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)

In rat aortic smooth muscle cells, platelet-derived growth factor (PDGF) stimulated a sustained activation of mitogen-activated protein kinase (MAP kinase) while the response to angiotensin II (AII) was transient. This was due to a relatively greater initial activation of MAP kinase kinase (MEK) and a correspondingly greater residual MEK activity at later time points. Pretreatment of cells with the novel MEK inhibitor PD 098059 reduced MEK activation at 5 min in response to each agonist by a similar proportion (70%); however, at this time point MAP kinase activation in response to PDGF was only marginally affected while the response to AII was substantially reduced. PD 098059 did, however, reduce PDGF-stimulated MEK activity after 30 min and this correlated with a loss in MAP kinase activity and DNA synthesis. Pretreatment with forskolin also caused a similar pattern of inhibition of agonist-stimulated MEK and MAP kinase activity. Only following protein kinase C down-regulation were both AII- and PDGF-stimulated MAP kinase activation substantially reduced and this correlated with the virtual loss of both MEK and c-Raf-1 activity in response to both agents. The differential inhibition of MAP kinase activation by forskolin was not due to specific activation of A-Raf by PDGF; both PDGF and AII stimulated A-Raf kinase and this activity was strongly inhibited by forskolin. These results suggest that the efficacy of MEK activation determines the duration of MAP kinase activation and the susceptibility of MAP kinase activation to inhibition by different agents. The results also argue against the selective activation of A-Raf by PDGF as a mechanism to explain the differences in the kinetics of MAP kinase activity stimulated by AII and PDGF.
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PMID:Efficacy of agonist-stimulated MEK activation determines the susceptibility of mitogen-activated protein (MAP) kinase to inhibition in rat aortic smooth muscle cells. 880 60

The function of the c-Raf-1 zinc finger domain in the activation of the Raf kinase was examined by the creation of variant zinc finger structures. Mutation of Raf Cys 165 and Cys 168 to Ser strongly inhibits the Ras-dependent activation of c-Raf-1 by epidermal growth factor (EGF). Deletion of the Raf zinc finger and replacement with a homologous zinc finger from protein kinase C gamma (PKC gamma) (to give gamma/Raf) also abrogates EGF-induced activation but enables a vigorous phorbol myristate acetate (PMA)-induced activation. PMA activation of gamma/Raf does not require endogenous Ras or PKCs and probably occurs through a PMA-induced recruitment of gamma/Raf to the plasma membrane. The impaired ability of EGF to activate the Raf zinc finger variants in situ is attributable, at least in part, to a major decrement in their binding to Ras-GTP; both Raf zinc finger variants exhibit decreased association with Ras (V12) in situ upon coexpression in COS cells, as well as diminished binding in vitro to immobilized, processed COS recombinant Ras(V12)-GTP. In contrast, Raf binding to unprocessed COS or prokaryotic recombinant Ras-GTP is unaffected by Raf zinc finger mutation. Thus, the Raf zinc finger contributes an important component to the overall binding to Ras-GTP in situ, through an interaction between the zinc finger and an epitope on Ras, distinct from the effector loop, that is present only on prenylated Ras.
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PMID:An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ. 897 84

More and more effectors for the Ras-related protein superfamily are being discovered and it is emerging that these GTP-binding proteins interact with more than one effector to generate more than one cellular signal. Atomic details for the interaction of Rap/Ras with one of the effectors, the protein kinase c-Raf-1, have recently become available by X-ray structure analysis. The implications for the specificity of the signal transduction pathway, and how the GTP-dependent switch mechanism modulates the interaction with effectors will be discussed here, using Ras as a paradigm.
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PMID:How Ras-related proteins talk to their effectors. 900 33

Invasive and metastatic cells require protease expression for migration through the extracellular matrix. Metastatic NIH 3T3 fibroblasts transformed by different activated ras genes showed two different protease phenotypes, rasuPA+/CL- and rasCL+/uPA- (Zhang, J-Y., and Schultz, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype rasuPA+/CL- is dependent on expression of the serine-type protease urokinase plasminogen activator (uPA) and the phenotype rasCL+/uPA- on the cystine-type protease cathepsin L (CL) for lung colonization in experimental metastasis. The existence of multiple invasive phenotypes on ras-isoform transformation implied the activation of alternative pathways downstream from Ras. We now show that c-Raf-1, extracellular signal-regulated protein kinase (ERK)-1, and ERK-2 are hyperphosphorylated, and the ERK activity is high in both the uPA- and CL-dependent ras-transformed invasive phenotypes. Levels of c-Jun and c-Jun NH2-terminal kinase (JNK) activity are also high in the uPA-dependent phenotype, but they are almost undetectable in the CL-dependent phenotype. The uPA Ras-response element is a PEA3/URTF element, and mobility shift assays show a strong PEA3/URTF protein band in the uPA-dependent phenotype. This band is competed by a consensus AP-1 DNA sequence and by antibodies to PEA3 and c-Jun. Thus, the uPA-invasive phenotype appears to require the activation of Ets/PEA3 and c-Jun transcription factors activated by the ERK and JNK pathways, while the CL-invasive phenotype appears to require ERK activity with suppression of JNK and c-Jun activities. These postulates are supported by the introduction of a dominant negative c-Jun, TAM67, into cells of phenotype rasuPA+/CL-, which down-regulated the high uPA mRNA levels characteristic of this phenotype to basal levels and up-regulated basal levels of CL mRNA to levels similar to those observed in cells of phenotype rasCL+/uPA-. We conclude that the JNK pathway acts as a switch between two distinct protease phenotypes that are redundant in their abilities to grow tumors and metastasize.
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PMID:Characterization of downstream Ras signals that induce alternative protease-dependent invasive phenotypes. 903 12

