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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.
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PMID:Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells. 915 3

The catalytic domains of the Raf family of protein kinases (deltaRaf) differ in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to significantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these differences in amino acid sequence influence the relative catalytic activity of the Raf kinase domains we constructed forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 that encode either aspartic acid [DD], phenylalanine [FF] or tyrosine [YY] at these positions. These proteins were expressed both in mammalian cells as fusions with the hormone binding domain of the estrogen receptor and as epitope-tagged proteins in Sf9 insect cells to test their oncogenic and catalytic potentials. When expressed in Rat1 or 3T3 cells in the presence of hormone all of the deltaRaf-1:ER and deltaA-Raf:ER proteins were transforming with the exception of the [FF] form of deltaA-Raf. In general the [DD] forms of the deltaRaf-1:ER and deltaA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of deltaA-Raf:ER and deltaRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the deltaB-Raf:ER proteins were highly transforming and deltaB-Raf catalytic activity was largely unaffected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.
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PMID:Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1. 928 56

We conditionally overexpressed a MEK1 mutant that contains triple mutations in the regulatory and kinase domains, and investigated its effects on the MAP kinase cascade in Swiss 3T3 cells. Expression of the mutant produced a 60% blockade in MAP kinase activity. However, only a modest blockade in DNA synthesis was observed, without any reductions in the phosphorylation of two proteins known to be substrates of MAP kinase. Moreover, the overexpression of MEK1(3A) failed to block endogenous MEK1 activation, although MEK1(3A) formed complexes with both c-Raf and B-Raf as well as p42/p44 MAPK. These results suggest that there may be multiple biochemical inputs into the MEK/MAPK pathway.
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PMID:Inducible expression of a mutant form of MEK1 in Swiss 3T3 cells. 936 Nov 91

Bailie et al. [In Vitro Cell Dev. Biol. (1992) 28A, 621-624] reported that primary cultures of rat hepatocytes possess low affinity binding sites for nerve growth factor (NGF). NGF treatment of primary cultures of rat hepatocytes with a maximally effective concentration of NGF (20 ng/ml, 0.8 nM) caused acute phasic activation of Raf-1 and p42(MAPkinase), and a smaller sustained activation of B-Raf. The transient increase in Raf-1 and p42(MAPkinase) activity returned to baseline within approximately 30 min. NGF treatment of hepatocytes did not induce expression of cyclin dependent kinase (cdk) inhibitor proteins, but instead stimulated cdk2 activity and increased [3H]thymidine incorporation into DNA. In contrast to hepatocytes, NGF treatment of PC12 pheochromocytoma cells caused large sustained activations of B-Raf and p42(MAPkinase), and a lower phasic activation of Raf-1. The sustained activations of B-Raf and p42(MAPkinase) were for more than 5 h. Treatment of PC12 cells with NGF increased p21(Cip1/WAF-1) expression, reduced cdk2 activity and inhibited DNA synthesis, the opposite to the effects of NGF treatment of hepatocytes. However when p42(MAPkinase) was chronically activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased. Equally, treatment of hepatocytes with 50 mM ethanol elevated the basal activity of p42(MAPkinase) and temporally extended the ability of NGF treatment to activate p42(MAPkinase). Ethanol and NGF co-treatment increased expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins and decreased hepatocyte DNA synthesis. These data demonstrate that NGF can cause either acute/phasic or sustained activation of the MAP kinase cascade in different cell types. Acute activation of the MAP kinase cascade correlated with increased DNA synthesis. In contrast, sustained activation of the MAP kinase cascade correlated with increased expression of cdk inhibitor proteins, a reduction in cdk activity, and an inhibition of DNA synthesis. These data suggest a general mechanism exists where acute activation of the MAP kinase cascade promotes G1 progression/S phase entry and that chronic activation of the MAP kinase cascade inhibits this process.
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PMID:The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. 949 19

Activation of mitogen-activated protein (MAP) kinase (also known as extracellular-signal-regulated kinase, or ERK) by growth factors can trigger either cell growth or differentiation. The intracellular signals that couple growth factors to MAP kinase may determine the different effects of growth factors: for example, transient activation of MAP kinase by epidermal growth factor stimulates proliferation of PC12 cells, whereas they differentiate in response to nerve growth factor, which acts partly by inducing a sustained activation of MAP kinase. Here we show that activation of MAP kinase by nerve growth factor involves two distinct pathways: the initial activation of MAP kinase requires the small G protein Ras, but its activation is sustained by the small G protein Rap1. Rap1 is activated by CRK adaptor proteins and the guanine-nucleotide-exchange factor C3G, and forms a stable complex with B-Raf, an activator of MAP kinase. Rap1 is required for at least two indices of neuronal differentiation by nerve growth factor: electrical excitability and the induction of neuron-specific genes. We propose that the activation of Rap1 by C3G represents a common mechanism to induce sustained activation of the MAP kinase cascade in cells that express B-Raf.
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PMID:Rap1 mediates sustained MAP kinase activation induced by nerve growth factor. 956 Jan 48

