not neutrophils. Insulin had no independent effect but primed neutrophils for aggregation in response to FMLP. Our studies support a p2lras-, Raf-1-dependent pathway for MAPK activation in neutrophils and suggest that neutrophil adhesion may be regulated, in part, by MAPK
— id: 8311, year: 1996, vol: 271, page: 12049, stat: Journal Article,

Translocation of p21rac2 from cytosol to plasma membrane is neither necessary nor sufficient for neutrophil NADPH oxidase activity
Philips MR; Feoktistov A; Pillinger MH; Abramson SB
1995 May 12;270(19):11514-11521, Journal of biological chemistry
Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically
— id: 6712, year: 1995, vol: 270, page: 11514, stat: Journal Article,

Prenylcysteine-directed carboxyl methyltransferase activity in human neutrophil membranes
Philips MR; Pillinger MH
1995 ;256:49-63, Methods in enzymology
— id: 12854, year: 1995, vol: 256, page: 49, stat: Journal Article,

Activation-dependent carboxyl methylation of neutrophil G-protein gamma subunit
Philips MR; Staud R; Pillinger M; Feoktistov A; Volker C; Stock JB; Weissmann G
1995 Mar 14;92(6):2283-2287, Proceedings of the National Academy of Sciences of the United States of America
The gamma subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins (G gamma) are post-translationally processed at their C termini by prenylation, proteolysis, and carboxyl methylation. Whereas prenylation of G gamma is required for membrane association of G proteins, the role of carboxyl methylation is unknown. Here we show that human neutrophils express G gamma 2 but not G gamma 3 or G gamma 7 and that carboxyl methylation of G gamma 2 is associated with signal transduction. In a reconstituted cell-free system, neutrophil G gamma 2 was labeled by the methyl donor S-[methyl-3H]adenosyl-L-methionine. Carboxyl methylation was confirmed by alkaline hydrolysis and quantitation of volatile [3H]methanol. Neutrophil G gamma 2 methylation was stimulated by activation of G protein with guanosine 5'-[beta, gamma-thio]triphosphate. We estimate that after 1 hr of G-protein activation at least 6% of the total pool of G gamma 2 was carboxyl-methylated. The inflammatory agonist fMet-Leu-Phe stimulated guanosine 5'-[beta,gamma-thio]triphosphate-dependent carboxyl methylation. Methylation of G gamma 2 was inhibited by the carboxyl methyltransferase inhibitor N-acetyl-S-trans,trans-farnesylcysteine at concentrations that affected signal transduction in neutrophils. These results demonstrate that activation of neutrophil Gi is associated with alpha-carboxyl methyl esterification of G gamma 2 and suggest that carboxyl methylation of G gamma may play a role in signal transduction
— id: 6713, year: 1995, vol: 92, page: 2283, stat: Journal Article,

CARBOXYL METHYLATION OF RHO-FAMILY RAS-RELATED PROTEINS IN HUMAN NEUTROPHILS IS ASSOCIATED WITH ACTIN POLYMERIZATION
PHILIPS, MR; FEOKTISTOV, AS; PILLINGER, MH
1995 SEP ;38(9):159-159, Arthritis & rheumatism
— id: 86687, year: 1995, vol: 38, page: 159, stat: Journal Article,

Crosstalk in signal transduction via EP receptors: prostaglandin E1 inhibits chemoattractant-induced mitogen-activated protein kinase activity in human neutrophils
Pillinger MH; Philips MR; Feoktistov A; Weissmann G
1995 ;23:311-316, Advances in prostaglandin thromboxane & leukotriene research
— id: 6714, year: 1995, vol: 23, page: 311, stat: Journal Article,

CROSSTALK IN SIGNAL-TRANSDUCTION VIA EP RECEPTORS - PROSTAGLANDIN E(1) INHIBITS CHEMOATTRACTANT-INDUCED MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY IN HUMAN NEUTROPHILS
PILLINGER, MH; PHILIPS, MR; FEOKTISTOV, A; WEISSMANN, G
1995 FEB ;23(2):311-316, Advances in prostaglandin & thromboxane research
— id: 87404, year: 1995, vol: 23, page: 311, stat: Journal Article,

