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T Pawson

Publications and source records attributed to T Pawson.

At least 127 records · Page 7Linked to original sources
PubMed1995

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps.

The gene coding for the tyrosine protein kinase domain of v-fps was subcloned into a plasmid vector expressing glutathione-S-transferase (GST). This new vector expresses a fusion protein in Escherichia coli composed of the kinase domain linked with GST at the N-terminus (GST-kin). A portion of the total expressed protein was soluble upon cell lysis and was purified by affinity chromatography using glutathione cross-linked agarose. GST-kin (M(r) 57,000) is a phosphoprotein as judged by 32P autoradiography, consistent with the known autophosphorylation site within the kinase core [Weinmaster et al. (1984) Cell, 37, 559-568]. Cleavage of the fusion protein with thrombin and purification on phosphocellulose resin yielded the pure kinase domain (M(r) 33,000). The activity of the kinase domain is indistinguishable from that of GST-kin using the peptide substrate EEEIYEEIE, indicating that N-terminal fusion has no effect on the kinase domain. GST-kin phosphorylates a second peptide, EAEIYEAIE, with improved catalytic efficiency. Initial velocity data are consistent with a random bireactant mechanism with no substrate synergism observed in the ternary complex. Steady-state kinetic analyses reveal that this peptide is phosphorylated, with a kcat of 3.6 s-1, a Kpeptide of 500 microM and a KATP of 250 microM. The expression, purification and preliminary kinetic analysis of the kinase domain of v-fps provide the first step in the application of structure-function studies for this oncoprotein.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.

Amino Acid Sequence↗
PubMed1995

A Drosophila shc gene product is implicated in signaling by the DER receptor tyrosine kinase.

Antibodies to the human Shc adaptor protein were used to isolate a cDNA encoding a Drosophila Shc protein (dShc) by screening an expression library. The dshc gene, which maps to position 67B-C on the third chromosome, encodes a 45-kDa protein that is widely expressed throughout the Drosophila life cycle. In flies, the dShc protein physically associates with the activated Drosophila epidermal growth factor receptor homolog (DER) and is inducibly phosphorylated on tyrosine by DER. The 45-kDa dShc protein is closely related both in overall organization and in amino acid sequence (46% identity) to the 52-kDa mammalian Shc isoform. In addition to a C-terminal Src homology 2 (SH2) domain, dShc contains an N-terminal phosphotyrosine-binding (PTB) domain, which associates in vitro with the autophosphorylated DER receptor tyrosine kinase and with phosphopeptides containing an Asn-Pro-X-pTyr motif, where pTyr stands for phosphotyrosine. A potential binding site for the dShc PTB domain is located at Tyr-1228 of DER. These results indicate that the shc gene has been conserved in evolution, as have the binding properties of the Shc PTB and SH2 domains. Despite the close relationship between the Drosophila and mammalian Shc proteins, dShc lacks the high-affinity Grb2-binding site found in mammalian Shc, suggesting that Shc proteins may have functions in addition to regulation of the Ras pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity.

The EPH-related transmembrane tyrosine kinases constitute the largest known family of receptor-like tyrosine kinases, with many members displaying specific patterns of expression in the developing and adult nervous system. A family of cell surface-bound ligands exhibiting distinct, but overlapping, specificities for these EPH-related kinases was identified. These ligands were unable to act as conventional soluble factors. However, they did function when presented in membrane-bound form, suggesting that they require direct cell-to-cell contact to activate their receptors. Membrane attachment may serve to facilitate ligand dimerization or aggregation, because antibody-mediated clustering activated previously inactive soluble forms of these ligands.

Amino Acid Sequence↗
PubMed1994

The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.

Adaptor Proteins, Signal Transducing↗
PubMed1994

SH2 domain specificity and activity modified by a single residue.

