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J Schlessinger

Publications and source records attributed to J Schlessinger.

At least 181 records · Page 10Linked to original sources
PubMed1993

Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism.

Amides↗
PubMed1993

Tyrosine 785 is a major determinant of Trk--substrate interaction.

Interaction of the nerve growth factor (NGF) receptor/Trk with cellular substrates was investigated by transient co-overexpression in human 293 fibroblasts using ET-R, a chimeric receptor consisting of the epidermal growth factor receptor (EGF-R) extracellular ligand binding domain and the Trk transmembrane and intracellular signal-generating sequences. The chimera was fully functional, and associated with and phosphorylated phospholipase C gamma (PLC gamma), ras GTPase-activating protein (GAP) and the non-catalytic subunit of phosphatidylinositol-3'-kinase, p85, in a ligand-dependent manner. Deletion of 15 C-terminal amino acids, including tyrosine 785 (Y-785) abrogated receptor and substrate phosphorylation activities. Mutation of Y-785 to phenylalanine somewhat impaired receptor phosphorylation activity, which was reflected in reduced GAP and p85 phosphorylation. In contrast, ET-YF phosphorylation of PLC gamma was significantly reduced, while the high affinity association potential with this substrate was abrogated by this point mutation in vitro and in intact cells. Furthermore, a tyrosine-phosphorylated synthetic C-terminal peptide competitively inhibited Trk cytoplasmic domain association with PLC gamma. Thus, the short C-terminal tail appears to be a crucial structural element of the Trk cytoplasmic domain, and phosphorylated Y-785 is a major and selective interaction site for PLC gamma.

Amino Acid Sequence↗
PubMed1993

Phosphatidylinositol 3'-kinase associates with p145c-kit as part of a cell type characteristic multimeric signalling complex.

p145c-kit is expressed in cell lineages of diverse origin and appears to regulate distinct cell type characteristic functions. Independent mutations at the murine Dominant White Spotting (W) locus result in the alteration of p145c-kit tyrosine kinase activity and signalling potential, which differentially affects melanocyte migration, germ cell regeneration and hematopoietic cell differentiation. Molecules that may be involved in mediation and definition of p145c-kit signalling pathways were investigated in cell lines of hematopoietic, melanogenic and central nervous system origin. High-affinity association of endogenous cellular proteins with activated p145c-kit was limited to a characteristic set of molecules that correlated with the presence of phosphatidylinositol (PtdIns) 3'-kinase activity. The observed association pattern of proteins was cell type characteristic, and all of the proteins were displaced from the receptor by competition with excess receptor binding subunit of PtdIns 3'-kinase. Our data indicate that PtdIns 3'-kinase associates with p145c-kit as part of a multimeric signalling complex, and suggest that the cell type characteristic composition of this complex influences the signalling potential of p145c-kit in the diverse cell types in which it is expressed and thereby defines its cell type-specific functions.

Amino Acid Sequence↗
PubMed1993

Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor.

We have previously shown that an active epidermal growth factor receptor (EGF-R) kinase is necessary for efficient sorting of the EGF-R to the lysosome, and we have shown that this occurs in the multivesicular body (MVB), where EGF-R are sorted away from recycling receptors by being removed to the internal vesicles of the MVB. The aim of the present study was to identify substrates of the EGF-R kinase associated with MVBs which might play a role in this sorting process. We used a density shift technique to isolate MVBs and show that the major substrates phosphorylated in vitro within MVBs which contain an active EGF-R kinase are the EGF-R itself and annexin I. Annexin I is associated with both plasma membrane and MVBs in a calcium-independent manner but can be phosphorylated in vitro only in MVBs. Phosphorylation of calcium-independent annexin I in isolated MVBs converts it to a form that requires calcium for membrane association. In cells with an active EGF-R kinase the amount of calcium-independent annexin I in MVBs is reduced, suggesting that a phosphorylation-induced conversion of the calcium independent to the calcium-dependent form also occurs in vivo. Our observations, together with the known properties of annexin I in mediating membrane fusion, suggest that inward vesiculation in MVBs is induced by the EGF-R and is mediated by phosphorylated annexin I.

3T3 Cells↗
PubMed1993

The human GRB2 and Drosophila Drk genes can functionally replace the Caenorhabditis elegans cell signaling gene sem-5.

