Jan M Sap, Ph.D.

Regulatory Effects of Nitric Oxide on Src Kinase, FAK, p130Cas, and Receptor Protein Tyrosine Phosphatase Alpha (PTP-alpha): A Role for the Cellular Redox Environment
Curcio, MF; Batista, WL; Linares, E; Nascimento, FD; Moraes, MS; Borges, RE; Sap, J; Stern, A; Monteiro, HP
2010 JUL ;13(2):109-125, Antioxidants & Redox Signaling
The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts 'knockout'' for PTP-alpha (PTP-alpha(-/-) cells), and 'rescued'' 'knockout'' fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration. Antioxid. Redox Signal. 13, 109-125
— id: 110121, year: 2010, vol: 13, page: 109, stat: Journal Article,

Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTP alpha Is Substrate-specific and Compatible with Lipid Raft Localization
Vacaresse, N; Moller, B; Danielsen, EM; Okada, M; Sap, J
2008 DEC 19 ;283(51):35815-35824, Journal of biological chemistry
Src family tyrosine kinases (SFKs) function in multiple signaling pathways, raising the question of how appropriate regulation and substrate choice are achieved. SFK activity is modulated by several protein-tyrosine phosphatases, among which RPTP alpha and SHP2 are the best established. We studied how RPTP alpha affects substrate specificity and regulation of c-Src and Fyn in response to epidermal growth factor and platelet-derived growth factor. We find that RPTP alpha, in a growth factor-specific manner, directs the specificity of these kinases toward a specific subset of SFK substrates, particularly the focal adhesion protein Paxillin and the lipid raft scaffolding protein Cbp/PAG. A significant fraction of RPTP alpha is present in lipid rafts, where its targets Fyn and Cbp/PAG reside, and growth factor-mediated SFK activation within this compartment is strictly dependent on RPTP alpha. Forced concentration of RPTP alpha into lipid rafts is compatible with activation of Fyn. Finally, RPTP alpha-induced phosphorylation of Paxillin and Cbp/PAG induces recruitment of the SFK inhibitory kinase Csk, indicative of negative feedback loops limiting SFK activation by RPTP alpha . Our findings indicate that individual SFK-controlling PTPs play important and specific roles in dictating SFK substrate specificity and regulatory mechanism
— id: 91386, year: 2008, vol: 283, page: 35815, stat: Journal Article,

Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage-gated potassium channels in Schwann cells
Tiran, Zohar; Peretz, Asher; Sines, Tal; Shinder, Vera; Sap, Jan; Attali, Bernard; Elson, Ari
2006 Oct;17(10):4330-4342, Molecular biology of the cell
Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPepsilon and PTPalpha and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPalpha inhibits Kv channels more strongly than PTPepsilon; this correlates with constitutive association of PTPalpha with Kv2.1, driven by membranal localization of PTPalpha. PTPalpha, but not PTPepsilon, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPepsilon and PTPalpha differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo
— id: 102593, year: 2006, vol: 17, page: 4330, stat: Journal Article,

Essential domain of receptor tyrosine phosphatase beta (R
Yahiro, K; Wada, A; Yamasaki, E; Nakayama, M; Nishi, Y; Hisatsune, J; Morinaga, N; Sap, J; Noda, M; Moss, J; Hirayama, T
2004 DEC 3 ;279(49):51013-51021, Journal of biological chemistry
Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, R
— id: 47213, year: 2004, vol: 279, page: 51013, stat: Journal Article,

Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation
Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng; Dusa, Adina; Su, Jing; Zagzag, David; Bianchi, Riccardo; Casaccia-Bonnefil, Patrizia; Arancio, Ottavio; Sap, Jan
2003 Aug 15;22(16):4121-4131, EMBO journal
Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra(-/-) mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPalpha also underperform in the radial-arm water-maze test. These studies identify RPTPalpha as a key mediator of neuronal migration and synaptic plasticity
— id: 38440, year: 2003, vol: 22, page: 4121, stat: Journal Article,

