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Biomedical subjects

T Pawson

Publications and source records attributed to T Pawson.

At least 109 records · Page 6Linked to original sources
PubMed1996

Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.

3T3 Cells↗
PubMed1996

Ras-GTP levels are elevated in human NF1 peripheral nerve tumors.

Neurofibromin, the gene product of NF1, is a Ras GTPase Activating Protein. The absence of neurofibromin leads to increased levels of Ras-GTP, which contributes to the proliferation of NF1 neurogenic sarcoma cell lines. Whether this pathogenic mechanism is applicable to benign and malignant peripheral nerve tumours from NF1 and non NF1 patients is not known, due to lack of a tissue based assay. We have adapted a colorimetric enzymatic assay for determining levels of Ras bound guanine nucleotides in tissues. Ras-GTP levels were increased in NF1 neurogenic sarcomas (15 times) and benign NF1 neurofibromas (four times), compared to non NF1 schwannomas. Neurofibromin was not expressed in NF1 sarcomas, in support of its important negative Ras regulatory role in the pathogenesis of NF1 peripheral nerve tumors.

Base Sequence↗
PubMed1996

The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution.

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors.

Amino Acid Sequence↗
PubMed1995

Syk tyrosine kinase required for mouse viability and B-cell development.

The Syk cytoplasmic protein-tyrosine kinase has two amino-terminal SH2 domains and a carboxy-terminal catalytic domain. Syk, and its close relative ZAP-70, are apparently pivotal in coupling antigen- and Fc-receptors to downstream signalling events. Syk associates with activated Fc receptors, the T cell receptor complex and the B-cell antigen-receptor complex (BCR) in immature and mature B lymphocytes. On receptor activation, the tandem SH2 domains of Syk bind dual phosphotyrosine sites in the conserved ITAM motifs of receptor signalling chains, such as the immunoglobulin alpha and beta-chains of the BCR, leading to Syk activation. Here we have investigated Syk function in vivo by generating a mouse strain with a targeted mutation in the syk gene. Homozygous syk mutants suffered severe haemorrhaging as embryos and died perinatally, indicating that Syk has a critical role in maintaining vascular integrity or in wound healing during embryogenesis. Analysis of syk-/- lymphoid cells showed that the syk mutation impaired the differentiation of B-lineage cells, apparently by disrupting signalling from the pre-BCR complex and thereby preventing the clonal expansion, and further maturation, of pre-B cells.

Animals↗
PubMed1995

A single point mutation switches the specificity of group III Src homology (SH) 2 domains to that of group I SH2 domains.

Src homology 2 (SH2) domains recognize phosphotyrosine-containing sequences, and thereby mediate the association of specific signaling proteins in response to tyrosine phosphorylation (Pawson, T., and Schlessinger, J. (1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2 domains to tyrosine-phosphorylated sites is determined by sequences adjacent to the phosphotyrosine. Based on the phosphopeptide specificity and crystal structures, SH2 domains were classified into four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778). The beta D5 residues of SH2 domains were predicted to be critical in distinguishing these groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778; Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993) Nature 362, 87-91). We report here that replacing the aliphatic residues at the beta D5 positions of two Group III SH2 domains (phosphoinositide 3-kinase N-terminal SH2 domain and phospholipase C-gamma C-terminal SH2 domain) with Tyr (as found in Group I SH2 domains) results in a switch in phosphopeptide selectivity, consistent with the specificities of Group I SH2 domains. These results establish the importance of the beta D5 residue in determining specificities of SH2 domains.

Amino Acid Sequence↗
PubMed1995

Vascular system defects and neuronal apoptosis in mice lacking ras GTPase-activating protein.

The gene encoding p120-rasGAP, a negative regulator of Ras, has been disrupted in mice. This Gap mutation affects the ability of endothelial cells to organize into a highly vascularized network and results in extensive neuronal cell death. Mutati ons in the Gap and Nf1 genes have a synergistic effect, such that embryos homozygous for mutations in both genes show an exacerbated Gap phenotype. Thus rasGAP and neurofibromin act together to regulate Ras activity during embryonic development.

