Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand-dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation.
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PMID:Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase. 172 91

Transformation by activated pp60c-src has been correlated by genetic analysis with the tyrosine phosphorylation of a 120 kilodalton (kDa) protein, p120. We now demonstrate tyrosine phosphorylation of p120 following stimulation of cells by growth factors whose receptors have intrinsic tyrosine-specific protein kinase activity. Stimulation of quiescent NIH3T3 cells with platelet-derived growth factor (PDGF) resulted in the tyrosine phosphorylation of p120 that was maximal by 5 min and returned to background levels by 30 min. p120 was also phosphorylated on tyrosine after addition of colony-stimulating factor 1 (CSF-1) or epidermal growth factor (EGF) to NIH3T3 cells engineered to express high levels of their respective receptors. Two additional src substrates, p110 and p85, were analysed under identical assay conditions. PDGF, CSF-1, and EGF induced only a minimal increase in the tyrosine phosphorylation of p85 and no change in the phosphorylation of p110. Thus, the marked ligand-induced tyrosine phosphorylation of p120 was a property not shared by the other src substrates examined. Immunoblotting with antibodies to p120 and the ras GTPase activating protein, GAP, suggests that p120 and GAP are unrelated. In addition, the amino acid sequences of four cyanogen bromide peptides derived from p120 showed no homology to GAP or to sequences in either the PIR or Swiss-Prot databases. These data suggest that tyrosine phosphorylation of p120 may contribute to both signal transduction through growth factor receptors and pp60src induced transformation.
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PMID:PDGF, CSF-1, and EGF induce tyrosine phosphorylation of p120, a pp60src transformation-associated substrate. 185 49

We have reported earlier the isolation of two recessive, serum- and anchorage-dependent revertants from an NIH 3T3 line which had been transformed with multiple copies of a c-H-ras oncogene. In both revertants the oncogene was fully expressed and fusion of either revertant with normal (untransformed) cells or of the two revertants with one another resulted in transformed progeny. These, and other data indicate that the transforming activity of the c-H-ras oncogene is impaired in the two revertants, in consequence of defects in distinct genes needed to mediate this activity. Here, we describe some of the biochemical features of the revertants. In both of these (as in the transformed line) the bulk of the ras-p21 protein was found in the membrane fraction. This suggests proper posttranslational processing. Furthermore, no difference was detected either in the ras-p21 protein GTPase stimulating activity of GAP or in the extent of GAP-tyrosine phosphorylation among growing cultures of the two revertants, the transformed line and the parental NIH 3T3 line. The level of glucose transporter mRNA was severalfold higher in the transformed line than in the NIH 3T3 line. In the two revertants, however, the level was as low as that in the NIH 3T3 line. This indicates that the reversion impaired the effect of the c-H-ras oncogene on transcription. The raf oncogene (proposed to increase transcription factor activity) could retransform both revertants. Moreover, as revealed in experiments with growing cultures, neither transformation by the c-H-ras oncogene nor reversion from the transformed state altered the electrophoretic mobility of the raf protein or the level of its actin kinase activity. These results suggest that transformation by the c-H-ras oncogene is not mediated by the activation of raf protein kinase. The tyrosine phosphorylation of the p34cdc2 protein kinase (a cell cycle regulatory enzyme) was severalfold higher in the transformed line than in the NIH 3T3 line. The level of p34cdc2 protein kinase phosphorylation was as high in the R260 revertant as in the transformed line and as low in the R116 revertant as in the NIH 3T3 line. We are attempting to identify the defective mediator genes impairing the transforming activity of the c-H-ras oncogene in the two revertants.
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PMID:Characterization of recessive (mediator-) revertants from NIH 3T3 cells transformed with a c-H-ras oncogene. 199 48

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.
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PMID:Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae. 749 91

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

Raf-1 is a 74-kDa serine-threonine kinase which serves as the immediate downstream target of Ras in the cell growth signal transduction pathway. Recent genetic and biochemical experiments have demonstrated that (1) Ras interacts directly with the amino-terminal domain of Raf and (2) residues 51-131 of the Raf sequence are sufficient to mediate this interaction [Vojtek, A. B., Hollenberg, S. M., & Cooper, J. A. (1993) Cell 74, 205-214]. We have expressed a corresponding segment of the human Raf sequence (Raf55-132) in Escherichia coli as a fusion with maltose binding protein. The fusion protein was purified by affinity chromatography and cleaved at a pre-engineered site with factor Xa protease to liberate the 78-residue fragment of Raf. Raf55-132 bound to Ras with high affinity in a competition assay with GAP. An unlabeled version of Raf55-132 was studied by 2D homonuclear NMR, and uniformly 15N- and 13C/15N-labeled versions of Raf55-132 were studied by 2D and 3D heteronuclear NMR. Nearly complete sequence-specific assignments were made for the backbone HN, H alpha, 15N, and 13C alpha resonances. NOEs were used to determine regions of secondary structure and the overall folding topology. Raf55-132 is an independently folded domain composed of a five-stranded beta-sheet, a three-turn alpha-helix, and possibly an additional one-turn helix. Its structure resembles that of ubiquitin, even though there is no more than 11% sequence homology between the two proteins.
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PMID:Chemical shift assignments and folding topology of the Ras-binding domain of human Raf-1 as determined by heteronuclear three-dimensional NMR spectroscopy. 801 39