Many intracellular targets of protein-tyrosine kinases possess Src homology 2 (SH2) domains that directly recognize phosphotyrosine-containing sites on autophosphorylated growth factor receptors and cytoplasmic proteins, and thereby mediate the activation of biochemical signalling pathways. SH2 domains possess relatively well conserved residues that form the phosphotyrosine-binding pocket, and more variable residues that are implicated in determining binding specificity by recognition of the three amino acids carboxy-terminal to phosphotyrosine (the +1 to +3 positions). One such residue, occupying the EF1 position of the +3-binding pocket, is a Thr in the SH2 domain of the Src tyrosine kinase, but is predicted to be a Trp in the SH2 domain of the Sem-5/drk/Grb2 adaptor protein. Here we report that changing this residue in the Src SH2 domain from Thr to Trp switches its selectivity to resemble that of the Sem-5/drk/Grb2 SH2 domain. Furthermore, this mutant Src SH2 domain effectively substitutes for the SH2 domain of the Sem-5 protein in activation of the Ras pathway in vivo. These results identify a residue that can modify SH2 selectivity, and indicate that the biological activity of an SH2 domain correlates with its binding specificity.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Direct interaction between Shc and the platelet-derived growth factor beta-receptor.

The Src homology 2 (SH2) domain-containing Shc proteins p52shc and p46shc become phosphorylated upon activation of several tyrosine kinases and are implicated in mitogenic signal transduction. Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to autophosphorylation of tyrosine residues, which is known to mediate interactions with several SH2 domain-containing signaling molecules. In this study, we have characterized the interaction between the PDGF beta-receptor and Shc. PDGF beta-receptor coprecipitation in Shc immunoprecipitates was dependent on stimulation with PDGF-BB. The Shc SH2 domain expressed as a bacterial fusion protein bound the autophosphorylated PDGF beta-receptor. Moreover, the Shc SH2 domain could bind the autophosphorylated purified baculovirus-expressed PDGF beta-receptor intracellular domain, which indicates a direct association of Shc with the PDGF beta-receptor. Activation of the PDGF beta-receptor induced the preferential phosphorylation of p52shc. Tyrosine-phosphorylated Shc, in turn, formed a complex with the signaling molecule Grb2. Synthetic peptide analysis revealed that certain autophosphorylation sites in the PDGF beta-receptor (Tyr-579, Tyr-740, Tyr-751, and Tyr-771) were able to mediate the specific binding of the Shc SH2 domain as well as intact Shc proteins. A mutant PDGF beta-receptor in which Tyr-579 was replaced with phenylalanine showed 40% impaired association of Shc in vivo, but phosphorylation of Shc proteins was not affected. We conclude that multiple autophosphorylation sites in the PDGF beta-receptor are responsible for the binding of Shc. This is in contrast to previously characterized interactions between the PDGF beta-receptor and SH2 domain-containing proteins, which generally involve one high affinity binding site in the receptor.

Adaptor Proteins, Signal Transducing↗
PubMed1994

SH2-containing phosphotyrosine phosphatase Syp is a target of p210bcr-abl tyrosine kinase.

The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction and neoplastic transformation. These mechanisms are regulated by the activities of both protein-tyrosine kinases and protein-tyrosine phosphatases. Recent studies have identified a novel protein-tyrosine phosphatase, termed Syp, that is widely expressed in various tissues. Syp encodes a cytoplasmic phosphatase that contains two Src homology 2 (SH2) domains. Since SH2 domains have been shown to target the association of signal-transducing molecules to activated tyrosine kinases, experiments were performed to determine whether Syp might form specific complexes with p210bcr-abl, a fusion protein believed to be involved in the pathogenesis of chronic myelogenous leukemia and, thus, possibly alter or mediate p210bcr-abl tyrosine kinase activity. We found that Syp was highly and constitutively tyrosine phosphorylated in three different murine cell lines transfected with a p210bcr-abl expression vector. Furthermore, p210bcr-abl, Syp, and Grb2 formed stable complexes in BCR-ABL-expressing cells. Complex formation between p210bcr-abl and Syp was mediated in vitro by the NH2-terminal SH2 domain of Syp. Last, p210bcr-abl tyrosine kinase was effectively dephosphorylated by Syp in vitro. These results suggest an interaction between Syp and BCR-ABL protein, which might play a role in cellular transformation of BCR-ABL.

Animals↗
PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗
PubMed1994

Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide.

