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T Pawson

Publications and source records attributed to T Pawson.

At least 145 records · Page 8Linked to original sources
PubMed1994

The Fps/Fes protein-tyrosine kinase promotes angiogenesis in transgenic mice.

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase known to be highly expressed in hematopoietic cells. To investigate fps/fes biological function, an activating mutation was introduced into the human fps/fes gene which directs amino-terminal myristylation of the Fps/Fes protein. This mutant, myristylated protein induced transformation of Rat-2 fibroblasts. The mutant fps/fes allele was incorporated into the mouse germ line and was found to be appropriately expressed in transgenic mice, in a tissue-specific pattern indistinguishable from that of the endogenous mouse gene. These mice displayed widespread hypervascularity, progressing to multifocal hemangiomas. High levels of both the transgenic human and endogenous murine fps/fes transcripts were detected in vascular tumors by using RNase protection, and fps/fes transcripts were localized to endothelial cells of both the vascular tumors and normal blood vessels by in situ RNA hybridization. Primary human umbilical vein endothelial cultures were also shown to express fps/fes transcripts and the Fps/Fes tyrosine kinase. These results indicate that fps/fes expression is intrinsic to cells of the vascular endothelial lineage and suggest a direct role of the Fps/Fes protein-tyrosine kinase in the regulation of angiogenesis.

Amino Acid Sequence↗
PubMed1994

Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.

Amino Acid Sequence↗
PubMed1994

Structure and function of SH2 domains.

In order for cells to respond to their environment, a series of regulated molecular events has to take place. External signalling molecules bind to cellular receptors and thereby trigger the activation of multiple intracellular pathways, which modify cellular phenotypes. The cell-surface receptors for a wide range of polypeptide hormones possess protein tyrosine kinase activity, which is induced by binding of the appropriate extracellular ligand. Tyrosine phosphorylation can act as a molecular switch, by initiating the recruitment of cytoplasmic effector molecules containing Src homology (SH) 2 domains, to activated receptors. These SH2-containing proteins, in turn, regulate intracellular signalling pathways. Here, we discuss the role of tyrosine phosphorylation in triggering signalling pathways, as well as the functions of SH2 domains, which mediate these events through phosphotyrosine-dependent protein-protein interactions.

Amino Acid Sequence↗
PubMed1994

Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins.

The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which is implicated in the regulation of Ras, suggesting that Shc is involved in the intracellular transmission of growth signals from activated tyrosine kinases to Ras. Overexpression of Shc proteins in cultured fibroblasts induces a transformed phenotype. We now report that, in vitro, the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole Shc high affinity binding site for Grb2. Mutant Shc proteins with substitution of the Tyr317 by Phe lose the capacity to be highly phosphorylated on tyrosine upon growth factor receptor activation, to bind Grb2 and to induce neoplastic transformation. In contrast, Shc proteins that have an extensive aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc and support the notion that Shc proteins can deliver activation signals to RAS.

3T3 Cells↗
PubMed1994

Immunolocalization of the Nuk receptor tyrosine kinase suggests roles in segmental patterning of the brain and axonogenesis.

Neural kinase (Nuk) encodes a murine receptor-like tyrosine kinase belonging to the Eph/Elk/Eck family. Protein localization studies indicate that during early embryogenesis Nuk is confined to the developing nervous system, where it marks segments along the axis of the neural tube in the hindbrain (rhombomeres r2, r3 and r5) and specific morphological bulges of the midbrain and forebrain. Subcellular localization of Nuk indicates that this receptor is concentrated at sites of cell-cell contact, often involving migrating neuronal cells or their extensions. Most notably, high levels of Nuk protein are found within initial axon outgrowths and associated nerve fibers. The axonal localization of Nuk is transient and is not detected after migrations have ceased, suggesting a role for this tyrosine kinase during the early pathfinding and/or fasciculation stages of axonogenesis. The subcellular localization of Nuk, as well as the presence of fibronectin type III and immunoglobulin-like adhesive domains on the extracellular region, suggest this receptor tyrosine kinase may function to regulate specific cell-cell interactions during early development of the murine nervous system.

Amino Acid Sequence↗
PubMed1994

Receptor-binding, tyrosine phosphorylation and chromosome localization of the mouse SH2-containing phosphotyrosine phosphatase Syp.