Prenylcysteine analogs to study function of carboxylmethylation in signal transduction
Volker C; Pillinger MH; Philips MR; Stock JB
1995 ;250(19):216-225, Methods in enzymology
Carboxylmethylation of ras-related proteins is stimulated immediately on exposure of myeloid cells to inflammatory agonists. When the methylation reaction is inhibited with prenylcysteine analogs, G-protein-mediated signal transduction responses are disrupted, but responses to phorbol ester, calcium ionophore, and phospholipase C (PLC) remain intact. Furthermore, prenylcysteine analogs block GTP gamma S-induced aggregation of permeabilized platelets. Together, these results suggest that protein prenylcysteine methylation can play a role in signal transduction. A number of studies with AdoMet antagonists have suggested a role for methylation in cell-cycle regulation and stimulus-response coupling. Because the compounds generally inhibit all cellular methylation events, however, their effects have been difficult to interpret. On the other hand, prenylcysteine analogs have proved to be specific inhibitors of protein prenylcysteine methylation, as opposed to other types of methylation reactions. This enables the segregation of the role of methylation at C-terminal prenylcysteine residues from methylation at other sites, such as the carboxyl terminus of the catalytic subunit of PP2A. It should be emphasized, however, that prenylcysteine tails of proteins may interact with other target sites in addition to the methyltransferase enzyme(s), and prenylcysteine analogs may compete for these sites as well. One cannot assume that the inhibition of a response by the drugs necessarily implicates the involvement of a prenylcysteine methylation reaction. Studies with the analogs must be interpreted in conjunction with other results to ascertain the locus of their effects
— id: 23914, year: 1995, vol: 250, page: 216, stat: Journal Article,

Inhibition of neutrophil function by aspirin-like drugs (NSAIDS): requirement for assembly of heterotrimeric G proteins in bilayer phospholipid
Abramson SB; Leszczynska-Piziak J; Clancy RM; Philips M; Weissmann G
1994 Feb 9;47(3):563-572, Biochemical pharmacology
Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms that are independent of their effects on prostaglandin biosynthesis. We examined the effects of sodium salicylate and piroxicam on GTP/GDP exchange by a regulatory G protein (G alpha i). Plasma membrane and cytosol of human neutrophils were prepared by nitrogen cavitation and discontinuous sucrose density centrifugation. Salicylate (3 mM) and piroxicam (50 microM) reduced [35S]GTP gamma S binding to purified plasma membranes [65 +/- 3.7 and 75 +/- 5.3% (P < 0.003) of control, respectively]. Membrane-associated G alpha/beta gamma was solubilized by treatment of plasma membranes with sodium cholate. NSAIDs did not inhibit binding of GTP to solubilized G alpha/beta gamma derived from detergent-treated plasma membranes. Lipid reconstitution was achieved by detergent dialysis followed by the addition of bilayer liposomes (phosphatidylcholine). Salicylate and piroxicam inhibited GTP gamma S binding to G alpha/beta gamma derived from solubilized plasma membranes reconstituted in phosphatidylcholine vesicles (bilayer structures) but had no effect when phosphatidylethanolamine (hexagonal phase II structure) was used for reconstitution. Salicylate and piroxicam had no effect on GTP binding to cytosolic fractions in which soluble G alpha i exists as a free subunit, suggesting that the effect required either assembly of G alpha i/beta gamma heterotrimer or the presence of a lipid bilayer. Although the addition of purified bovine beta gamma subunits to dialyzed cytosol increased both the total GIP binding capacity and the pertussis toxin-dependent ADP-ribosylation of G alpha i, consistent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrimeric G alpha cytosol/beta gamma bovine when the complex was inserted into bilayer liposomes. The data indicate that salicylate and piroxicam disrupt neutrophil function via their capacity to interfere with GTP/GDP exchange at an alpha subunit of a regulatory G protein, an effect which requires assembly of the active heterotrimer G alpha i/beta gamma in a phospholipid bilayer
— id: 6306, year: 1994, vol: 47, page: 563, stat: Journal Article,

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils
Pillinger MH; Volker C; Stock JB; Weissmann G; Philips MR
1994 Jan 14;269(2):1486-1492, Journal of biological chemistry
Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state
— id: 6485, year: 1994, vol: 269, page: 1486, stat: Journal Article,

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils
Philips MR; Pillinger MH; Staud R; Volker C; Rosenfeld MG; Weissmann G; Stock JB
1993 Feb 12;259(5097):977-980, Science
In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins
— id: 13250, year: 1993, vol: 259, page: 977, stat: Journal Article,

Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors
Cronstein BN; Levin RI; Philips M; Hirschhorn R; Abramson SB; Weissmann G
1992 Apr 1;148(7):2201-2206, Journal of immunology
We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role)
— id: 9751, year: 1992, vol: 148, page: 2201, stat: Journal Article,

LIGATION OF THE NEUTROPHIL FORMYL PEPTIDE RECEPTOR INDUCES CARBOXYL METHYLATION OF G-GAMMA
STAUD, R; PILLINGER, MH; WEISSMANN, G; PHILIPS, MR
1992 SEP ;35(9):S137-S137, Arthritis & rheumatism
— id: 74222, year: 1992, vol: 35, page: S137, stat: Journal Article,

Low molecular weight GTP-binding proteins in human neutrophil granule membranes
Philips MR; Abramson SB; Kolasinski SL; Haines KA; Weissmann G; Rosenfeld MG
1991 Jan 15;266(2):1289-1298, Journal of biological chemistry
Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments
— id: 8314, year: 1991, vol: 266, page: 1289, stat: Journal Article,

Nonsteroidal antiinflammatory drugs exert differential effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes
Abramson SB; Cherksey B; Gude D; Leszczynska-Piziak J; Philips MR; Blau L; Weissmann G
1990 Feb;14(1):11-30, Inflammation
Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C]glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity
— id: 9760, year: 1990, vol: 14, page: 11, stat: Journal Article,

Constitutive and induced phosphorylation of the alpha- and beta-chains of the CD11/CD18 leukocyte integrin family. Relationship to adhesion-dependent functions
Buyon JP; Slade SG; Reibman J; Abramson SB; Philips MR; Weissmann G; Winchester R
1990 Jan 1;144(1):191-197, Journal of immunology
We sought to determine whether the activation event which renders the CD11/CD18 leukocyte integrin/Leu-CAM glycoproteins capable of promoting cell to cell adhesion was associated with the induced posttranslational modification of phosphorylation. In neutrophils, two species of alpha-chains, a predominant CD11b 165-kDa subunit and a minor 150-kDa CD11c subunit were found to be constitutively phosphorylated. However, the 95-kDa CD18 common beta-chain was not phosphorylated in resting cells but became strongly phosphorylated in cells incubated with PMA. The beta-chain was phosphorylated in a dose-related manner within 1 min of the addition of PMA, reached maximal intensity between 4 to 10 min, and remained fully phosphorylated at 30 min. The similarities of the kinetics of homotypic aggregation induced by PMA to the time course of phosphorylation suggest that phosporylation may be relevant to at least this type of Leu-CAM-dependent adhesion. In contrast, in the presence of FMLP which induces aggregation with different kinetics than PMA, no phosphorylation of the common beta-chain was observed over a time interval of from 30 s to 10 min further emphasizing the apparent differences in the two modes of activation to an adhesive state. The phosphorylated species on neutrophils were readily detected by immunoprecipitation with each CD18 mAb and most but not all CD11b mAb which otherwise precipitated 125I-labeled CD11b species suggesting that the CD11b alpha-chain labelled with 32P may differ slightly from the 125I-labeled species in terms of its recognition by certain CD11b mAb. In mononuclear cells, similar constitutive phosphorylation of the CD11a and CD11c alpha-chains was observed that remained unchanged in the presence of either FMLP or PMA. As was demonstrated in neutrophils, phosphorylation of the CD18 beta-chains of mononuclear cells was not constitutive but was induced in the presence of PMA and not FMLP. Taken together these data suggest the existence of specific recognition sites on beta-chains for a regulatory kinase-phosphatase system
— id: 9762, year: 1990, vol: 144, page: 191, stat: Journal Article,

Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase
Haines KA; Reibman J; Callegari PE; Abramson SB; Philips MR; Weissmann G
1990 Jun 15;144(12):4757-4764, Journal of immunology
Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses
— id: 9758, year: 1990, vol: 144, page: 4757, stat: Journal Article,

