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

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. Here, we investigated the functional activity of RGS3 and a truncated form of RGS3 on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses revealed the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) was shown recently to function as a negative regulator of pheromone signaling in yeast (Druey, K. M., Blumer, K. J., Kang, V. R., and Kehrl, J. H. (1996) Nature 379, 742-746). Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibited a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates were reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA showed no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 was impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity.
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PMID:A truncated form of RGS3 negatively regulates G protein-coupled receptor stimulation of adenylyl cyclase and phosphoinositide phospholipase C. 918 81

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.
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PMID:G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras. 923 98

Most mammalian cells have the capacity to migrate. When placed into culture, cells will generally display a set rate of basal, unstimulated locomotion. The cells will begin to move in one direction and, after some time, change directions resulting in a random walk. External stimuli can influence cell motility in several ways to either enhance or retard the rate of migration (chemokinesis), to change the average amount of cell migration observed before the cell turns (persistence), or to increase the directionality of movement by limiting the number of turns made by the cells. Several factors have been identified that stimulate cell movement, but the signaling mechanisms that mediate this induced cell movement have only recently begun to be studied. In this review, we discuss the signals that support the directional movement of fibroblasts and epithelial cells in response to chemoattractant gradients. The work will emphasize studies carried out by our laboratory and others on the stimulation of cell motility by the PDGF. These results indicate that at least two sets of signaling molecules cooperate to regulate cell motility in vivo. These include phospholipase C-gamma, phosphoinositide-3' kinase and the Ras-GTPase activating protein Ras-GAP. The first set are those which bind to the intracellular domain of the receptor tyrosine kinase and bring about the phosphorylation and/or activation of intracellular effectors proximal to the receptor. The second is a set of down-stream effectors that regulate either the rate of cell movement or the directionality of that movement depending on the cell type. These include Ras and the Ras-related GTPase Rac along with free phosphoinositides and calcium ions that regulate the actin polymerization machinery. Signals that mediate nuclear changes leading to cell proliferation, such as elements of the MAP kinase pathway, do not appear to play a role in PDGF-stimulated cell migration. Current work thus suggests that a coordinated spatial regulation of signaling elements that interact with the cell membrane and cytoskeleton but not necessarily with nuclear elements is the controlling mediator of directional cell motility.
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PMID:Signaling mechanisms in growth factor-stimulated cell motility. 925 9

Small G proteins transduce signals from plasma-membrane receptors to control a wide range of cellular functions. These proteins are clustered into distinct families but all act as molecular switches, active in their GTP-bound form but inactive when GDP-bound. The Rho family of G proteins, which includes Cdc42Hs, activate effectors involved in the regulation of cytoskeleton formation, cell proliferation and the JNK signalling pathway. G proteins generally have a low intrinsic GTPase hydrolytic activity but there are family-specific groups of GTPase-activating proteins (GAPs) that enhance the rate of GTP hydrolysis by up to 10(5) times. We report here the crystal structure of Cdc42Hs, with the non-hydrolysable GTP analogue GMPPNP, in complex with the GAP domain of p50rhoGAP at 2.7A resolution. In the complex Cdc42Hs interacts, mainly through its switch I and II regions, with a shallow pocket on rhoGAP which is lined with conserved residues. Arg 85 of rhoGAP interacts with the P-loop of Cdc42Hs, but from biochemical data and by analogy with the G-protein subunit G(i alpha1), we propose that it adopts a different conformation during the catalytic cycle which enables it to stabilize the transition state of the GTP-hydrolysis reaction.
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PMID:Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP. 926 6

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family led us to examine the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants are strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggested that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 has previously been demonstrated to stimulate the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE).
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PMID:Cooperative transformation of NIH3T3 cells by G alpha12 and Rac1. 926 13

Exposure of mammalian cells to stressful stimuli results in activation of the c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinases (SAPKs), a family of protein kinases related to mitogen-activated protein (MAP) kinase. JNK/SAPKs are activated by specific MAP kinase kinases (MKKs), one of which, MKK4/SEK1, has been characterized extensively. In Drosophila, the JNK/SAPK Basket (Bsk) and the MKK Hemipterous (Hep), are important for embryonic development. Loss of function of either gene inhibits dorsal closure, a morphogenetic movement in which the edges of the embryonic ectoderm move together over the amnioserosa. There is evidence that the Rho GTPases Rac and Cdc42 are also required for dorsal closure, suggesting that Rac or Cdc42 may regulate Hep and Bsk. We have identified MKK7, a murine homolog of Hep. MKK7 functionally rescues hep mutant flies. In fibroblasts, MKK7 is activated by stress and by the GTPase Rac1. MKK7 directly phosphorylates and activates JNK/SAPK. Thus, MKK7 is a homolog of hep and functions in a conserved signaling pathway involving JNK/SAPK and the GTPase Rac1.
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PMID:MKK7 is a stress-activated mitogen-activated protein kinase kinase functionally related to hemipterous. 931 5