Animals↗
PubMed1995

MAP kinase phosphorylation of mSos1 promotes dissociation of mSos1-Shc and mSos1-EGF receptor complexes.

The mouse protein mSos1 has a central Ras guanine nucleotide exchange domain, and a long proline-rich C-terminal tail which contains several potential binding sites for the SH3 domains of the adaptor protein, Grb2. In fibroblasts, growth factor stimulation results in the recruitment of Grb2-mSos1 into complexes with activated receptors and cytoplasmic phosphoproteins such as Shc, which are apparently involved in Ras activation, and subsequently to an increase in mSos1 phosphorylation on serine and threonine. The catalytic and C-terminal domains of mSos1 contain several potential sites for phosphorylation by mitogen-activated protein kinases. In vitro, purified p42/p44 MAP-kinase selectively phosphorylated the C-terminal tail of mSos1. Comparative tryptic phosphopeptide mapping of mSos1 phosphorylated in vitro by MAP kinase and of mSos1 immunoprecipitated from EGF-stimulated cells, revealed several phosphopeptides in common. These common phosphorylation sites have been mapped to a region encompassing the first three proline (pro)-rich motifs in the tail of mSos1. Furthermore, a region of mSos1 containing the first two pro-rich motifs could associate with MBP kinase activity in vitro. Phosphorylation of mSos1 did not affect binding of Grb2 to mSos1, but appeared to decrease binding of the mSos1-Grb2 complex to Shc and the EGF-receptor. These findings suggest a potential inhibitory role for MAP-kinase in attenuating nucleotide exchange on Ras, by uncoupling mSos1 from membrane-bound receptor complexes that lead to Ras activation.

3T3 Cells↗
PubMed1995

Biochemical and genetic analysis of the Drk SH2/SH3 adaptor protein of Drosophila.

The Drk SH3-SH2-SH3 adaptor protein has been genetically identified in a screen for rate-limiting components acting downstream of the Sevenless (Sev) receptor tyrosine kinase in the developing eye of Drosophila. It provides a link between the activated Sev receptor and Sos, a guanine nucleotide release factor that activates Ras1. We have used a combined biochemical and genetic approach to study the interactions between Sev, Drk and Sos. We show that Tyr2546 in the cytoplasmic tail of Sev is required for Drk binding, probably because it provides a recognition site for the Drk SH2 domain. Interestingly, a mutation at this site does not completely block Sev function in vivo. This may suggest that Sev can signal in a Drk-independent, parallel pathway or that Drk can also bind to an intermediate docking protein. Analysis of the Drk-Sos interaction has identified a high affinity binding site for Drk SH3 domains in the Sos tail. We show that the N-terminal Drk SH3 domain is primarily responsible for binding to the tail of Sos in vitro, and for signalling to Ras in vivo.

Amino Acid Sequence↗
PubMed1995

Functional interaction between c-Src and its mitotic target, Sam 68.

The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src approximately Ras GTPase activating protein > p85 alpha (amino-terminal) > Grb2 >> p85 alpha (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 alpha SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68.

3T3 Cells↗
PubMed1995

Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins.

The molecular interactions of the Src homology 2 (SH2) domain and the N-terminal proline-rich sequence motifs (pro-1 to pro-5) of the SH2 protein Shb with other components were presently characterised. Using a degenerate phosphopeptide library the preferred binding site for the Shb SH2 domain was determined to pTyr-Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue. Experiments with competing peptides and platelet-derived growth factor (PDGF) beta-receptor mutants with Y to F substitutions in autophosphorylation sites revealed multiple binding sites for the Shb SH2 domain in the receptor. The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth factor receptor-1 (FGFR-1) mainly through position Y776. The receptor experiments suggest that other residues besides the +1 to +3 positions may also be of significance for Shb binding. The pro-4/pro-5 motif of Shb binds in vitro particularly well to the Src, p85 alpha PI3-kinase and Eps8 SH3 domains expressed as GST fusion proteins. However, the GST-SH3 domain fusion proteins tested bind in vitro to peptides corresponding to the pro-1 to pro-5 motifs of Shb with low affinity and selectivity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions. In vivo association between Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunoprecipitation. PDGF treatment did not affect the association between Eps8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins.