Dissociation between increased surface expression of gp165/95 and homotypic neutrophil aggregation
Buyon JP; Abramson SB; Philips MR; Slade SG; Ross GD; Weissmann G; Winchester RJ
1988 May 1;140(9):3156-3160, Journal of immunology
Whether homotypic neutrophil aggregation depends on the quantitative increase of gp165/95 molecules (Mac 1, CR3) recruited to the cell surface during activation was studied using mAb of the CD11b group that recognize distinct epitopes encoded by the alpha-subunit of this glycoprotein. After the addition of antibody MN41, neutrophils did not aggregate in response to a chemoattractant, FMLP. Blockade of preexisting surface gp165/95 by mAb MN41, followed by removal of the excess antibody from the mixture, was used to show that the molecules of gp165/95 newly expressed in response to stimulation by a chemoattractant were incapable of effectively mediating the induced cell-cell interactions of aggregation. Flow cytometry studies confirmed that binding of unlabeled antibody MN41 did not block further increases in surface expression of gp165/95 after stimulation with FMLP. These data suggest that molecules of gp165/95 exhibit two functionally distinct forms, one, present on the surface of freshly isolated neutrophils, that becomes competent to mediate the aggregation response upon activation by a stimulus and a second form that can be translocated to the cell surface by the stimulus but is greatly diminished if not lacking in the ability to participate in that aggregation event
— id: 9768, year: 1988, vol: 140, page: 3156, stat: Journal Article,

Increased levels of plasma anaphylatoxins in systemic lupus erythematosus predict flares of the disease and may elicit vascular injury in lupus cerebritis
Hopkins P; Belmont HM; Buyon J; Philips M; Weissmann G; Abramson SB
1988 May;31(5):632-641, Arthritis & rheumatism
We measured levels of complement anaphylatoxin split products, C3a and C5a, in the circulation of patients with systemic lupus erythematosus (SLE). In 23 SLE patients who were followed serially, the mean C3a value was 179 ng/ml during stable disease and 550 ng/ml during a disease flare. In 10 patients, C3a levels predicted disease activity, with the C3a value rising from a mean of 183 ng/ml at a time of stable disease to a mean of 242 ng/ml 1-2 months prior to a clinical exacerbation of disease. The mean C3a level in 5 patients with acute dysfunction of the central nervous system (CNS) was 1,297 ng/ml, which is significantly higher than that observed in patients with active disease but without CNS involvement (P less than 0.01). C5a levels were also significantly elevated in 4 patients with acute CNS disease. Pathologic specimens from 2 patients who died during an acute lupus flare revealed neutrophils occluding the cerebral and intestinal vessels. Fluorescein angiography in a patient with CNS lupus revealed vasoocclusive retinopathy. In 5 of 7 SLE patients who were pregnant, C3a levels were elevated, with a group mean value of 310 ng/ml. There was a negative correlation (r = -0.59) between C3a and C3 levels in pregnant patients with SLE, and this finding is consistent with complement activation as the cause of decreasing C3 levels. We suggest that serial measurements of C3a can predict flares of disease in lupus patients and can demonstrate complement activation during pregnancy in women with SLE. In addition, release of C3a and C5a (mediators of inflammation) into the circulation may elicit vascular injury, particularly in patients with lupus cerebritis
— id: 9769, year: 1988, vol: 31, page: 632, stat: Journal Article,

Up-regulation of the iC3b receptor (CR3) is neither necessary nor sufficient to promote neutrophil aggregation
Philips MR; Buyon JP; Winchester R; Weissmann G; Abramson SB
1988 Aug;82(2):495-501, Journal of clinical investigation
The iC3b receptor (CR3) is required for neutrophil adhesive functions, including homotypic aggregation. Because stimuli that enhance neutrophil adhesion also induce up-regulation of surface CR3, it is widely held that these two responses are causally related. We have dissociated CR3 display (immunofluorescence) from CR3 function (aggregation). Neutrophils isolated at 4 degrees C and rewarmed to 37 degrees C up-regulated surface CR3 twofold, but did not aggregate. The kinetics of FMLP-induced CR3 up-regulation were discordant with those of aggregation. In the absence of extracellular divalent cations, CR3 expression increased twofold after exposure to FMLP, but neutrophils did not aggregate. FMLP elicited 3.5-fold more aggregation than the ionophore A23187, yet less than one-half as much CR3 up-regulation. 3 mM sodium salicylate inhibited aggregation 55 +/- 4%, but had no effect on CR3 up-regulation. Conversely, 1 mM tetracaine completely inhibited CR3 up-regulation, while significantly enhancing aggregation. Neutroplasts expressed CR3, but did not up-regulate the receptor; in contrast, FMLP induced CR3-dependent aggregation of neutroplasts. We conclude that, although constitutive surface CR3 is required for neutrophil aggregation, the up-regulation of CR3 is neither necessary nor sufficient to promote cell-cell adhesion
— id: 9767, year: 1988, vol: 82, page: 495, stat: Journal Article,