Protein kinases of the Raf family act as signal-transducing elements downstream of activated cell surface receptors and are involved in the regulation of proliferation, differentiation, and cell survival. Whereas the role of c-Raf-1 as a mitogen-activated protein/extracellular signal-regulated kinase activator within the mitogenic cascade is well established, less is known about the mammalian Raf isoforms A-Raf and B-Raf. Here we report that B-Raf binds to PA28alpha, one of two subunits of the 11S regulator of proteasomes. PA28alpha was isolated as a B-Raf-binding protein in a yeast two-hybrid screen of a PC12 cDNA library. Both proteins can be coimmunoprecipitated after transient expression in 293 cells. No association could be found between PA28alpha and A-Raf or c-Raf-1. B-Raf binds to a region in PA28alpha that is important for its proteasome-activating function.
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PMID:Interaction between the protein kinase B-Raf and the alpha-subunit of the 11S proteasome regulator. 967 60

Upon binding to its G protein-coupled transmembrane receptors, the actions of PGF2alpha on the corpus luteum are initiated by the phospholipase C/diacylglycerol-inositol 1,4,5-trisphosphate (InsP3)/Ca2+-protein kinase C (PKC) pathway. However, little is known about the downstream intracellular signaling events that can lead to transcriptional activation in response to PGF2alpha. The present study was conducted to examine the involvement of the mitogen-activated protein kinase (MAPK) signaling cascade in the corpus luteum. Three isoforms of the Raf family of oncoprotein kinases (A-Raf, B-Raf, and Raf-1 or c-Raf) were detected in bovine luteal cells. Raf-1 and B-Raf, but not A-Raf, were activated by PGF2alpha (1 microM) and the pharmacological PKC activator phorbol myristate acetate (PMA, 20 nM). Kinetic analysis revealed that PGF2alpha rapidly and transiently activated Raf-1. In vitro protein kinase assays demonstrated that activation of Raf-1 and B-Raf resulted in the phosphorylation and activation of MAPK kinase (MEK1), which subsequently phosphorylated p42mapk. As determined by hyperphosphorylation, tyrosine phosphorylation, and enzymatic activity, p42mapk and p44mapk were rapidly and transiently activated by both PGF2alpha (1 microM) and PMA (20 nM). Additionally, both PGF2alpha (1 microM) and PMA (20 nM) stimulated phosphorylation of Raf-1, MEK1, and p42mapk in 32P-labeled cells. Our data demonstrate that PGF2alpha activates the Raf/MEK1/p42/44mapk signaling cascade in bovine luteal cells and that the actions of PGF2alpha are mimicked by the PKC activator PMA. Activation of the Raf/MEK1/MAPK signaling cascade by PGF2alpha in luteal cells provides a mechanism to transduce signals initiated by PGF2alpha receptors on the cell surface into the nucleus. Activation of the Raf/MEK1/MAPK signaling cascade may be associated with transcriptional activation of luteal genes possessing activator protein-1-binding sites.
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PMID:Prostaglandin F2alpha stimulates the Raf/MEK1/mitogen-activated protein kinase signaling cascade in bovine luteal cells. 972 43

Rap1 and Ras are homologous GTPases that are implicated in cell proliferation and differentiation. At present, little is known about the regulation of Rap1 activity. Using a recently developed assay with activation-specific probes, we found increased activity of endogenous Rap1 in NIH3T3 cells after stimulation with the neuropeptide growth factor bombesin in a concentration- and time-dependent manner. The activity of endogenous Ras was unaffected. Analysis of putative effectors showed no activation of c-Raf-1 or B-Raf after bombesin stimulation. However, MAPK/Erk-phosphorylation and the proliferation rate was increased. In addition, Rap1 was activated during cell adhesion to coated and uncoated tissue culture plates, as well as in response to various mitogens. Surprisingly, the basal Rap1 activity was observed to be cell density-dependent, with low levels when cells were reaching confluency. The results suggest that Rap1 acts as an important mediator of mitogenic signals distinct to Ras activation.
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PMID:Activity of Rap1 is regulated by bombesin, cell adhesion, and cell density in NIH3T3 fibroblasts. 973 13

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.
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PMID:Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf. 973 1

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


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