RPTP-alpha acts as a transducer of mechanical force on alphav/beta3-integrin-cytoskeleton linkages
von Wichert, Gotz; Jiang, Guoying; Kostic, Ana; De Vos, Kurt; Sap, Jan; Sheetz, Michael P
2003 Apr 14;161(1):143-153, Journal of cell biology
Cell motility on ECM critically depends on the cellular response to force from the matrix. We find that force-dependent reinforcement of alphav/beta3-integrin-mediated cell-matrix connections requires the receptor-like tyrosine phosphatase alpha (RPTPalpha). RPTPalpha colocalizes with alphav-integrins at the leading edge during early spreading, and coimmunoprecipitates with alphav-integrins during spreading on fibronectin and vitronectin. RPTPalpha-dependent activation of Src family kinases, in particular activation of Fyn, is required for the force-dependent formation of focal complexes and strengthening of alphav/beta3-integrin-cytoskeleton connections during the initial phase of ECM contact. These observations indicate that Src family kinases have distinct functions during adhesion site assembly, and that RPTPalpha is an early component in force-dependent signal transduction pathways leading to the assembly of focal complexes on both fibronectin and vitronectin
— id: 34653, year: 2003, vol: 161, page: 143, stat: Journal Article,

A possible molecular mechanism of action for the potential tumor suppressor gene INT6 provided by studies in fission yeast
Yen, HCS; Ren, G; Nelson, L; Sha, Z; Sap, J; Chang, E
2003 OCT 28 ;82(17):S172-S172, Breast cancer research & treatment
— id: 42532, year: 2003, vol: 82, page: S172, stat: Journal Article,

A complex of RPTP alpha and alpha(v)/beta(3)-integrins acts as transducer of mechanical force and initiates focal contact assembly through activation of Fyn
Kostic, A; von Wichert, G; Sap, J; Sheetz, MP
2002 NOV ;13(4):335A-335A, Molecular biology of the cell
— id: 37190, year: 2002, vol: 13, page: 335A, stat: Journal Article,

The differentiation of skeletal muscle cells involves a protein-tyrosine phosphatase-alpha-mediated C-Src signaling pathway
Lu, Huogen; Shah, Poonam; Ennis, David; Shinder, Gail; Sap, Jan; Le-Tien, Hoang; Fantus, I George
2002 Nov 29;277(48):46687-46695, Journal of biological chemistry
Protein-tyrosine phosphatase-alpha (PTPalpha) plays an important role in various cellular signaling events, including proliferation and differentiation. In this study, we established L6 cell lines either underexpressing or overexpressing PTPalpha by stable transfection of cells with antisense PTPalpha or with full-length wild-type human or mouse or double catalytic site Cys --> Ala mutant (DM8) PTPalpha cDNA. Expression of PTPalpha in these cell lines was determined by immunoblotting and immunofluorescence. Cells harboring antisense PTPalpha exhibited a significantly reduced growth rate and thymidine incorporation when compared with the wild-type L6 cells. In contrast, cells overexpressing PTPalpha showed more rapid (2-fold) proliferation. Myoblasts with diminished PTPalpha failed to undergo fusion and did not form myotubes in reduced serum whereas overexpression of PTPalpha promoted myogenesis 2 days earlier than wild-type L6 cells. Overexpression of phosphatase-inactive mutant PTPalpha recapitulated the phenotype of the antisense cells. The different myogenic activities of these cell lines were correlated with the expression of myogenin and creatine kinase activity. Consistent with previous reports, PTPalpha positively regulated the activity of the protein-tyrosine kinase Src. Treatment of L6 cells with PP2 or SU6656, specific inhibitors of Src family kinases, and transient transfection of dominant-inhibitory Src inhibited the formation of myotubes and expression of myogenin. Moreover, enhanced expression of PTPalpha and activation of Src was detected during myogenesis. Together, these data indicate that PTPalpha is involved in the regulation of L6 myoblast growth and skeletal muscle cell differentiation via an Src-mediated signaling pathway
— id: 34654, year: 2002, vol: 277, page: 46687, stat: Journal Article,

c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation
Yang, Liang-Tung; Alexandropoulos, Konstantina; Sap, Jan
2002 May 17;277(20):17406-17414, Journal of biological chemistry
SRC family kinases have been consistently and recurrently implicated in neurite extension events, yet the mechanism underlying their neuritogenic role has remained elusive. We report that epidermal growth factor (EGF) can be converted from a non-neuritogenic into a neuritogenic factor through moderate activation of endogenous SRC by receptor-protein-tyrosine phosphatase alpha (a physiological SRC activator). We show that such a qualitative change in the response to EGF is not accompanied by changes in the extent or kinetics of ERK induction in response to this factor. Instead, the pathway involved relies on increased tyrosine phosphorylation of, and recruitment of Crk to, the SRC substrate Sin/Efs. The latter is a scaffolding protein structurally similar to the SRC substrate Cas, tyrosine phosphorylation of which is critical for migration in fibroblasts and epithelial cells. Expression of a dominant negative version of Sin interfered with receptor-protein-tyrosine phosphatase alpha/EGF- as well as fibroblast growth factor-induced neurite outgrowth. These observations uncouple neuritogenic signaling in PC12 cells from sustained activation of ERK kinases and for the first time identify an effector of SRC function in neurite extension
— id: 34655, year: 2002, vol: 277, page: 17406, stat: Journal Article,

