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)

In KB epidermoid cells, we previously showed that interleukin-1 alpha (IL-1) and various mitogens activate the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate both myelin basic protein (MBP) and a peptide containing Thr669 of the epidermal growth factor receptor. In cell-free extracts made from gingival fibroblasts treated with platelet-derived growth factor or HepG2 hepatoma cells stimulated with phorbol myristate acetate, MBP and Thr669 kinase were both elevated 4-fold, and ERK1 and ERK2 were tyrosine-phosphorylated. In these cells IL-1 activated a kinase(s) that phosphorylated Thr669 peptide but not MBP and failed to cause tyrosine phosphorylation of ERK1/ERK2. Ceramide has been proposed as an intracellular mediator of IL-1 action, but C2-ceramide or sphingosine stimulated predominantly MBP-specific kinase activity in fibroblasts and had no effect in HepG2 cells. p54 MAP kinase (also called stress-activated protein kinase) is a c-Jun kinase first isolated from livers of cycloheximide-treated rats. After IL-1 stimulation, immunoprecipitates of lysates made from all three cell types with specific anti-p54 MAP kinase serum contained Thr669 and c-Jun phosphorylating activity, whereas precipitates from unstimulated cells contained no detectable p54 kinase activity. The major peak of IL-1-stimulated HepG2 Thr669 kinase activity co-chromatographed on Mono Q and phenyl-Superose with immunodetectable p54 MAP kinase. IL-1 did not cause p21ras activation in any cell type. Induction of Thr 669 kinase activity was not abrogated by elevation of cAMP levels, which has been shown to interfere with the activation of Raf-1. We could not detect MAP kinase kinase phosphorylating activity in unfractionated lysates made from IL-1-stimulated fibroblasts or HepG2 cells. KB cells contained a small amount of this activity, but it was not precipitated with an anti-Raf-1 antibody. We conclude that most of the IL-1-activated Thr669 kinase activity in fibroblasts and HepG2 cells, and a portion in KB cells, is due to p54 MAP kinase and that its activation is Ras-, Raf-, and MAP kinase kinase-independent.
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PMID:Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase. 752 98

Signaling via the fibroblast growth factor receptor 1 (FGFR1, flg) was analyzed in Ba/F3 hematopoietic cells expressing either wild-type or a mutant FGF receptor (Y766F) unable to activate phospholipase C-gamma (PLC-gamma) and stimulate phosphatidylinositol (PI) hydrolysis. Stimulation of cells expressing wild-type or mutant FGFR with acidic FGF (aFGF) caused similar activation of Ras. However, an approximately 3-fold reduced activation of Raf-1 and MAP kinase was observed in aFGF-stimulated cells expressing mutant receptors as compared to cells expressing wild-type FGF receptors. Comparison of phosphopeptide maps of Raf-1 immunoprecipitated from the two cell types activated by either aFGF or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate) suggests that Raf-1 is phosphorylated by both Ras-dependent and PLC-gamma-dependent mechanisms. In spite of the differential effect on Raf-1 and MAP kinase activation, aFGF stimulated similar proliferation of cells expressing wild-type or mutant receptors indicating that Ras-dependent activation of Raf-1 and MAP kinase is sufficient for transduction of FGFR mitogenic signals. Ras may also activate signal transduction pathways that are complementary or parallel to the MAP kinase pathway to stimulate cell proliferation.
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PMID:Reduced activation of RAF-1 and MAP kinase by a fibroblast growth factor receptor mutant deficient in stimulation of phosphatidylinositol hydrolysis. 753 87

Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.
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PMID:The MEK kinase activity of the catalytic domain of RAF-1 is regulated independently of Ras binding in T cells. 753 98

Mitogenic stimulation of Swiss 3T3 fibroblasts with bombesin results in receptor-mediated activation of a complex array of effectors, including phospholipase C beta and mitogen-activated protein (MAP) kinase. Incubation of Swiss 3T3 fibroblasts with the 11-amino acid [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide inhibited bombesin-stimulated cell proliferation and phospholipase C beta activation even at high bombesin concentrations. The peptide did not inhibit the activation of phospholipase C beta by a GTPase-deficient form of the Gq-like protein, G16, indicating that the peptide does not inhibit phospholipase C beta and is acting at a point upstream of the activated form of the G protein alpha subunit. The peptide inhibited MAP kinase activation at low bombesin concentrations, but unlike phospholipase C beta, this inhibition could be overcome with 30 nM bombesin. In control Swiss 3T3 cells, bombesin did not measurably activate Ras or Raf-1 above basal levels. Following incubation of the cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, 50 nM bombesin activated Raf-1 4-6-fold over basal levels. Platelet-derived growth factor-stimulated activities of PLC, Ras, Raf-1, and MAP kinase were unaltered after incubation of Swiss 3T3 cells with the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P peptide, as was platelet-derived growth factor-stimulated growth of the Swiss 3T3 cells. Thus, the peptide behaves as an antagonist that differentially inhibited phospholipase C beta and MAP kinase signal transduction pathways. The growth arrest observed with the peptide indicates that the bombesin-stimulated activation of MAP kinase is not sufficient to support mitogenesis in Swiss 3T3 cells.
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PMID:Differential modulation of bombesin-stimulated phospholipase C beta and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P. 753 38