Mutations in the Caenorhabditis elegans gene sem-5 affect cell signaling processes involved in guiding a class of cell migrations and inducing vulval cell fates. The sem-5 sequence encodes a protein comprised almost exclusively of SH2 and SH3 domains (SH, src homology region) that are found together in many signaling proteins and nonreceptor tyrosine kinases. A human protein, GRB2, was identified by its ability to associate with the activated human epidermal growth factor receptor (hEGFR). The GRB2 and Sem-5 proteins share an identical architecture of their SH2 and SH3 domains and 58% amino acid sequence identity. Here we demonstrate that GRB2 and a Drosophila sem-5-like gene Drk can specifically rescue sem-5 mutants. We also show that Sem-5, like GRB2, can bind to the activated hEGFR in vitro. We further correlate the abilities of several mutant variants of GRB2 and Sem-5 to bind to the hEGFR in vitro with their abilities to functionally replace sem-5 in vivo. These data indicate that GRB2 and Drk are functional homologues of Sem-5 and demonstrate the high degree of conservation of both structure and function between signaling systems throughout evolution.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

Amino Acid Sequence↗
PubMed1993

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

Amino Acid Sequence↗
PubMed1993

SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange.

src homology 2 (SH2) domains of intracellular signaling molecules such as phospholipase C-gamma and phosphatidylinositol 3'-kinase-associated protein p85 represent recognition motifs for specific phosphotyrosine-containing regions on activated growth factor receptors. The binding of SH2 domains to activated growth factor receptors controls the interaction with signaling molecules and the regulation of their activities. In this report, we describe the kinetic parameters and binding affinities of SH2 domains of p85 toward short phosphotyrosine-containing peptides with the amino acid sequence motif YMXM, derived from a major insulin receptor substrate, IRS-1, by using real time biospecific interaction analysis (BIAcore). Associations were specific and of very high affinity, with dissociation constants of 0.3 to 3 nM, between phosphopeptides and the two separate SH2 domains contained within p85. Nonphosphorylated peptides showed no measurable binding, and the interactions were specific for the primary sequence very close to the phosphotyrosine residue. Moreover, the interactions between phosphopeptides and SH2 domains of other signaling molecules were of much lower affinity. Interestingly, the binding of the SH2 domains to the tyrosine-phosphorylated peptides was of high affinity as a result of a very high on rate, of 3 x 10(7) to 40 x 10(7)/M/s; at the same time, the rate of dissociation, of 0.11 to 0.19/s, was rapid, allowing for rapid exchange of associating proteins with the tyrosine phosphorylation sites.

Amino Acid Sequence↗
PubMed1993

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.

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.

Aging↗
PubMed1993

Retroviral gene transfer of epidermal growth factor receptor into HL60 cells results in a partial block of retinoic acid-induced granulocytic differentiation.

HL60 cells are devoid of endogenous epidermal growth factor receptor (EGFR). They respond to retinoic acid and undergo terminal granulocytic differentiation. EGFR complementary DNA was introduced into HL60 cells by retroviral gene transfer. Scatchard plot showed that the binding characteristics are identical to those of A431 cells. HL60-EGFR cells were estimated to express 34,000 EGFR/cell (Kd = 5 nM). The tyrosine phosphorylation upon ligand binding is the first step of signal transduction. The dominant phosphotyrosyl proteins in epidermal growth factor-stimulated HL60-EGFR cells include a 170 kDa protein (EGFR itself), and 125 and 53 kDa proteins. The EGFR signal results in the induction of 92 kDa gelatinase/matrix metalloproteinase in HL60-EGFR cells, thereby providing evidence of the function of the exogenous EGFR and a semiquantitative measure of the EGFR signal. These HL60-EGFR cells offer a unique opportunity to examine the potentially important role of EGFR (c-erbB) in maintaining homeostasis between self-renewal and differentiation. c-erbB has been shown to play a physiological role in the self-renewal of the very early avian stem cells which do express EGFR. The v-erbB (double truncated EGFR) has been shown to cause avian erythroblastosis. We found that these HL60-EGFR cells responded to retinoic acid differently from the HL60-control cells. A partial block of only 45% granulocytic differentiation and concomitant proliferation was noted, consistent with a shift of balance between self-renewal and differentiation toward the former.