Expression of protein tyrosine phosphatase alpha (RPTPalpha) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo
Ardini E; Agresti R; Tagliabue E; Greco M; Aiello P; Yang LT; Menard S; Sap J
2000 Oct 12;19(43):4979-4987, Oncogene
Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPalpha is implicated in the activation of Src family kinases, and regulation of integrin signaling, cell adhesion, and growth factor responsiveness. To explore its potential contribution to human neoplasia, we surveyed RPTPalpha protein levels in primary human breast cancer. We found RPTPalpha levels to vary widely among tumors, with 29% of cases manifesting significant overexpression. High RPTPalpha protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPalpha in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed tumor growth and metastasis. To our knowledge, this is the first example of a study correlating expression level of a specific bona fide PTP with neoplastic disease status in humans
— id: 17815, year: 2000, vol: 19, page: 4979, stat: Journal Article,

Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?
Petrone A; Sap J
2000 Jul;113(Pt 13):2345-2354, Journal of cell science
Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity
— id: 11649, year: 2000, vol: 113, page: 2345, stat: Journal Article,

Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells
Cong LN; Chen H; Li Y; Lin CH; Sap J; Quon MJ
1999 Feb 16;255(2):200-207, Biochemical & biophysical research communications
Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two transmembrane PTPases) in this metabolic action of insulin. Rat adipose cells were transfected with either PTP-alpha or PTP-kappa and effects of these PTPases on the translocation of a cotransfected epitope-tagged GLUT4 were studied. Cells overexpressing wild-type PTP-alpha had significantly lower levels of cell surface GLUT4 in response to insulin and a threefold decrease in insulin sensitivity when compared with control cells expressing only tagged GLUT4. Co-overexpression of PTP-alpha and PTP1B did not have additive effects, suggesting that these PTPases share common substrates. Cells overexpressing either wild-type PTP-kappa or catalytically inactive mutants of PTP-alpha had dose-response curves similar to those of control cells. Since overexpression of PTP-alpha, but not PTP-kappa, had effects on translocation of GLUT4, our data suggest that PTPalpha may be a specific negative modulator of insulin-stimulated glucose transport
— id: 17817, year: 1999, vol: 255, page: 200, stat: Journal Article,

Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-alpha
Jiang G; den Hertog J; Su J; Noel J; Sap J; Hunter T
1999 Oct 7;401(6753):606-610, Nature
Protein-tyrosine phosphatases (PTPs) are vital for regulating tryosine phosphorylation in many processes, including growth and differentiation. The regulation of receptor-like PTP (RPTP) activity remains poorly understood, but based on the crystal structure of RPTPalpha domain 1 we have proposed that dimerization can negatively regulate activity, through the interaction of an inhibitory 'wedge' on one monomer with the catalytic cleft of domain 1 in the other monomer. Here we show that dimerization inhibits the activity of a full-length RPTP in vivo. We generated stable disulphide-bonded full-length RPTPalpha homodimers by expressing mutants with single cysteines at different positions in the ectodomain juxtamembrane region. Expression of wild-type RPTPalpha and Phe135Cys and Thr141Cys mutants in RPTPalpha-null mouse embryo cells increased dephosphorylation and activity of Tyr 529 in the protein tyrosine kinase c-Src; in contrast, expression of a Pro137Cys mutant did not. Mutation of Pro 210/211 to leucine in the inhibitory wedge of the Pro137Cys mutant restored its ability to activate c-Src, indicating that dimerization may inhibit full-length RPTPalpha activity in a manner stereochemically consistent with RPTPalpha crystal structures. Our results suggest that RPTPalpha activity can in principle be negatively regulated by dimerization in vivo
— id: 17816, year: 1999, vol: 401, page: 606, stat: Journal Article,

Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse
Shen P; Canoll PD; Sap J; Musacchio JM
1999 May 1;826(2):157-171, Brain research
Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions in vitro [Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, J. Sap, Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region, Mol. Cell. Biol. 13 (1993) 2942-2951; J. Sap, Y.-P. Jiang, D. Friedlander, M. Grumet, J. Schlessinger, Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding, Mol. Cell. Biol. 14 (1994) 1-9]. The homology of RPTP-kappa's ectodomain to neural cell adhesion molecules indicates potential roles in developmental processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene, the consequent loss of RPTP-kappa's enzymatic activity does not produce any obvious phenotypic defects [W.C. Skarnes, J.E. Moss, S.M. Hurtley, R.S.P. Beddington, Capturing genes encoding membrane and secreted proteins important for mouse development, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6592-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells throughout the CNS that display RPTP-kappa promoter activity. Several neural systems highly expressed the transgene-most notably cortical, olfactory, hippocampal, hypothalamic, amygdaloid and visual structures. These well-characterized brain regions may provide a basis for future studies of RPTP-kappa function
— id: 56429, year: 1999, vol: 826, page: 157, stat: Journal Article,

Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts
Su J; Muranjan M; Sap J
1999 May 20;9(10):505-511, Current biology. CB
BACKGROUND: Fyn and c-Src are two of the most widely expressed Src-family kinases. Both are strongly implicated in the control of cytoskeletal organization and in the generation of integrin-dependent signalling responses in fibroblasts. These proteins are representative of a large family of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect of inactivation of the c-src gene. In response to adhesion on a fibronectin substrate, RPTPalpha-/- fibroblasts also exhibited characteristic deficiencies in integrin-mediated signalling responses, such as decreased tyrosine phosphorylation of the c-Src substrates Fak and p 130(cas), and reduced activation of extracellular signal regulated (Erk) MAP kinases. CONCLUSIONS: These observations demonstrate that RPTPalpha functions as a physiological upstream activator of Src-family kinases in fibroblasts and establish this tyrosine phosphatase as a newly identified regulator of integrin signalling
— id: 56441, year: 1999, vol: 9, page: 505, stat: Journal Article,

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression
Jacob KK; Sap J; Stanley FM
1998 Feb 20;273(8):4800-4809, Journal of biological chemistry
A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent. Experiments with inhibitors of phosphatidylinositol 3-kinase suggest that insulin-increased prolactin-CAT expression is phosphatidylinositol 3-kinase-independent. These results suggest that RPTPalpha may be a physiological regulator of insulin action
— id: 7617, year: 1998, vol: 273, page: 4800, stat: Journal Article,

Association of PTPk with other cellular proteins
Amankular, N; Yang, LT; Sap, J
1997 NOV ;8(5):129-129, Molecular biology of the cell
— id: 53156, year: 1997, vol: 8, page: 129, stat: Journal Article,

Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences
Su J; Yang LT; Sap J
1996 Nov 8;271(45):28086-28096, Journal of biological chemistry
Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2 domain in Grb2 (, ). We show here that association of Grb2 with RPTPalpha also involves a critical function for the C-terminal SH3 domain of Grb2. Furthermore, Grb2 SH3 binding peptides interfere with RPTPalpha-Grb2 association in vitro, and the RPTPalpha protein can dissociate the Grb2-Sos complex in vivo. These observations constitute a novel mode of Grb2 association and suggest a model in which association with a tyrosine-phosphorylated protein restricts the repertoire of SH3 binding proteins with which Grb2 can simultaneously interact. The function of the Tyr798 tyrosine phosphorylation/Grb2 binding site in RPTPalpha was studied further by expression of wild type or mutant RPTPalpha proteins in PC12 cells. In these cells, wild type RPTPalpha interferes with acidic fibroblast growth factor-induced neurite outgrowth; this effect requires both the catalytic activity and the Grb2 binding Tyr798 residue in RPTPalpha. In contrast, expression of catalytically active RPTPalpha containing a mutated tyrosine phosphorylation/Grb2 association site enhances neurite outgrowth. Our observations associate a functional effect with tyrosine phosphorylation of, and ensuing association of signaling proteins with, a receptor protein-tyrosine phosphatase and raise the possibility that RPTPalpha association may modulate Grb2 function and vice versa
— id: 12475, year: 1996, vol: 271, page: 28086, stat: Journal Article,