The murine phosphotyrosine phosphatase, Syp, is a widely-expressed cytoplasmic enzyme that contains two SH2 domains. Syp is physically associated with activated receptors for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), apparently through its SH2 domains. This phosphatase is rapidly phosphorylated in cells treated with PDGF or EGF, and is constitutively phosphorylated in v-src transformed cells. Here we report that either the N-terminal or C-terminal Syp SH2 domain alone bound to the activated beta PDGF receptor or EGF-receptor in vitro, and that the two SH2 domains linked together exhibited synergistic binding. Substitution of the Tyr1009 autophosphorylation site in the C-terminal tail of activated beta PDGFR with Phe abolished the in vitro binding of either SH2 domain to the activated receptor. A 9 amino acid phosphopeptide corresponding to the Tyr1009 autophosphorylation site of the beta PDGFR inhibited association of the Syp SH2 domains with the receptor. These results indicate that the Syp SH2 domains have an intrinsic specificity for the Tyr1009 autophosphorylation site of the beta PDGFR that dictates binding of the intact Syp phosphatase, and suggest that both SH2 domains have a related binding specificity. Phosphoamino acid analysis of Syp from PDGF-stimulated cells indicated that PDGF primarily induces Syp phosphorylation on tyrosine residues. The mouse Syp gene has been mapped to chromosome 5F region by the fluorescence in situ hybridization. These findings suggest specific functions for Syp in signal transduction downstream of receptor tyrosine kinases.

Animals↗
PubMed1994

Substrate specificities and identification of a putative binding site for PI3K in the carboxy tail of the murine Flt3 receptor tyrosine kinase.

Flt3 is a receptor tyrosine kinase (RTK) structurally related to the CSF-1R encoded by the c-fms locus, Kit and the PDGFR which is restricted in its expression to hematopoietic precursor populations and several distinct cell types within the central nervous system. Although the ligand for Flt3 has recently been identified, the developmental function of Flt3 within these tissues has not yet been described. In order to examine the signalling properties of this receptor, we previously constructed a chimeric molecule containing the extracellular domain of CSF-1R fused to the transmembrane and cytoplasmic domain of mouse Flt3 (FF3). The ability of the FF3 to directly associate with or tyrosine phosphorylate specific cytoplasmic signalling molecules in vivo was examined. GAP, Vav, Shc, and to a lesser extent PLC gamma become tyrosine-phosphorylated but no in vivo association with the receptor was detectable. FF3 associates with PI3K activity and the SH2 domains of p85 and Grb-2. Phosphopeptide competition experiments suggest that the PI3K binding site is located outside of the kinase insert in the carboxy tail of the receptor.

Amino Acid Sequence↗
PubMed1994

Phosphorylation of the SHC proteins on tyrosine correlates with the transformation of fibroblasts and erythroblasts by the v-sea tyrosine kinase.

The S13 avian erythroblastosis viral genome encodes an oncogenic tyrosine kinase, termed env-sea, that is capable of transforming fibroblasts and erythroblasts. Although the tyrosine kinase activity of the env-sea protein has been shown to be necessary for transformation, no substrates for this enzyme have been detected in vivo. Here we demonstrate that the recently described shc proteins are phosphorylated on tyrosine residues in both S13 transformed fibroblasts and erythroblasts. Furthermore, using an S13 temperature sensitive mutant, we show that the phosphorylation of the shc proteins occurs concomitantly with the activation of the tyrosine kinase activity of the env-sea protein. These observations make the phosphorylation of the shc proteins a good candidate for being involved in oncogenic signaling by the env-sea oncoprotein.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Tyrosine kinase signalling pathways.

Protein-tyrosine kinases act as receptors for a wide range of external signals that control the growth and differentiation of normal cells. Additionally, many retroviral and cellular oncogenes encode tyrosine kinase variants that are constitutively active. Recent evidence suggests that the intracellular targets of tyrosine kinases contain a protein module of approximately 100 amino acids, the Src homology 2 (SH2) domain. SH2 domains directly recognize tyrosine phosphorylation sites, and are thereby recruited to activated, autophosphorylated growth factor receptors. These interactions, in turn, stimulate the biochemical signalling pathways that control gene expression, cytoskeletal architecture, and cell metabolism. SH2-containing proteins frequently contain a distinct element of approximately 50 residues, the SH3 domain, that recognizes proline-rich motifs. Proteins with SH2 and SH3 domains can act as adaptors to couple tyrosine kinases to downstream targets with SH3-binding sites. A specific example of the synergistic action of SH2 and SH3 domains involves regulation of the Ras pathway by the adaptor protein Sem-5/drk/Grb2, which links tyrosine kinases to the Ras guanine nucleotide releasing protein Sos, which converts Ras to the active GTP-bound state.

Amino Acid Sequence↗
PubMed1993

Expression and catalytic activity of the tyrosine phosphatase PTP1C is severely impaired in motheaten and viable motheaten mice.

Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.

Animals↗
PubMed1993

The 64-kDa protein that associates with the platelet-derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp.