Raf-1 is a 74-kD serine/threonine kinase located in the cell cytoplasm that is activated by phosphorylation in cells stimulated with a variety of mitogens and growth factors, including hematopoietic growth factors. Using c-raf antisense oligonucleotides to block Raf-1 expression, we have established that Raf-1 is required for hematopoietic growth factor-induced proliferation of murine cell lines stimulated by growth factors whose receptors are members of several different structural classes: (a) the hematopoietin receptor family, including interleukin (IL)-2, IL-3, IL-4, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor (GM-CSF), and erythropoietin; (b) the tyrosine kinase receptor class, including Steel factor and CSF-1; and (c) IL-6, leukemia inhibitory factor, and oncostatin M, whose receptors include the gp130 receptor subunit. Although results of previous experiments had suggested that IL-4 does not phosphorylate or activate the Raf-1 kinase, c-raf antisense oligonucleotides inhibited IL-4-induced proliferation of both myeloid and T cell lines, and IL-4 activated Raf-1 kinase activity in an IL-4-dependent myeloid cell line. In colony assays, c-raf antisense oligonucleotides completely inhibited colony formation of unseparated normal murine bone marrow cells stimulated with either IL-3 or CSF-1 and partially inhibited cells stimulated with GM-CSF. In addition, c-raf antisense oligonucleotides completely inhibited both IL-3- and GM-CSF-induced colony formation of CD34+ purified human progenitors stimulated with these same growth factors. Thus, Raf-1 is required for growth factor-induced proliferation of leukemic murine progenitor cell lines and normal murine and human bone marrow-derived progenitor cells regardless of the growth factor used to stimulate cell growth.
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PMID:Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. 753 43

Raf-1 is a serine/threonine kinase poised at a key relay point in mitogenic signal transduction pathways from the cell surface to the nucleus. Activation of the transforming potential of Raf-1 has been associated with N-terminal truncation and/or fusion to other proteins, suggesting that the Raf-1 N-terminal half harbors a negative regulatory domain. Seven internal deletion mutants that together scan the entire N-terminal half of human Raf-1 protein were generated to map functional regions in this regulatory domain. Effects of the deletion mutations on kinase activity of Raf-1 were evaluated using a baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1, mitogen-activated protein kinase kinase. Deletion of amino acids 276-323 in the unique sequence between conserved regions 2 and 3 leads to modest elevation of Raf-1 basal kinase activity, whereas deletion of amino acids 133-180 in conserved region 1 results in diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal deletion mutants, including a truncated version that is transforming in rodent fibroblasts, exhibits greatly increased levels of basal kinase activity. In addition, while activation of Raf-1 kinase by Ras requires sequences in conserved region 1, only the C-terminal half containing the kinase domain of Raf-1 is required for activation by Src. These findings demonstrate that N-terminal deletions in Raf-1 do not necessarily result in constitutively elevated basal kinase activity and that the N-terminal regulatory domain is completely dispensable for Raf-1 activation by Src.
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PMID:Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase. 753 98

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 zeta isoform of protein kinase C (zeta PKC) has been shown to be an important step in mitogenic signal transduction. Using a yeast interaction screen to search for potential novel substrates of zeta PKC, we identified the heterogeneous ribonucleoprotein A1 (hnRNPA1). This protein specifically interacts with the catalytic domain of zeta PKC but not with its regulatory region or with the full-length protein, or with a kinase-defective mutant of the zeta PKC catalytic domain. In addition, no interaction was detected with other kinases such as Raf-1 or Mos, that, like zeta PKC, are critically involved in signal transduction, or with the catalytic domain of epsilon PKC, which is the PKC isotype with the highest homology to zeta PKC. hnRNPA1 is directly phosphorylated by both recombinant and native zeta PKC, and this phosphorylation is increased when zeta PKC is immunoprecipitated from mitogen-activated fibroblasts. As an additional control, hnRNPA1 is not phosphorylated appreciably by catalytic epsilon PKC or by a mixture of highly purified classical PKC isotypes maximally activated by phosphatidylserine and Ca2+. Treatment of quiescent cell cultures with a potent mitogen such as platelet-derived growth factor promotes a significant phosphorylation of hnRNPA1 in vivo that is impaired by expression of a dominant negative mutant of zeta PKC. Furthermore, expression of a catalytically active zeta PKC mutant phosphorylates hnRNPA1 in vivo. These findings suggest that zeta PKC could be critically involved in a novel pathway that connects membrane signaling to nuclear regulatory events, at the level of RNA transport and processing. Results also shown here by using different zeta PKC mutants suggesting the control of the cytoplasmic localization of hnRNPA1 by zeta PKC. Also of potential functional relevance are the results demonstrating that the phosphorylation by zeta PKC severely impairs both hnRNPA1 RNA binding and its ability to promote strand annealing in vitro.
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PMID:Identification of heterogeneous ribonucleoprotein A1 as a novel substrate for protein kinase C zeta. 754 Oct 49

A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.
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PMID:Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. 754 86

We have attempted to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus by reconstituting them in the baculovirus/Sf9 cell system. We have used this system to coexpress different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 and assayed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos. We observe that activation of ERK-1 via Raf-1 and p21ras dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v-ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. Our observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. Our data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors.
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PMID:Reconstitution of signal transduction from the membrane to the nucleus in a baculovirus expression system: activation of Raf-1 leads to hypermodification of c-jun and c-fos via multiple pathways. 754 94


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