Stimulation of receptor protein-tyrosine phosphatase alpha activity and phosphorylation by phorbol ester
den Hertog J; Sap J; Pals CE; Schlessinger J; Kruijer W
1995 Mar;6(3):303-307, Cell growth & differentiation
Receptor Protein-Tyrosine Phosphatase alpha (RPTP alpha) is a transmembrane protein with two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains and a relatively short (123 amino acids) extracellular domain. Here we report that treatment of transfected cells that express RPTP alpha with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, a direct activator of protein kinase C, induced a rapid, transient increase in RPTP alpha activity due to a 2- to 3-fold increase in substrate affinity. A transient increase in RPTP alpha serine phosphorylation was concomitant with the enhanced activity. Tryptic phosphopeptide mapping of RPTP alpha demonstrated that phosphorylation of three tryptic peptides was enhanced in response to phorbol ester. In vitro dephosphorylation of RPTP alpha from phorbol ester-treated cells reduced RPTP alpha activity to prestimulation levels, indicating that enhanced serine phosphorylation directly accounted for the increase in activity. Our results demonstrate that serine phosphorylation may play a key role in the regulation of the activity of transmembrane PTPs
— id: 17819, year: 1995, vol: 6, page: 303, stat: Journal Article,

Homophilic interactions mediated by receptor tyrosine phosphatases mu and kappa. A critical role for the novel extracellular MAM domain
Zondag GC; Koningstein GM; Jiang YP; Sap J; Moolenaar WH; Gebbink MF
1995 Jun 16;270(24):14247-14250, Journal of biological chemistry
The receptor-like protein tyrosine phosphatases (RPTP) mu and RPTP kappa have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTP mu and RPTP kappa can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTP mu and RPTP kappa fail to interact in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTP mu lacking the MAM domain in insect Sf9 cells. Truncated RPTP mu is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored in a chimeric RPTP mu molecule containing the MAM domain of RPTP kappa. However, this chimeric RPTP mu does not interact with either RPTP mu or RPTP kappa. These results indicate that the MAM domain of RPTP mu and RPTP kappa is essential for homophilic cell-cell interaction and helps determine the specificity of these interactions
— id: 17818, year: 1995, vol: 270, page: 14247, stat: Journal Article,

Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin
Barnea G; Grumet M; Milev P; Silvennoinen O; Levy JB; Sap J; Schlessinger J
1994 May 20;269(20):14349-14352, Journal of biological chemistry
The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system
— id: 6318, year: 1994, vol: 269, page: 14349, stat: Journal Article,

Close similarity between receptor-linked tyrosine phosphatase and rat brain proteoglycan
Barnea G; Grumet M; Sap J; Margolis RU; Schlessinger J
1994 Jan 28;76(2):205-205, Cell
— id: 17822, year: 1994, vol: 76, page: 205, stat: Journal Article,

Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation
Daum G; Regenass S; Sap J; Schlessinger J; Fischer EH
1994 Apr 8;269(14):10524-10528, Journal of biological chemistry
Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7000-7004). Using the polymerase chain reaction technique, simultaneous expression of both isoforms was demonstrated in human T-cell and vascular smooth muscle libraries, as well as in the A431 human epidermal cancer cell line. Following transient expression in COS-1 cells, each isoform gave rise to two proteins of 100 and 130 kDa, respectively. Endoglycosidase treatment showed that the 100-kDa species corresponded to a molecule exclusively glycosylated on N-residues, whereas the 130-kDa species contained both, N- and O-linked carbohydrates. Pulse-chase experiments demonstrated that the smaller RPTP alpha protein is a precursor of the larger one. A high affinity antibody was generated that recognizes the immature protein only; however, both proteins can be detected by Western blot analysis after a simple chemical hydrolysis. Following Superose 12 chromatography, the 100- and 130-kDa species of RPTP alpha emerged as 200- and 340-kDa proteins, respectively. Both species exhibited similar enzymatic activities as determined with a peptide substrate in immunoprecipitates
— id: 17821, year: 1994, vol: 269, page: 10524, stat: Journal Article,

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation
Desai DM; Sap J; Silvennoinen O; Schlessinger J; Weiss A
1994 Sep 1;13(17):4002-4010, EMBO journal
Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules
— id: 17820, year: 1994, vol: 13, page: 4002, stat: Journal Article,

Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding
Sap J; Jiang YP; Friedlander D; Grumet M; Schlessinger J
1994 Jan;14(1):1-9, Molecular & cellular biology
Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation
— id: 6511, year: 1994, vol: 14, page: 1, stat: Journal Article,

Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2
Su J; Batzer A; Sap J
1994 Jul 22;269(29):18731-18734, Journal of biological chemistry
Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function
— id: 6530, year: 1994, vol: 269, page: 18731, stat: Journal Article,

CD45 TYROSINE PHOSPHATASE REGULATION OF TYROSINE KINASES THAT INTERACT WITH THE T-CELL ANTIGEN RECEPTOR
WEISS, A; CHAN, A; IWASHIMA, M; STRRAUS, D; SIEH, M; SAP, J; SCHLESSINGER, J; DESAI, DM
1994 JAN 4 ;269(6):156-156, Journal of cellular biochemistry
— id: 52584, year: 1994, vol: 269, page: 156, stat: Journal Article,

CD45 TYROSINE PHOSPHATASE REGULATION OF TYROSINE KINASES THAT INTERACT WITH THE T-CELL ANTIGEN RECEPTOR Abstract]
WEISS, A; CHAN, A; IWASHIMA, M; STRRAUS, D; SIEH, M; SAP, J; SCHLESSINGER, J; DESAI, DM
1994 JAN 4 ;269(6):129-129, Journal of cellular biochemistry
— id: 52583, year: 1994, vol: 269, page: 129, stat: Journal Article,

The expression of a novel receptor-type tyrosine phosphatase suggests a role in morphogenesis and plasticity of the nervous system
Canoll PD; Barnea G; Levy JB; Sap J; Ehrlich M; Silvennoinen O; Schlessinger J; Musacchio JM
1993 Oct 15;75(2):293-298, Brain research. Developmental brain research
Analysis of the localization of receptor-type protein tyrosine phosphatase-beta (RPTP-beta) by in situ hybridization and immunocytochemistry indicates that it is predominantly expressed in the developing central nervous system (CNS). RPTP-beta is highly expressed in radial glia and other forms of glial cells that play an important role during development. The immunoreactivity localizes to the radial processes of these cells, which act as guides during neuronal migration and axonal elongation. The pattern of RPTP-beta expression changes with the progression of glial cell differentiation. In the adult, high levels of RPTP-beta are seen in regions of the brain where there is continued neurogenesis and neurite outgrowth. The spatial and temporal patterns of RPTP-beta expression suggest that this receptor phosphatase plays a role in morphogenesis and plasticity of the nervous system
— id: 56505, year: 1993, vol: 75, page: 293, stat: Journal Article,

The expression of receptor type tyrosine phosphatase-beta (RPTP-beta): Evidence for a role in morphogenesis and development of the nervous system
Canoll, P. D.; Barnea, G.; Levy, J. B.; Sap, J.; Silvennoinen, O.; Ehrlich, M.; Schlessinger, J.; Musacchio, J. M.
1993 ;19(1-3):1301-1301, Abstracts (Society for Neuroscience)
— id: 92600, year: 1993, vol: 19, page: 1301, stat: Journal Article,

Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase
Desai DM; Sap J; Schlessinger J; Weiss A
1993 May 7;73(3):541-554, Cell
CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein in which the extracellular and transmembrane domains of CD45 are replaced with those of the EGF receptor (EGFR) is able to restore TCR signaling in a CD45-deficient cell. Thus, the cytoplasmic domain of CD45 is necessary and sufficient for TCR signal transduction. Moreover, EGFR ligands functionally inactivate the EGFR-CD45 chimera in a manner that is dependent on dimerization of the chimeric protein. Inactivation of EGFR-CD45 chimera function results in the loss of TCR signaling, indicating that CD45 function is continuously required for TCR-mediated proximal signaling events. These results suggest that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases
— id: 17823, year: 1993, vol: 73, page: 541, stat: Journal Article,

Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region
Jiang YP; Wang H; D'Eustachio P; Musacchio JM; Schlessinger J; Sap J
1993 May;13(5):2942-2951, Molecular & cellular biology
We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene maps to chromosome 10, at approximately 21 centimorgans from the centromere
— id: 13177, year: 1993, vol: 13, page: 2942, stat: Journal Article,