Amino Acid Sequence↗
PubMed1995

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

BACKGROUND: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. RESULTS: We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. CONCLUSION: These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

3T3 Cells↗
PubMed1995

Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.

Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.

Amino Acid Sequence↗
PubMed1995

Overexpression of Shc proteins potentiates the proliferative response to the granulocyte-macrophage colony-stimulating factor and recruitment of Grb2/SoS and Grb2/p140 complexes to the beta receptor subunit.

The high affinity receptor for GM-CSF consists of a unique alpha subunit and a beta subunit that is shared with receptors for IL-3 and IL-5. Activation of GM-CSF receptor (GMR) triggers two distinct cytoplasmic signalling pathways, JAK2 and Ras, and is sufficient to maintain proliferation of growth factor-dependent cell lines. Shc proteins are phosphorylated upon activation of GMR and may be involved in the transmission of GM-CSF signals to Ras. To define the role of Shc proteins in cells stimulated with GM-CSF, we investigated both the network of interactions that involve Shc after GM-CSF stimulation and the effects of overexpressing Shc proteins on the proliferative response to GM-CSF. Two cytoplasmic complexes, Grb2/Sos and Grb2/p140 bind through the Grb2 SH2 domain to phosphorylated Shc, and are thereby recruited to the beta subunit. Both complexes are stable, even in the absence of ligand, and depend on the direct association of p140 and Sos respectively with the SH3 domains of Grb2. p140 is an uncharacterized protein constitutively phosphorylated on tyrosine and, in its Grb2-bound form, expressed only in hematopoietic cells, the oligomeric complex formed by phosphorylated beta subunit-phosphorylated Shc-Grb2-SoS-p140 is also induced by IL-3 and L-5 stimulation of growth-factor dependent cell lines. Overexpression of wild-type Shc proteins in growth factor-dependent cells increases both MAP kinase activation and proliferation in response to GM-CSF. These effects require the association of Shc with Grb2. Taken together these results indicate that phosphorylation of Shc proteins is a crucial step in the transmission of GM-CSF proliferative stimuli, since it creates a high affinity binding site for the Grb2/SoS complex, whose function is to activate Ras and, for the Grb2/p140 complex, whose function remains unknown.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Construction of an SH2 domain-binding site with mixed specificity.

SH2 domains bind to specific phosphotyrosine-containing sites in a fashion dictated by the amino acids flanking the phosphotyrosine. Attention has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr1021 in the tail of the beta-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholipase C (PLC)-gamma 1. We show that the residues 4 and 5 amino acids COOH-terminal to Tyr1021 (+4 Leu and +5 Pro) are required for efficient PLC-gamma 1 binding, and that their replacement with the corresponding residues from a phosphatidylinositol 3'-kinase binding site abrogates stable association with PLC-gamma 1. In contrast, replacement of the +3 Pro with Met produces a Tyr1021 site with mixed specificity that binds both PLC-gamma 1 and phosphatidylinositol 3'-kinase. This motif is rendered specific for phosphatidylinositol 3'-kinase by further substitution of the +4 Leu. These results indicate that the +4 and +5 residues are important for the selective binding of specific SH2 domains. This study suggests that phosphotyrosine sites can be tailored to bind one or more SH2 domains with high affinity, depending on the combination of residues in the +1 to +5 positions.

Amino Acid Sequence↗
PubMed1995

Protein modules and signalling networks.

Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.

Amino Acid Sequence↗
PubMed1995

The PTB domain: a new protein module implicated in signal transduction.

Src homology 2 (SH2) domains have been identified in a large number of proteins involved in signal transduction downstream of receptor tyrosine kinases. They allow cytoplasmic signalling proteins to bind specifically to other polypeptides that are phosphorylated on tyrosine in response to growth factor stimulation. A novel phosphotyrosine-binding (PTB) domain has been identified recently in the amino terminus of Shc, an adaptor molecule that appears to be involved in Ras activation PTB domains are longer than SH2 domains, and recognize phosphotyrosine in the context of amino-terminal residues, in contrast to SH2 domains, which recognize them in the context of carboxy-terminal residues.

Amino Acid Sequence↗