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)

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

S49 cells expressed type 2 somatostatin receptors (sstr2) by immunoblotting. Analysis by reverse transcription and polymerase chain reaction (RT-PCR) methodologies showed that S49 cells express predominantly sstr2A and sstr2B mRNAs; other subtypes were either not detected, in the case of sstr1, sstr3, sstr4, or variably detected, in the case of sstr5. No mutations were present in S49 cells at codon 12, 13, or 61 of the N-, K-, or H-ras genes. Nevertheless, randomly growing S49 cells contained Raf-1 activity by specific immune complex kinase assays. Treatment of S49 cells with somatostatin transiently inactivated the basal activity of Raf-1, but not that of B-Raf. Addition of somatostatin plus guanyl-5'-yl imidodiphosphate (GMPPNP) to S49 membranes stimulated PTPase activity. The concentration dependence for stimulation of PTPase activity correlated with high affinity binding of [125I-Tyr11]somatostatin-14. Both the effect of somatostatin to stimulate PTPase activity and to inactivate Raf-1 were abrogated by PTx. PTPase activity stimulated by somatostatin plus GMPPNP was recovered in a peak of high apparent M(r) (670,000) after solubilisation with Triton X-100 and Superose 6 chromatography. Furthermore, addition of activated, brain G alpha i/o subunits to fractions from control membranes stimulated PTPase activity in the high M(r) peak. Thus, S49 membranes contain a G-protein regulated PTPase (PTPase-G), and PTPase-G in these cells may reside in a high molecular weight complex.
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PMID:S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ. 941 18

FKBP65 is a member of the FK506-binding protein class of immunophilins and is the only member reported to contain four peptidylprolyl cis-trans isomerase domains and an unrelated COOH-terminal domain. In this report, we show that the heat shock protein hsp90 and the serine/threonine protein kinase c-Raf-1 are components of FKBP65 immune complexes. The NH2-terminal regulatory domain of c-Raf-1 appears to be required for its interaction with FKBP65. Using GST-FKBP65 fusion protein and purified Raf proteins, we show that full-length FKBP65 can interact with c-Raf-1 but not B-Raf. The activation kinetics of c-Raf-1 after v-H-RasV12 injection of Xenopus oocytes appear to correlate with FKBP65/c-Raf-1 interaction, suggesting that FKBP65 may preferentially associate with forms of c-Raf-1 that are more posttranslationally modified. The interaction of FKBP65 with the c-Raf-heat shock protein 90 heterocomplex implicates this immunophilin in signal-transduction processes.
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PMID:The immunophilin FKBP65 forms an association with the serine/threonine kinase c-Raf-1. 943 87

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

Raf-1, A-Raf and B-Raf comprise a small family of highly conserved serine/threonine protein kinases, whose activities play a fundamental role in the control of proliferation and differentiation. The best studied family member, Raf-1, is expressed ubiquitously and constitutively, and its activity is regulated by post-translational mechanisms. Raf-1 can be activated by many signals that include growth factors, tumor promoters, inflammatory cytokines, calcium mobilization, DNA damaging agents, and oxygen radicals. Ras-mediated translocation of Raf-1 to the plasma membrane is a crucial step in its activation process, and is thought to facilitate phosphorylation by membrane-bound kinases. Raf-1 has also been reported to undergo intracellular redistribution following its activation: to the perinuclear space in murine NIH3T3 cells and rat hepatic Ito cells, and into the nucleus in gerbil hippocampal pyramidal cells and human MO7 leukemia cells. In contrast to the translocation to the plasma membrane, the perinuclear and/or nuclear translocation of Raf-1 has not been investigated in detail. In this paper, we report an examination of the subcellular localization of endogenous Raf-1 in a fibroblastic cell line (Rat-1) commonly used in transformation assays. Using the methods of cellular fractionation as well as in situ immunofluorescence, we show that no detectable movement of Raf-1 to the perinuclear or nuclear space can be observed. Tethering of activated Raf to the plasma membrane does not interfere with its transforming activity.
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PMID:Studies of perinuclear and nuclear translocation of the Raf-1 protein in rodent fibroblasts. 955 Oct 81

We studied the G protein alpha-subunit Galpha12 in various tissues and cell lines. Significant amounts of Galpha12 were detected by immunoblots in liver, chromaffin cells, RINm5F cells, 3T3-F442A cells, and preadipocytes, but not in adipocytes, sperm, kidney, NB2A cells, or brain. To study the role of Galpha12 in adipose tissue differentiation, the preadipocyte cell line 3T3-F442A was transfected with wild-type Galpha12 or a constitutively activated mutant of Galpha12. Stable expression of the activated mutant of Galpha12 stimulated cell growth and inhibited preadipocyte differentiation. In contrast, wild-type Galpha12 overexpression inhibited preadipocyte differentiation, without any effect on cell proliferation. The role of Galpah12 on the Raf/MEK/mitogen-activating protein kinase (MAPK) cascade was studied. In confluent preadipocytes, expression of the activated mutant of Galpha12 induced an increase in B-Raf expression, but no change in MAPK activity. Differentiation was associated with a decrease in MAPK activity in control 3T3-F442A cells. Wild-type Galpha12 overexpression prevented the decrease in MAPK activity and induced MEK1, but not B-Raf, expression. Moreover, the activated mutant of Galpha12 induced an increase in MAPK activity and in the expression of both MEK1 and B-Raf. These data indicate that the activated mutant of Galpha12 stimulates the proliferation of 3T3-F442A preadipocytes, possibly through an increase in B-Raf expression, independently of the MEK/MAPK pathway, but prevents differentiation, probably through an increase in MEK1 expression and MAPK activity.
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PMID:Mutant alpha-subunit of the G protein G12 activates proliferation and inhibits differentiation of 3T3-F442A preadipocytes. 960 99

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

Neurotransmitter biosynthesis is regulated by environmental stimuli, which transmit intracellular signals via second messengers and protein kinase pathways. For the catecholamine biosynthetic enzymes, dopamine beta-hydroxylase and tyrosine hydroxylase, regulation of gene expression by cyclic AMP, diacyl glycerol, and Ca2+ leads to increased neurotransmitter biosynthesis. In this report, we demonstrate that the cAMP-mediated regulation of transcription from the dopamine beta-hydroxylase promoter is mediated by the AP1 proteins c-Fos, c-Jun, and JunD. Following treatment of cultured cells with cAMP, protein complexes bound to the dopamine beta-hydroxylase AP1/cAMP response element element change from consisting of c-Jun and JunD to include c-Fos, c-Jun, and JunD. The homeodomain protein Arix is also a component of this DNA-protein complex, binding to the adjacent homeodomain recognition sites. Transfection of a dominant negative JunD expression plasmid inhibits cAMP-mediated expression of the dopamine beta-hydroxylase promoter construct in PC12 and CATH.a cells. In addition to the role of c-Fos in regulating dopamine beta-hydroxylase gene expression in response to cAMP, a second pathway, involving Rap1/B-Raf is involved. These experiments illustrate an unusual divergence of cAMP-dependent protein kinase signaling through multiple pathways that then reconverge on a single element in the dopamine beta-hydroxylase promoter to elicit activation of gene expression.
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PMID:AP1 proteins mediate the cAMP response of the dopamine beta-hydroxylase gene. 972 25

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


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