The gene for receptor-linked protein-tyrosine-phosphatase (PTPA) is assigned to human chromosome 20p12-pter by in situ hybridization (ISH and FISH)
Rao VV; Loffler C; Sap J; Schlessinger J; Hansmann I
1992 Jul;13(3):906-907, Genomics
— id: 17824, year: 1992, vol: 13, page: 906, stat: Journal Article,

v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII
Disela C; Glineur C; Bugge T; Sap J; Stengl G; Dodgson J; Stunnenberg H; Beug H; Zenke M
1991 Nov;5(11):2033-2047, Genes & development
The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein
— id: 17825, year: 1991, vol: 5, page: 2033, stat: Journal Article,

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector
Fuerstenberg S; Beug H; Introna M; Khazaie K; Munoz A; Ness S; Nordstrom K; Sap J; Stanley I; Zenke M; et al.
1990 Dec;64(12):5891-5902, Journal of virology
A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells
— id: 17826, year: 1990, vol: 64, page: 5891, stat: Journal Article,

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells
Munoz A; Hoppner W; Sap J; Brady G; Nordstrom K; Seitz HJ; Vennstrom B
1990 Feb;4(2):312-320, Molecular endocrinology
To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon nuclear expression of the cTR-alpha protein the cells become responsive to thyroid hormone, as detected by expression of a number of genes (malic enzyme, phosphoenolpyruvate carboxykinase, and Na+/K(+)-ATPase) reported to be indirectly induced by the hormone in vivo. In addition, our data show that the c-erbA product directly activates the Moloney murine leukemia virus promoter in a ligand-dependent manner. The data show that the chicken c-erbA-alpha protein can modulate the expression of rat genes under either direct or indirect control by thyroid hormone
— id: 17829, year: 1990, vol: 4, page: 312, stat: Journal Article,

Cloning and expression of a widely expressed receptor tyrosine phosphatase
Sap J; D'Eustachio P; Givol D; Schlessinger J
1990 Aug;87(16):6112-6116, Proceedings of the National Academy of Sciences of the United States of America
We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid extracellular domain (including signal peptide) of R-PTP-alpha is marked by a high serine/threonine content (32%) as well as eight potential N-glycosylation sites but displays no similarity to known proteins. Genetic mapping assigns the gene for R-PTP-alpha to mouse chromosome 2, closely linked to the Il-1a and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA
— id: 17244, year: 1990, vol: 87, page: 6112, stat: Journal Article,

A major thyroid hormone response element in the third intron of the rat growth hormone gene
Sap J; de Magistris L; Stunnenberg H; Vennstrom B
1990 Mar;9(3):887-896, EMBO journal
The rat growth hormone (RGH) gene constitutes a well-documented model system for the direct regulation of transcription by thyroid hormones. In order to analyse its interaction with sequences in the RGH gene, we have overproduced the thyroid hormone receptor-alpha (c-erbA) protein using a vaccinia virus expression system. The expressed protein bound T3 and DNA-cellulose with expected affinities, and the major binding site for the receptor protein was found to be located in the third intron of the RGH gene. This site displayed significantly higher affinity for the receptor protein than a previously described thyroid hormone response element (TRE) in the promoter of this gene, and also conferred stronger hormone responsiveness in vivo to a heterologous promoter. The data suggest that this novel TRE plays a major role in the regulation of rat growth hormone gene expression by thyroid hormones
— id: 17828, year: 1990, vol: 9, page: 887, stat: Journal Article,

v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA
Zenke M; Munoz A; Sap J; Vennstrom B; Beug H
1990 Jun 15;61(6):1035-1049, Cell
The v-erbA oncogene, one of the two oncogenes of the avian erythroblastosis virus, efficiently blocks erythroid differentiation and suppresses erythrocyte-specific gene transcription. Here we show that the overexpressed thyroid hormone receptor c-erbA effectively modulates erythroid differentiation and erythrocyte-specific gene expression in a T3-dependent fashion, when introduced into erythroid cells via a retrovirus. In contrast, the endogenous thyroid hormone receptor does not detectably affect erythroid differentiation. The analysis of a series of chimeric v-/c-erbA proteins suggests that the v-erbA oncoprotein has lost one type of thyroid hormone receptor function (regulating erythrocyte gene transcription in response to T3), but constitutively displays another function: it represses transcription in the absence of T3. The region responsible for the loss of hormone-dependent regulator activity of v-erbA has been mapped to the very C-terminus of c-erbA, encompassing a cluster of highly conserved amino acid residues with the potential to form an amphipathic alpha-helix
— id: 17827, year: 1990, vol: 61, page: 1035, stat: Journal Article,

