Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.
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PMID:Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium. 751 36

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.
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PMID:Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. 752 76

Tyrosine kinase are important mediators of signal transduction in eukaryotic cells. In order to better understand the mechanism of catalysis we studied a set of mutants of the prototype tyrosine kinase, the c-Src protein, a homologue of the Rous Sarcoma virus oncogene. Based on an X-ray structure of cAMP-dependent protein kinase (cAPK) we mutated an arginine residue conserved in subdomain VI of all known kinases to a non-charged residue. This residue coordinates phosphate of the autophosphorylation site located in subdomain VII of cAPK and this interaction has been proposed to be crucial for substrate binding. The mutant R385A of c-Src had low kinase activity towards exogenous substrates yet was able to autophosphorylate at tyrosine 416. When introduced into an activated v-src gene the R385A mutation totally blocked cell transformation. Our data suggest that the function of the conserved arginine 385 is to coordinate the phosphate of the autophosphorylation site and to provide in this way a stable template for substrate binding.
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PMID:Catalytic activity and transformation potential of v-Src require arginine 385 in the substrate binding pocket. 752 17

We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.
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PMID:The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase. 753 95

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.
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PMID:Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways. 753 26

Angiogenesis, the formation of new microvasculature by capillary sprouting, is crucial for tumour development. Hypoxic regions of solid tumours produce the powerful and directly acting angiogenic protein VEGF/VPF (vascular endothelial growth factor/vascular permeability factor). We now investigate the signal transduction pathway involved in hypoxic induction of VEGF expression. Hypoxia is known to induce a tyrosine kinase cascade that results in the activation of nitrogen-fixation genes in Rhizobium meliloti, and activation of tyrosine kinases is critical in signalling triggered by growth factors and ultraviolet light. We show here that genistein, an inhibitor of protein tyrosine kinase, blocks VEGF induction. Hypoxia increases the kinase activity of pp60c-src and its phosphorylation on tyrosine 416 but does not activate Fyn or Yes. Expression of either a dominant-negative mutant form of c-Src or of Raf-1 markedly reduces VEGF induction. VEGF induction by hypoxia in c-src(-) cells is impaired, although there is a compensatory activation of Fyn. Our results provide an insight into hypoxia-triggered intracellular signalling, define VEGF as a new downstream target for c-SRC, and suggest a role for c-SRc in promoting angiogenesis.
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PMID:Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. 754 Jul 25

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.
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PMID:The K protein domain that recruits the interleukin 1-responsive K protein kinase lies adjacent to a cluster of c-Src and Vav SH3-binding sites. Implications that K protein acts as a docking platform. 759 45

Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src.
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PMID:The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src. 768 Nov 47

Undifferentiated crypt cells from chicken small intestine contain 15-fold higher levels of tyrosine-phosphorylated proteins than do differentiated enterocytes located at the villus apex. The tyrosine kinase activity and the tyrosine-phosphorylated proteins are associated with the Triton-insoluble cytoskeleton. To determine whether: (1) pp60c-src is an active tyrosine kinase in crypt cell cytoskeletons and (2) cytoskeletal-associated pp60c-src activity decreases as crypt cells differentiate, we isolated pp60c-src from subcellular fractions of cells along the crypt-villus axis of chicken small intestine and measured its protein kinase activity. We observed that pp60c-src activity in crypt cytoskeleton was higher (on average, fourfold as measured by enolase phosphorylation or sevenfold as measured by autophosphorylation) than that in cytoskeletons from differentiated enterocytes. Moreover, nearly 70% of pp60c-src activity in crypt cells, like that of pp60v-src, pp60c-src mutants with elevated kinase, activity or pp60v-src from activated platelets, localized to the cellular cytoskeleton. In contrast, less than 20% of pp60c-src activity in differentiated enterocytes, like that of kinase-inactive pp60v-src or pp60c-src from fibroblasts or resting platelets, associated with the cytoskeleton. Furthermore, in crypt cells, unlike differentiated enterocytes, cytoskeletal-associated pp60c-src appeared to have higher specific protein tyrosine kinase activity than did soluble pp60c-src. The data suggest that a kinase-active form of pp60c-src located in the cytoskeleton of crypt cells may be responsible for phosphorylating proteins on tyrosine and regulating growth and differentiation of the cells.
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PMID:Intestinal crypt cells contain higher levels of cytoskeletal-associated pp60c-src protein tyrosine kinase activity than do differentiated enterocytes. 768 Nov 58

The src family of protein-tyrosine kinases has long been implicated in signal transduction by growth factor receptors. In particular, pp60c-src, the product of the protooncogene c-src, has been shown to associated with the activated platelet-derived growth factor (PDGF) receptor. We demonstrate that, following stimulation of quiescent cells with PDGF, pp60c-src is translocated from the plasma membrane to the cytosol. This phenomenon was better defined utilizing an isolated plasma membrane system. The release of pp60c-src from the membrane in response to PDGF is accompanied by a 4-fold activation of its kinase activity and by phosphorylation at the amino terminus on serine/threonine residues as well as tyrosine residues. This amino-terminal phosphorylation appears to be responsible for a change in hydrophobicity of the pp60c-src molecule and hence for its release from the membrane. The kinase responsible for the serine/threonine phosphorylation and the concomitant release of pp60c-src has been identified as the cAMP-dependent protein kinase. Thus, PDGF stimulation activates at least two membrane-associated kinases (pp60c-src and cAMP-dependent protein kinase) very early in its signal transduction pathway.
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PMID:Translocation of pp60c-src from the plasma membrane to the cytosol after stimulation by platelet-derived growth factor. 769 37


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