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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases.
Mol Cell Biol 1994 Jul
PMID:Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages. 800 65

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.
Mol Endocrinol 1994 May
PMID:The level of insulin receptor tyrosine kinase activity modulates the activities of phosphatidylinositol 3-kinase, microtubule-associated protein, and S6 kinases. 805 65

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.
Mol Cell Biol 1994 Sep
PMID:Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity. 806 26

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.
Mol Cell Biol 1994 Apr
PMID:Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity. 813 39

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.
Mol Cell Biol 1994 May
PMID:Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase. 816 50

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.
Mol Cell Biol 1993 Dec
PMID:Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase. 824 87

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.
Mol Cell Biol 1994 Jan
PMID:Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits. 826 9

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10- to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.
Mol Cell Biol 1994 Jan
PMID:Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members. 826 17

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biol 1993 Mar
PMID:In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase. 838 74

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.
Mol Cell Biol 1993 Jun
PMID:Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach. 838 38


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