Repression of transcription mediated at a thyroid hormone response element by the v-erb-A oncogene product
Sap J; Munoz A; Schmitt J; Stunnenberg H; Vennstrom B
1989 Jul 20;340(6230):242-244, Nature
Several recent observations, such as the identification of the cellular homologue of the v-erb-A oncogene as a thyroid-hormone receptor, have strongly implicated nuclear oncogenes in transcriptional control mechanisms. The v-erb-A oncogene blocks the differentiation of erythroid cells, and changes the growth requirements of fibroblasts and erythroblasts. Mutations in v-erb-A protein have led to the loss of its affinity for thyroid hormones but do not affect its DNA-binding ability, a property required for biological activity. We report here the identification of a novel thyroid-hormone response element (TRE) in the long terminal repeat of Moloney murine leukaemia virus that binds the c-erb-A-alpha protein. The v-erb-A protein abolishes the responsiveness of this TRE to thyroid hormone, although it has a lower affinity than the normal receptor for the TRE. The data indicate that overexpressed v-erb-A protein negatively interferes with normal transcriptional-control mechanisms, and that amino-acid substitutions have altered its DNA-binding properties
— id: 17830, year: 1989, vol: 340, page: 242, stat: Journal Article,

Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein
Goldberg Y; Glineur C; Gesquiere JC; Ricouart A; Sap J; Vennstrom B; Ghysdael J
1988 Aug;7(8):2425-2433, EMBO journal
The c-erbA proto-oncogene encodes a nuclear receptor for thyroid hormone (T3), which is believed to stimulate transcription from specific target promoters upon binding to cis-acting DNA sequence elements. The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent version of this nuclear receptor. The v-erbA product inhibits terminal differentiation of avian erythroblasts, presumably by affecting the transcription of specific genes. We show here that the c-erbA-encoded nuclear receptor (p46c-erbA) is phosphorylated on serine residues on two distinct sites. One of these sites, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v-erbA-encoded P75gag-v-erbA protein. This site is located in the amino-terminal domain of these molecules, 21 amino acids upstream of the DNA-binding region. Phosphorylation of this site in both p46c-erbA and P75gag-v-erbA is enhanced 10-fold following treatment of cells with activators of either protein kinase C or cAMP-dependent protein kinase. Since cAMP-dependent protein kinase phosphorylates both p46c-erbA and P75gag-v-erbA in vitro at the same site as that observed in vivo, at least part of the cAMP-dependent phosphorylation of erbA molecules in cells could result from direct phosphorylation by this enzyme. The possible role phosphorylation may play in the function of the erbA-encoded transcriptional factors is discussed
— id: 17831, year: 1988, vol: 7, page: 2425, stat: Journal Article,

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha
Lax I; Johnson A; Howk R; Sap J; Bellot F; Winkler M; Ullrich A; Vennstrom B; Schlessinger J; Givol D
1988 May;8(5):1970-1978, Molecular & cellular biology
The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors
— id: 17832, year: 1988, vol: 8, page: 1970, stat: Journal Article,

Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein
Munoz A; Zenke M; Gehring U; Sap J; Beug H; Vennstrom B
1988 Jan;7(1):155-159, EMBO journal
To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone-independent activity of P75gag-v-erbA provided a selective advantage to the avian erythroblastosis virus during the original selection for a highly oncogenic strain of the virus
— id: 17833, year: 1988, vol: 7, page: 155, stat: Journal Article,

Biological effects of the v-erbA oncogene in transformation of avian erythroid cells
Vennstrom B; Beug R; Damm K; Engel D; Gehring U; Graf T; Munoz A; Sap J; Zenke M
1987 ;17(1):14-19, Hormone & metabolic research. Supplement series
— id: 17834, year: 1987, vol: 17, page: 14, stat: Journal Article,

The c-erb-A protein is a high-affinity receptor for thyroid hormone
Sap J; Munoz A; Damm K; Goldberg Y; Ghysdael J; Leutz A; Beug H; Vennstrom B
1986 Dec 18-31;324(6098):635-640, Nature
Hormone binding and localization of the c-erb-A protein suggest that it is a receptor for thyroid hormone, a nuclear protein that binds to DNA and activates transcription. In contrast, the product of the viral oncogene v-erb-A is defective in binding the hormone but is still located in the nucleus
— id: 17835, year: 1986, vol: 324, page: 635, stat: Journal Article,