Leukocyte tyrosine kinase (ltk) is a receptor-type tyrosine kinase which is suggested to be expressed in hematopoietic cells and neuronal cells in human. Recently we have cloned a full sized human ltk cDNA which has a 423 amino acid extracellular domain which may bind to unknown ligand(s), and a 415 amino acid cytoplasmic domain which contains a tyrosine kinase domain. To identify the cellular signal transducer proteins binding to the ltk protein, we have analysed the recombinant ltk protein transiently expressed in COS cells. By an in vitro immune complex kinase assay, a major 140 kDa phosphoprotein and other cellular phosphoproteins were co-immunoprecipitated with the 100 kDa ltk protein using anti-ltk monoclonal antibodies. Western blot analysis revealed that the wild-type ltk protein was tyrosine-phosphorylated in vivo and associated with SH2 containing proteins, PLC-gamma 1, p85 subunit of PI3-K and GAP, in vivo. Furthermore, the wild-type ltk protein also binds to a serine/threonine kinase, Raf-1, in vivo. In contrast, none of these signal transducer proteins were associated with a kinase-negative ltk mutant (K544M-ltk) in which methionine at the putative ATP binding site was replaced with lysine. These results suggest that the associations of the ltk protein with those signaling molecules depend on the tyrosine kinase activity of the ltk protein. This is the first detection of cytoplasmic signal transducers that bind to the ltk protein in vivo.
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PMID:Human ltk receptor tyrosine kinase binds to PLC-gamma 1, PI3-K, GAP and Raf-1 in vivo. 808 3

Horizontal cells, which are second-order neurons of the vertebrate retina, exhibit synaptic plasticity governed by light and dark adaptation. We have investigated the alterations in the protein phosphorylation patterns of isolated carp (Cyprinus carpio) horizontal cells in relation to their state of light adaptation by using an in vitro phosphorylation assay and compared the resulting data with protein synthesis patterns of the whole retina. Phosphoproteins and [35S]methionine-labelled proteins were analysed by one- and two-dimensional gel electrophoresis followed by autoradiography. The state of light adaptation significantly affected the in vitro phosphorylation of horizontal cell proteins with molecular weights of 68, 56/58, 47, 28 and 15 kDa, but had no effect on the protein synthesis of retinal proteins. In the light the most prominent increase of 32P incorporation was observed in the 47 kDa protein. The biochemical properties of this protein closely resembled those of the growth-associated GAP-48, found in the fish retina. In addition, the phosphorylation of horizontal cell homogenates in the presence of protein kinase activators such as cyclic AMP, calcium, calmodulin and phospholipids revealed that horizontal cells of the fish retina contain cyclic AMP-, calcium/calmodulin- and calcium/phospholipid-dependent protein kinase activity resulting in the phosphorylation of several horizontal cell proteins, including the phosphoproteins which were affected by the state of light adaptation.
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PMID:In vitro phosphorylation in isolated horizontal cells of the fish retina: effects of the state of light adaptation. 826 Nov 33

In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.
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PMID:Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. 833 87

Ras proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H-ras, K-ras, and N-ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643-654). Transmission of signals from Ras to Raf is regulated by the Ras-related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell. The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411-414; Stokoe et al., 1994: Science 264:1463-1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14-3-3 family that appear necessary for kinase activation. The 14-3-3 proteins interact with all three conserved regions of Raf, including the kinase domain. In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras-related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility. The protein R-Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez-Sarabia and Bischoff, 1993: Nature 366:274-275). On the other hand, it has recently been shown that R-Ras binds to the protooncogene product Bcl-2, a protein that transforms B cells by blocking apoptosis. R-Ras is regulated by the same GAP molecules as H-Ras and the other Ras protooncogene products, and may therefore be activated in a manner co-ordinate with these growth-promoting proteins. The possible connection between R-Ras and apoptosis will be discussed.
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PMID:Ras-related proteins in signal transduction and growth control. 860 82


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