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library we detected an interaction between A-Raf and CK2beta subunit. This binding was specific, as no interaction between CK2beta and B-Raf or c-Raf-1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity was enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.
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PMID:The regulatory subunit of protein kinase CK2 is a specific A-Raf activator. 904 66

Mutants of human neurofibromin and c-Raf-1 genes were fused to the 3' end of the hemagglutinin (HA) gene of influenza A virus by oligonucleotide-directed polymerase chain reaction (PCR). The two resulting chimeric genes, HA (1-534)/NF1 (1441-1518) and HA (1-534)/Raf-1 (51-132) which we designated HN and HR, respectively, were cloned in a vaccinia virus expression vector (pTMI) under the control of a T7 RNA polymerase promoter. The clones were expressed in a monkey cell line (CV-1) and the resulting chimeric proteins analysed. We found that expression levels of the chimeric proteins were similar to that of wild-type HA protein. Comparative endoglycosidase treatment revealed that the expressed chimeric proteins HN and HR were processed as wild-type HA, and FACS-analysis showed that both chimeric expression products localised in the cell membrane as the wild-type control. HN and HR expressing cells showed similar fusogenic activity as CV-1 cells transfected with wild-type HA indicating the correct topology of the fusion inducing portion (HA) of these chimera in the membrane. These findings show that the influenza virus hemagglutinin (HA) is a suitable vehicle to target foreign proteins with therapeutical potential into the cell membrane. In this respect HN and HR could potentially be used to block the abnormal signals generated by particular proteins in the cell membrane that lead to cell transformation.
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PMID:Targeted delivery of human neurofibromin and c-Raf-1 mutants to the cytoplasmic membrane by use of the influenza virus hemagglutinin. 912 41

The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.
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PMID:Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro. 917 52

In the developing eye of Drosophila the protein kinase D-Raf controls the specification of the R7 photoreceptor cells. We show that overexpression of wild-type D-Raf inhibits the formation of R7 cells in a dose-dependent manner. Conversely, overexpression of mutant D-Raf proteins in which the conserved S388 is replaced by A or by D promotes the formation of supernumerary R7 cells, indicating increased D-Raf activity in vivo. S388 in D-Raf corresponds to S259 in c-Raf; shown to be involved in binding of 14-3-3. We show that analogous substitutions of S259 in c-Raf prevent binding of 14-3-3 zeta to the amino terminus of c-Raf and cause a Ras-independent constitutively increased c-Raf kinase activity. Binding of 14-3-3 zeta to the second binding site at the carboxy terminal catalytic domain was unaffected by these mutations. These results suggest that the increased kinase activity of mutant D-Raf is caused by the selective loss of 14-3-3 binding to its amino terminus. Therefore, binding of 14-3-3 to the amino terminus of Raf appears to negatively regulate Raf kinase activity in vivo.
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PMID:Negative regulation of Raf activity by binding of 14-3-3 to the amino terminus of Raf in vivo. 923

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.
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PMID:GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction. 923 96

14-3-3 proteins mediate interactions between proteins involved in signal transduction and cell cycle regulation. Phosphorylation of target proteins as well as 14-3-3 are important for protein-protein interactions. Here, we describe the purification of a protein kinase from porcine brain that phosphorylates 14-3-3 zeta on Thr-233. This protein kinase has been identified as casein kinase Ialpha (CKIalpha) by peptide mapping analysis and sequencing. Among mammalian 14-3-3, only 14-3-3 tau possesses a phosphorylatable residue at the same position (Ser-233), and we show that this residue is also phosphorylated by CKI. In addition, we show that 14-3-3 zeta is exclusively phosphorylated on Thr-233 in human embryonic kidney 293 cells. The residue 233 is located within a region shown to be important for the association of 14-3-3 to target proteins. We showed previously that, in 293 cells, only the unphosphorylated form of 14-3-3 zeta associates with the regulatory domain of c-Raf. We have now shown that in vivo phosphorylation of 14-3-3 zeta at the CKIalpha site (Thr-233) negatively regulates its binding to c-Raf, and may be important in Raf-mediated signal transduction.
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PMID:14-3-3 is phosphorylated by casein kinase I on residue 233. Phosphorylation at this site in vivo regulates Raf/14-3-3 interaction. 936 Sep 56


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