The solution structure of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLC-gamma 1), in complex with a phosphopeptide corresponding to its Tyr-1021 high affinity binding site on the platelet-derived growth factor receptor, has been determined by nuclear magnetic resonance spectroscopy. The topology of the SH2-phosphopeptide complex is similar to previously reported Src and Lck SH2 complexes. However, the binding site for residues C-terminal to the phosphotyrosine (pTyr) is an extended groove that contacts peptide residues at the +1 to +6 positions relative to the pTyr. This striking difference from Src and Lck reflects the fact that the PLC-gamma 1 complex involves binding of a phosphopeptide with predominantly hydrophobic residues C-terminal to the pTyr and therefore serves as a prototype for a second class of SH2-phosphopeptide interactions.

Amino Acid Sequence↗
PubMed1994

Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway.

The cytosolic 185 and 210 kDa Bcr-Abl protein tyrosine kinases play important roles in the development of Philadelphia chromosome positive (Ph+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (Ph+ ALL). p185 and p210 Bcr-Abl contain identical abl-encoded sequences juxtaposed to a variable number of bcr-derived amino acids. As the mitogenic and transforming activities of tyrosine kinases involve stimulation of the Ras pathway, we analyzed Bcr-Abl oncoproteins for interactions with cytoplasmic proteins that mediate Ras activation. Such polypeptides include Grb2, which comprises a single Src homology 2 (SH2) domain flanked by two SH3 domains, and the 66, 52 and 46 kDa Shc proteins which possess an SH2 domain in their carboxy-terminus. Grb2 associates with tyrosine phosphorylated proteins through its SH2 domain, and with the Ras guanine nucleotide releasing protein mSos1 through its SH3 domains. mSos1 stimulates conversion of the inactive GDP-bound form of Ras to the active GTP-bound state. In bcr-abl-transformed cells, Grb2 and mSos1 formed a physical complex with Bcr-Abl. In vitro, the Grb2 SH2 domain bound Bcr-Abl through recognition of a tyrosine phosphorylation site within the amino-terminal bcr-encoded sequence (p.Tyr177-Val-Asn-Val), that is common to both Bcr-Abl proteins. These results suggest that autophosphorylation within the Bcr element of Bcr-Abl creates a direct physical link to Grb2-mSos1, and potentially to the Ras pathway, and thereby modifies the target specificity of the Abl tyrosine kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗
PubMed1994

Phosphotyrosine phosphatases with SH2 domains: regulators of signal transduction.

Among the rapidly growing family of protein tyrosine phosphatases (PTPs), a subfamily of cytoplasmic PTPs that contain SH2 domains has been identified in both mammals and Drosophila. These PTPs each contain two tandem SH2 domains at their amino-terminus, and a single phosphatase domain. The SH2-containing PTPs appear to be downstream targets of growth factor receptor tyrosine kinases, and have been shown genetically to be required for normal development in Drosophila and in the mouse. Accumulating biochemical and genetic data therefore suggest that SH2-containing PTPs might have a physiological role in intracellular signal transduction.

Animals↗
PubMed1994

Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses.

In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.

3T3 Cells↗
PubMed1994

Biochemistry of the Src protein-tyrosine kinase: regulation by SH2 and SH3 domains.

pp60c-Srs (c-Src) is the prototype for a family of cytoplasmic protein-tyrosine kinases involved in the control of signal transduction. In addition to the enzymatic kinase domain, c-Src has several noncatalytic domains which regulate Src tyrosine kinase activity in both a positive and a negative fashion. Phosphorylation of c-Src at Tyr527 in the noncatalytic C-terminal tail is a key mechanism for repression of c-Src tyrosine kinase activity. This inhibitory phosphorylation is apparently catalyzed by another cytoplasmic tyrosine kinase (Csk). Recent evidence suggests that the c-Src SH2 domain participates in this phosphorylation-dependent repression of kinase activity through an intramolecular association with the phosphotyrosine-containing C-terminus. The SH3 domain of c-Src also negatively regulates c-Src tyrosin kinase activity, although the mechanism is as yet unknown. However, in the background of constitutively active transforming Src variants, such as a c-Src mutant with an amino acid substitution eliminating Tyr527 (527F c-Src) or the retroviral oncogene v-src product pp60v-src (v-Src), both the SH2 and SH3 domains contribute positively to the enzymatic and biological activities of the Src tyrosine kinase through interactions with Src substrates and/or cellular regulators.

Amino Acid Sequence↗