During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. We have characterized two mechanisms that limit the extent of vulval induction. First, we found that gap-1 may directly inhibit the LET-60 Ras signaling pathway. We identified the gap-1 gene in a genetic screen for inhibitors of vulval induction. gap-1 is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit excess vulval induction. Second, we found that let-23 EGFR prevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1 signaling pathway. Using genetic mosaic analysis, we show that let-23 activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells.
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PMID:Inhibition of Caenorhabditis elegans vulval induction by gap-1 and by let-23 receptor tyrosine kinase. 933 33

During inflammation, P-selectin on activated platelets and endothelial cells initiates adhesion of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1). We investigated whether ligation of PSGL-1 also transmits signals into leukocytes. Neutrophils incubated with anti-PSGL-1 monoclonal antibodies, but not with Fab fragments of these antibodies, rapidly increased tyrosine phosphorylation of proteins with relative molecular masses of 105-120, 70-84, and 42-44 kDa. PSGL-1-dependent adhesion of neutrophils to P-selectin increased tyrosine phosphorylation of similarly sized proteins. Cytochalasin B did not prevent the tyrosine phosphorylation induced by ligation of PSGL-1, suggesting that an intact cytoskeleton is not required for signaling. Engagement of PSGL-1 activated the GTPase Ras through a mechanism that did not require tyrosine phosphorylation of PSGL-1 or association of the Shc.Grb2.Sos1 complex with PSGL-1. Engagement of PSGL-1 activated the 42-44-kDa extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinases through a pathway that required activation of the MAP kinase kinase. Ligation of PSGL-1 also stimulated secretion of interleukin-8. The tyrosine kinase inhibitor, genistein, blocked tyrosine phosphorylation and secretion of interleukin-8, whereas the MAP kinase kinase inhibitor PD98059 partially inhibited secretion of interleukin-8. Tyrosine phosphorylation stimulated through PSGL-1 on selectin-tethered leukocytes may propagate a signaling cascade that is integrated with signals generated by other mediators.
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PMID:Engagement of P-selectin glycoprotein ligand-1 enhances tyrosine phosphorylation and activates mitogen-activated protein kinases in human neutrophils. 935 45

Insulin-like growth factor-I (IGF-I) receptor plays an important role in normal cell cycle progression and tumor growth, and it is thought to be essential for cellular transformation. To test this hypothesis, we stably transfected a GTPase-deficient mutant human Galpha13, which is highly oncogenic when overexpressed in vitro, into R- fibroblasts derived from IGF-I receptor-deficient mice. Northern blots of multiple clones revealed the expression of a 1.8-kilobase pair mutant Galpha13 transcript in transfected cells, in addition to the 6-kilobase pair endogenous mRNA. The transfection resulted in a doubling of the expression of Galpha13 protein in these cells as assessed by Western blot analysis. The transforming ability of the mutant Galpha13 was tested using the soft agar assay. Nontransfected R- cells cultured with 10% fetal bovine serum failed to form colonies after 3 weeks. Most of the mutant Galpha13-expressing clones formed significant numbers of colonies (11-50 colonies/1000 cells plated). Overexpression of the IGF-I receptor enabled R- cells to form colonies (27 colonies), and co-transfection of the mutant Galpha13 caused a further increase in colony formation (117-153 colonies) in three of five clones analyzed. Apparently Galpha13 works through pathways other than mitogen-activated protein kinase and c-Jun N-terminal kinase in transforming R- cells, because their activities were not significantly altered by the mutant Galpha13 expression. These results demonstrate that Galpha13 can induce cellular transformation through pathways apparently independent of the IGF-I receptor and that activation of the IGF-I receptor signaling pathways, although not essential for the transforming phenotype, enhances the effect of other pathways.
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PMID:The constitutively active mutant Galpha13 transforms mouse fibroblast cells deficient in insulin-like growth factor-I receptor. 936 1

We have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.
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PMID:Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation. 939 39


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