Cell Differentiation↗
PubMed1992

Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases.

The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1992

IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

3T3 Cells↗
PubMed1992

High-efficiency expression/cloning of epidermal growth factor-receptor-binding proteins with Src homology 2 domains.

Src homology 2 domains bind to tyrosine-phosphorylated growth factor receptors and are found in proteins that serve as substrates for tyrosine kinases, such as phospholipase C-gamma 1 and ras GTPase-activating protein. We have previously described the cloning of phosphatidylinositol 3'-kinase-associated p85 from expression libraries with the tyrosine-phosphorylated epidermal growth factor receptor as a probe. We have now modified this technique by using T7 polymerase-based expression libraries, which significantly improves sensitivity of the method. In one screening of such a library, we identified five different murine Src homology 2 domain-containing proteins, which we call GRBs (growth factor receptor-bound proteins). Two of these proteins represented the tyrosine kinase fyn and the mouse homologue of phospholipase C-gamma 1, whereas two genes encoded proteins similar to v-crk and NCK. We also isolated the gene for GRB-7, which encodes a protein of 535 amino acids. In addition to a Src homology 2 domain, GRB-7 also has a region of similarity to the noncatalytic domain of ras GTPase-activating protein and is highly expressed in liver and kidney. Use of this expression/cloning system should increase our ability to identify downstream modulators of growth factor action.

Amino Acid Sequence↗
PubMed1992

Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis.

Stimulation of growth factor receptors with tyrosine kinase activity is followed by rapid receptor dimerization, tyrosine autophosphorylation and phosphorylation of signalling molecules such as phospholipase C gamma (PLC gamma) and the ras GTPase-activating protein. PLC gamma and GTPase-activating protein bind to specific tyrosine-phosphorylated regions in growth factor receptors through their src-homologous SH2 domains. Growth factor-induced tyrosine phosphorylation of PLC gamma is essential for stimulation of phosphatidylinositol hydrolysis in vitro and in vivo. We have shown that a short phosphorylated peptide containing tyrosine at position 766 from a conserved region of the fibroblast growth factor (FGF) receptor is a binding site for the SH2 domain of PLC gamma (ref. 8). Here we show that an FGF receptor point mutant in which Tyr 766 is replaced by a phenylalanine residue (Y766F) is unable to associate with and tyrosine-phosphorylate PLC gamma or to stimulate hydrolysis of phosphatidylinositol. Nevertheless, the Y766F FGF receptor mutant can be autophosphorylated, and can phosphorylate several cellular proteins and stimulate DNA synthesis. Our data show that phosphorylation of the conserved Tyr 766 of the FGF receptor is essential for phosphorylation of PLC gamma and for hydrolysis of phosphatidylinositol, but that elimination of this hydrolysis does not affect FGF-induced mitogenesis.

Animals↗
PubMed1992

The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling.

A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1992

Differential effects of W mutations on p145c-kit tyrosine kinase activity and substrate interaction.

The c-kit gene, mapped to the dominant white spotting (W) locus of the mouse (Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89; Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192), encodes a receptor tyrosine kinase, p145c-kit. Germline mutations at the W locus lead to loss of function alterations in p145c-kit, and result in mice with developmental defects of varying severity in the melanocytic, hematopoietic stem cell, and primordial germ cell lineages. To investigate in more detail the effect of W mutations on p145c-kit signaling, three mutations, W42, Wv, and W41, that confer severe, intermediate, and mild phenotypic characteristics, respectively, were introduced into the human p145c-kit tyrosine kinase domain. These mutations attenuated the intrinsic tyrosine kinase activity of the receptor to different degrees. In addition, they had differential effects on the interaction of the p145c-kit substrates, phospholipase C gamma, GTPase-activating protein, and the receptor-binding subunit of phosphatidylinositol 3'-kinase, p85. Notably, the Wv mutation, while retaining significant receptor tyrosine kinase activity, was unable to bind phospholipase C gamma and GTPase-activating protein, but could still associate with p85. These results suggest that the location of W mutations may be an important determinant of the specificity of substrate association and phosphorylation, and may explain, at least in part, the cell type-specific defects associated with certain W alleles.

Amino Acid Sequence↗