Ligand-stimulated autophosphorylation of the platelet-derived growth factor receptor (PDGFR) beta subunit creates a number of binding sites for SH2-containing proteins. One of the PDGFR-associated proteins is a 64-kDa protein of unknown identity and function. We present data indicating that the 64-kDa protein that associates with the activated PDGFR is Syp (also called SH-PTP2, PTP-1D, or SH-PTP3), the ubiquitously expressed 64-kDa SH2-containing protein-tyrosine phosphatase. Phosphorylation of Tyr-1009 in the C terminus of the PDGFR is required for the stable association of Syp, suggesting that phosphorylation of this residue creates a binding site for the Syp SH2 domains. Although Syp stably associates with the PDGFR, this event is not required for PDGF-stimulated tyrosine phosphorylation of Syp. These data raise the interesting possibility that protein-tyrosine phosphatases contribute to the intracellular relay of biological signals originating from receptor tyrosine kinases such as the PDGFR.

Humans↗
PubMed1993

Proteins with SH2 and SH3 domains couple receptor tyrosine kinases to intracellular signalling pathways.

The targets of receptor protein-tyrosine kinases are characterized by Src homology 2 (SH2) domains, that mediate specific interactions with receptor autophosphorylation sites. SH2-mediated interactions are important for the activation of biochemical signalling pathways in cells stimulated with growth factors. A distinct protein module, the SH3 domain, is frequently found in polypeptides that contain SH2 domains, and is also implicated in controlling protein-protein interactions in signal transduction. Evidence suggesting that SH2 and SH3 domains act synergistically in stimulation of the Ras pathway is discussed.

Amino Acid Sequence↗
PubMed1993

Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor.

The erythropoietin receptor (EpR) belongs to a family of hematopoietin receptors whose members lack tyrosine kinase activity. Nonetheless, within minutes of binding Ep, a number of cellular proteins become transiently phosphorylated on tyrosine residues. One of these proteins, as we and others have shown previously, is the EpR itself. To identify the remaining protein substrates, we have examined the antiphosphotyrosine immunoprecipitates of lysates from Ba/F3 cells expressing high levels of cell surface EpRs. We now present data showing that, in response to Ep, the 85-Kd regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) becomes immunoprecipitable with antiphosphotyrosine antibodies. This appears to be due, in large part, to the specific association of PI 3-kinase with the tyrosine-phosphorylated EpR, either directly or through a 93- or 70-Kd tyrosine-phosphorylated intermediate. The activity of this EpR associated PI 3-kinase, assessed in anti-EpR immunoprecipitates, is maximal within 2 minutes of incubation with Ep and returns almost to baseline levels by 10 minutes. In vitro studies suggest that the interaction between PI 3-kinase and the activated EpR is mediated by the N- and C-terminal SH2 domains of p85 and tyrosine-phosphorylated motifs on the EpR.

Amino Acid Sequence↗
PubMed1993

The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp.

The insulin receptor substrate 1 (IRS1) is a protein that is rapidly phosphorylated on tyrosine by the activated insulin receptor. Syp is a recently discovered, broadly expressed phosphotyrosine (Tyr(P)) phosphatase that contains two Src homology 2 (SH2) domains. We have found that insulin treatment of 3T3-L1 adipocytes leads to complex formation between IRS1 and Syp. Syp was detected in immunoadsorbates of IRS1 from extracts of insulin-treated but not basal cells by both immunoblotting and Tyr(P) phosphatase activity. The association of Syp with IRS1 apparently occurs between the SH2 domains of Syp and Tyr(P)-containing sequences of IRS1, since a fusion protein containing only the SH2 domains of Syp bound the Tyr(P) form of IRS1. Unlike the receptors for epidermal and platelet-derived growth factors, which in their activated state bind to the SH2 domains of Syp and elicit phosphorylation of Syp on tyrosine in intact cells, the Tyr(P) form of the insulin receptor did not bind to the SH2 domains of Syp, and no phosphorylation of Syp on tyrosine was detected in insulin-treated 3T3-L1 adipocytes. In combination with other findings these results indicate that IRS1 functions as a docking protein for SH2 domain-containing proteins participating in signaling from the insulin receptor.

3T3 Cells↗
PubMed1993

Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.

Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.

Amino Acid Sequence↗
PubMed1993

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

3T3 Cells↗
PubMed1993

Epidermal growth factor stimulates the tyrosine phosphorylation of SHC in the mouse.

Previous studies have demonstrated that the administration of epidermal growth factor (EGF) to neonatal mice results in the tyrosine phosphorylation of multiple substrates in all organs examined (Donaldson, R. W., and Cohen, S. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8477-8481). One of these substrates, a 55-kDa protein, was shown to associate with the receptor for EGF (EGFR). We now report the identification of this receptor-associated protein as SHC. Immunoprecipitation and Western blotting analyses have revealed that SHC associates only with the activated EGFR. In the absence of EGF stimulation, SHC exists in the liver as a cytoplasmic monomer. Intraperitoneal injection of EGF results, within minutes, in the translocation of 50-80% of SHC to the liver plasma membrane. The membrane-associated SHC was found to be tyrosine-phosphorylated; the subsequent release of SHC from the membrane correlated with a tyrosine dephosphorylation. We conclude that SHC is a physiological substrate that appears to participate in the in vivo signaling response to EGF.

Animals↗