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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)

Ras proteins play critical roles in regulating cell growth and differentiation, and mutated Ras genes are expressed in a variety of human cancers. Consequently, much interest has centered on the binding partners of Ras, including the Ras-binding domain (RBD) of Raf kinase. Here evidence is presented that domains homologous to the Raf RBD are present in tandem in RGS12, RGS14 and LOCO, and singly in molecules similar to mouse Tiam-1. In addition, RGS12, RGS14 and LOCO are shown to contain single "LGN motifs" that are guanine nucleotide exchange factors specific for the alpha-subunit of G proteins. These findings indicate "cross-talk" interactions between signalling pathways involving Ras and Rap and pathways involving Rho, Rac and G alpha GTPases.
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PMID:Raf-like Ras/Rap-binding domains in RGS12- and still-life-like signalling proteins. 1060 4

Localized disassembly of cortical F-actin has long been considered necessary for facilitation of exocytosis. Exposure of permeabilized mast cells to calcium/ATP induces cortical F-actin disassembly (calmodulin-dependent) and secretion (calmodulin-independent). The delay in the onset of secretion is characteristic for the calcium/ATP response and is abolished by GTP. Here we report that a constitutively active mutant of Rho (V14RhoA) enhanced both secretion and cortical F-actin disassembly. In addition, V14RhoA mimicked GTP by abolishing the delay in secretion. Inhibition of Rho by C3 transferase prevented both secretion ( approximately 80%) and F-actin disassembly (approximately 20%). Thus, both Rho GTPase and calcium/calmodulin contribute to the control of cortical F-actin disassembly. Stabilization of actin filaments by high concentrations of phalloidin or by a calmodulin-inhibitory peptide (based on the calmodulin-binding domain of myosin light chain kinase) did not affect the extent of secretion or the secretion-enhancing effects of V14RhoA. These results further support the existence of divergent, Rho-dependent, pathways regulating actin and exocytosis. Furthermore, compound Y-27632, a specific inhibitor of Rho-associated protein kinase (p160(ROCK)), attenuated the Rho-induced loss of cortical F-actin without affecting secretion. A model is presented in which Rho regulates secretion and cortical F-actin in a manner dependent on and/or synergistic with calcium.
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PMID:Rho controls cortical F-actin disassembly in addition to, but independently of, secretion in mast cells. 1060 85

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.
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PMID:RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism. 1064 58

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.
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PMID:Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop. 1065 53

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.
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PMID:Nuclear sequestration of the exchange factor Cdc24 by Far1 regulates cell polarity during yeast mating. 1065 92

Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant. Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.
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PMID:Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. 1069 72

We examined how (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632), an inhibitor of Rho-associated coiled coil-forming protein kinase (ROCK I) and Rho kinase (ROCK II), affects the relaxant responses to beta(2)-adrenoceptor agonists in bovine tracheal smooth muscle preparations precontracted with methacholine. Y-27632 (0.3-30 microM) caused a concentration-dependent attenuation of precontraction with methacholine (0.3-3 microM). Pretreatment with Y-27632 (1 microM) significantly (P<0.05) augmented salbutamol (0.3-100 nM) and terbutaline (0.3 nM-1 microM)-induced relaxations. These results suggest that the ROCK inhibitor could become a new type bronchodilator and its combination with beta(2)-adrenoceptor agonists may become a novel strategy for the long-term treatment of asthma.
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PMID:Y-27632 potentiates relaxant effects of beta 2-adrenoceptor agonists in bovine tracheal smooth muscle. 1068 2

Angiogenesis consists of multistep pathways such as the degradation of the matrix, proliferation of the endothelial cells, motility of the endothelial cells, formation of the cord structure and network formation of microvessels. The small GTPase Rho participates in cell motility through actin fiber polymerization. The role of the small GTPase Rho signal transduction pathway in regulating angiogenesis, however, is still unknown. In this study, we investigated the role of the small GTPase Rho signal transduction pathway in angiogenesis in vitro and in vivo using the exoenzyme, Clostridium botulinum C3 transferase, which specifically suppresses Rho and a compound, Y-27632, which suppresses p160ROCK (Rho-associated coiled-coil containing protein kinase). In this paper, we showed that the small GTPase Rho-p160ROCK signal transduction pathway played an important role in angiogenesis both in vitro and in vivo. These results suggest that inhibition of the small GTPase Rho signal transduction pathway by the p160ROCK inhibitor could be a possible new strategy for angiogenic diseases.
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PMID:The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo. 1070 6

Anchorage removal like growth factor removal induces apoptosis. In the present study we have characterized signaling pathways that can prevent this cell death using a highly growth factor- and anchorage-dependent line of lung fibroblasts (CCL39). After anchorage removal from exponentially growing cells, annexin V-FITC labeling can be detected after 8 h. Apoptosis was confirmed by analysis of sub-G1 DNA content and Western blotting of the caspase substrate poly (ADP-ribose) polymerase. Growth factor withdrawal accelerates and potentiates suspension-induced cell death. Activation of Raf-1 kinase in suspension cultures of CCL39 or Madin-Darby canine kidney cells stably expressing an estrogen-inducible activated-Raf-1 construct (DeltaRaf-1:ER) suppresses apoptosis induced by growth factor and/or anchorage removal. This protective effect appears to be mediated by the Raf, mitogen- or extracellular signal-regulated kinase kinase (MEK), and mitogen-activated protein kinase module because it is sensitive to pharmacological inhibition of MEK-1 and it can be mimicked by expression of constitutively active MEK-1 in CCL39 cells. Finally, apoptosis induced by disruption of the actin cytoskeleton with the Rho-directed toxin B (Clostridium difficile) is prevented by activation of the DeltaRaf-1:ER chimeric construct. These findings highlight the ability of p42/p44 mitogen-activated protein kinase to generate survival signals that counteract cell death induced by loss of matrix contact, cytoskeletal integrity, and extracellular mitogenic factors.
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PMID:The p42/p44 MAP kinase pathway prevents apoptosis induced by anchorage and serum removal. 1071 23

It has been well known that cell-anchorage and the cell cytoskeleton are deeply involved in the regulation of cell proliferation and cell cycle. However, the precise molecular mechanism involved in cell-anchorage and the cell cytoskeleton have remained be to elucidated. The recent great volume of information regarding cell cycle regulators such as cyclin, cyclin-dependent kinases (CDKs) and CDK inhibitors (CKI) has facilitated the understanding of the cell cycle in mammalian cells. In this review, we will focus on these cell cycle regulators to discuss the regulation of cell proliferation controlled by cell-anchorage and the cytoskeleton, and especially the roles of Rho family GTPases.
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PMID:Cell-anchorage, cell cytoskeleton, and Rho-GTPase family in regulation of cell cycle progression. 1074 Aug 11


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