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)

What do the recently determined crystal structures of 14-3-3 proteins and of a complex between part of the protein kinase Raf and the Ras-related protein Rap tell us about how 14-3-3 and Ras regulate the function of Raf?
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PMID:Protein-protein interactions. Putting the pieces together. 854 82

The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.
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PMID:Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. 860 12

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

Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1. Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms.
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PMID:Mechanisms regulating Raf-1 activity in signal transduction pathways. 860 83

Although Rafs play a central role in signal transduction, the mechanism(s) by which they become activated is poorly understood. Raf-1 activation is dependent on the protein's ability to bind Ras, but Ras binding is insufficient to activate Raf-1 tyrosine phosphorylation to this Ras-induced activation, in the absence of an over-expressed tyrosine kinase. We demonstrate that Raf-1 purified form Sf9 cells coinfected with baculovirus Ras but not Src could be inactivated by protein tyrosine phosphatase PTP-1B. 14-3-3 and Hsp90 proteins blocked both the tyrosine dephosphorylation and inactivation of Raf-1, suggesting that Raf-1 activity is phosphotyrosine dependent. In Ras-transformed NIH 3T3 cells, a minority of Raf-1 protein was membrane associated, but essentially all Raf-1 activity and Raf-1 phosphotyrosine fractionated with plasma membranes. Thus, the tyrosine-phosphorylated and active pool of Raf-1 constitute a membrane-localized subfraction which could also be inactivated with PTP-1B. By contrast, B-Raf has aspartic acid residues at positions homologous to those of the phosphorylated tyrosines (at 340 and 341) of Raf-1 and displays a high basal level of activity. B-Raf was not detectably tyrosine phosphorylated, membrane localized, or further activated upon Ras transformation, even though B-Raf has been shown to bind to Ras in vitro. We conclude that tyrosine phosphorylation is an essential component of the mechanism by which Ras activates Raf-1 kinase activity and that steady-state activated Ras is insufficient to activate B-Raf in vivo.
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PMID:Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. 862 47

The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.
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PMID:Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1. 863 18

Insulin-stimulated activity of Raf-1 kinase was examined in Rat-1 fibroblasts transfected with wild-type and mutant human insulin receptors. Insulin stimulated Raf-1 binding to p21Ras in HIRc (wild-type), delta CT (insulin receptor lacking a 43-amino acid C-terminal domain), and Y/F2 (tyrosine 1316 and 1322 replaced by phenylalanine) cells. Despite equal binding to p21Ras, the activity of Raf-1 kinase (measured by phosphorylation of its downstream substrate, mitogen-activated protein/extracellular receptor kinase (MEK) was significantly reduced in the delta CT cells. As an association of Raf-1 with p21Ras does not activate Raf-1 kinase, but merely targets Raf-1 to the plasma membrane, we examined the binding of Raf-1 to 14-3-3 proteins and to the insulin receptor itself. Raf-1 was detected in both 14-3-3 and insulin receptor immunoprecipitates. Association of Raf-1 with either 14-3-3 protein or insulin receptor was not influenced by insulin and was similar in all control and insulin-treated cell lines. These results indicate that the delta CT cells are deficient in stimulating Raf-1 activity despite normal binding of Raf-1 to p21Ras. Thus, an unidentified mechanism of Raf-1 activation at the plasma membrane must be impaired in these cells.
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PMID:Reduced phosphorylation of mitogen-activated protein kinase kinase in response to insulin in cells with truncated C-terminal domain of insulin receptor. 864 Nov 87

14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products. We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells. We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes. The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins.
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PMID:Activation-modulated association of 14-3-3 proteins with Cbl in T cells. 866 31

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.
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PMID:Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. 869 45

A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed.
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PMID:A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor. 875 86


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