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)

Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.
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PMID:Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation. 881 60

To elucidate the molecular basis for inhibition of B cell proliferation and differentiation by the Fc receptor for IgG (Fc(gamma)RII), we compared the signaling events in B cells stimulated by cross-linking surface Ig alone (positive signaling), or by co-cross-linking surface Ig and Fc(gamma)RII (negative signaling). Both modes of stimulation induced tyrosine kinase activation. Positive signaling induced activation of Ras, Raf-1 kinase, and mitogen-activated protein kinase; these events were significantly attenuated during negative signaling. Since Ras is activated by SOS and Vav, two known guanine nucleotide exchange factors, activation events associated with these molecules using the two different stimuli were examined. Results of these experiments indicated that tyrosine phosphorylation of Vav did not change upon co-cross-linking. In contrast, the association of Shc and Grb2 was abrogated under negative and induced under positive signaling conditions. Concomitantly, Shc was observed to associate with a tyrosine-phosphorylated 145-kDa protein, previously identified as Src homology 2-containing inositol phosphatase, only under conditions of negative signaling. Based on these results, we hypothesize that negative signaling via the Fc(gamma)RII in B cells is at least partly the result of a block in Ras activation, and that SOS, but not Vav, is the major guanine nucleotide exchange factor in B cells for Ras activation.
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PMID:Negative signaling in B cells causes reduced Ras activity by reducing Shc-Grb2 interactions. 901 51

Previous studies suggest that p56(lck) activity influences thymocyte development at a stage prior to TCR alphabeta expression. Transgenic mice that express high levels of p56(lck) activity during thymopoiesis develop thymic lymphomas consisting of cells with immature surface phenotypes. We have utilized cell lines derived from lck-induced thymic tumors to define biochemical pathways regulated by p56(lck) activity in immature thymocytes. Here we report that components of the Ras/Raf/MAPK pathway are constitutively activated in these lck-transformed immature thymoblasts. p56(lck) utilizes Shc and Grb2 adaptors to mediate activation of p21(ras) in the thymoblast lines by promoting tyrosine phosphorylation of the Shc protein and constitutive interaction between Shc and Grb2. The putative guanine nucleotide exchange factor p95(vav) is also maintained in constitutively tyrosine phosphorylated form as a result of elevated Lck activity. One target of activated Ras, the Raf-1 kinase, is hyperphosphorylated and downstream targets of activated Raf-1, Erk1 and Erk2, are hyperphosphorylated and activated in Lck-transformed thymocytes. Forskolin treatment reverses Raf-1 hyperphosphorylation in the cells and inhibits proliferation by blocking G1/S transition. In contrast, conventional protein tyrosine kinase inhibitors block proliferation by arresting Lck thymoblasts at G2/M. Lck-mediated stimulation of the Ras/Raf/MAPK pathway is also required to maintain cell viability by preventing programmed cell death. In summary, p56(lck) activity stimulates G1/S transition in immature thymoblasts and maintains cell viability via transduction of constitutive activation signals downstream to components of the Ras/Raf/MAPK pathway.
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PMID:Targets of p56(lck) activity in immature thymoblasts: stimulation of the Ras/Raf/MAPK pathway. 904 11

Ras proteins play the role of molecular switches by conformational change between a GTP and a GDP-bound state. In the yeast Saccharomyces cerevisiae, they are encoded by two partially redundant genes RAS1 and RAS2 with a different pattern of gene expression. They are essential for growth because they are required for the activation of the adenylate cyclase and thus the protein kinase A pathway. Other possible biological functions remains to be established. To achieve their biological function, they need to be processed after their synthesis, they are modified farnesylated and palmitoylated at their C-terminal end at their CaaX box. Palmitoylation, involved in membrane localization, is not essential for growth but required for glucose signaling whereas farnesylation appears to participate in adenylate cyclase activation. In the GTP-bound state ras proteins interact through their conserved effector domain with the adenylate cyclase, the product of the CYR1/CDC35 gene. They also interact with GTPase activating proteins encoded by IRA1 and IRA2. These proteins are specific for yeast ras. It has been shown that Ira2p recognizes specific residues of yeast ras not shared by mammalian ras. The interaction with the guanine nucleotide exchange factor (GEF) of the CDC25 family is enhanced by dominant negative mutations such as RAS2ala22. Using the two hybrid approach, we have showed the key role of position 80 in Ras2p and confirmed the involvement of the a2 helix, the other switching part of ras, in this interaction and the induced effect. As a counterpart we have identified positions in HGRF55 conserved in other GEF involved in ras interaction. The triggering elements of ras activation: the GEF Cdc25p and Sdc25p are limiting components of the ras system. Cdc25p is part of a multimolecular complex associated with the membrane. We have shown that it can form homodimers and heterodimers with Sdc25p. It is an unstable protein containing a cyclin destruction box. Therefore its activity on ras could be regulated by controlling its cellular content.
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PMID:[Ras proteins in Saccharomyces cerevisiae, their partners and their activation]. 925 49

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta- and epsilon-subunits of eIF2B, and eIF2B was shown to bind only to the beta-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta- or epsilon-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.
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PMID:Identification of interprotein interactions between the subunits of eukaryotic initiation factors eIF2 and eIF2B. 944 19

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
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PMID:GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor. 958 May 66

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca2+) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca2+ ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998) Science 280, 1082-1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca2+ and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca2+ and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.
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PMID:A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. 978 79

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.
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PMID:Regulation of RasGRP via a phorbol ester-responsive C1 domain. 981 87

cAMP (3',5' cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.
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PMID:A family of cAMP-binding proteins that directly activate Rap1. 985 55

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, showed that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras.GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.
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PMID:Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras. Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras. 985 97


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