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 28,634 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Whereas the molecular and cellular mechanisms governing astrocytoma invasion remain poorly understood, evidence in other cell systems has implicated a role for the Rho-GTPases in cell motility and invasion. Here, we examine how the inhibition or activation of Rho-kinase (ROCK) affects astrocytoma morphology, motility, and invasion. ROCK was inhibited in astrocytoma cells by using 5 to 100 mumol/L of Y27632 or by expressing the dominant-negative ROCK mutant, RB/PH TT. ROCK activation was achieved by expressing a constitutively active mutant, CAT. ROCK inhibition led to morphologic and cytoskeletal alterations characterized by an increase in the number and length of cell processes, increased membrane ruffling, and collapse of actin stress fibers. Using two-dimensional radial migration and Boyden chamber assays, we show that astrocytoma migration and invasion were increased at least 2-fold by ROCK inhibition. On the contrary, ROCK activation significantly inhibited migration and invasion of astrocytoma cells. Furthermore, using a Rac-GTP pull-down assay, we show that Rac1 is activated as a consequence of ROCK inhibition. Finally, we show that treatment of astrocytoma cells with small interfering RNA duplexes specific for Rac1-reversed stellation, prevented membrane ruffling formation and abrogated the increased motility observed following treatment with Y27632. Our data show that Rac1 plays a major role in astrocytoma morphology, motility, and invasion. These findings warrant further investigation to determine precisely how the modulation of Rac1 and ROCK can be exploited to inhibit glioma invasion.
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PMID:Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1. 1620 49

In epithelial cell lines, apical but not basolateral clathrin-mediated endocytosis has been shown to be affected by actin-disrupting drugs. Using electron and fluorescence microscopy, as well as biochemical assays, we show that the amount of actin dedicated to endocytosis is limiting at the apical surface of epithelia. In part, this contributes to the low basal rate of clathrin-dependent endocytosis observed at this epithelial surface. ARF6 in its GTP-bound state triggers the recruitment of actin from the cell cortex to the clathrin-coated pit to enable dynamin-dependent endocytosis. In addition, we show that perturbation of the apical endocytic system by expression of a clathrin heavy-chain mutant results in the collapse of microvilli. This phenotype was completely reversed by the expression of an ARF6-GTP-locked mutant. These observations indicate that concomitant to actin recruitment, the apical clathrin endocytic system is deeply involved in the morphology of the apical plasma membrane.
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PMID:Actin is required for endocytosis at the apical surface of Madin-Darby canine kidney cells where ARF6 and clathrin regulate the actin cytoskeleton. 1625 60

Eph receptor tyrosine kinases regulate the spatial organization of cells within tissues. Central to this function is their ability to modulate cell shape and movement in response to stimulation by the ephrin ligands. The EphB2 receptor was reported to inhibit cell-matrix adhesion by phosphorylating tyrosine 66 in the effector domain of R-Ras, a Ras family protein known to regulate cell adhesion and motility. Here, we further characterize the role of R-Ras downstream of both EphA and EphB receptors. Our data show that besides inhibiting R-Ras function through phosphorylation, Eph receptors can reduce R-Ras activity through the GTPase-activating protein, p120RasGAP. By using R-Ras mutants that cannot be inactivated by p120RasGAP and/or cannot be phosphorylated at tyrosine 66, we show that the two forms of R-Ras negative regulation - through increased GTP hydrolysis and phosphorylation - differentially contribute to various ephrin-mediated responses. Retraction of the COS cell periphery depends only on R-Ras inactivation through p120RasGAP. By contrast, both reduced R-Ras GTP levels and tyrosine 66 phosphorylation contribute to the ephrin inhibitory effects on COS cell migration and to ephrin-dependent growth cone collapse in primary neurons. Therefore, Eph receptors can regulate R-Ras in two different ways to achieve cell repulsion.
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PMID:Eph receptors inactivate R-Ras through different mechanisms to achieve cell repulsion. 1652 85

Neuronal outgrowth is a fundamental process for normal development of the nervous system. Despite recent advances, the molecular mechanisms governing neuronal motility are still poorly understood. To provide insight into the intracellular signaling mechanisms required for neuronal outgrowth, we have characterized the effects of a compound previously identified for its anti-motility effects on transformed cells. We show that this compound, motuporamine C, acts as a robust inhibitor of chick neurite outgrowth in a dose-dependent fashion. Furthermore, in the presence of motuporamine C, growth cone collapse is observed, followed by neurite retraction. After removal, growth cones re-extend lamellipodial and filopodial processes and re-establish motility. Neurons exposed to motuporamine C exhibit a significant upregulation of active Rho-GTP. Additionally, effector-blocking experiments using Rho and Rho-associated kinase inhibitors indicate that the Rho pathway plays a critical role in motuporamine C-mediated growth cone collapse. Thus, we have characterized a novel anti-motility compound that has a robust inhibitory effect on neuronal outgrowth and involves signaling through the Rho-Rho kinase collapse pathway. Due to these robust effects, motuporamine C may serve as a valuable tool in further examining the intracellular mechanisms associated with growth cone motility.
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PMID:The anti-invasive compound motuporamine C is a robust stimulator of neuronal growth cone collapse. 1656 36

Endocytosis of Eph receptors is critical for a number of biological processes, including modulating axon growth cone collapse response and regulating cell surface levels of receptor in epithelial cells. In particular, ephrin-A ligand stimulation of tumor cells induces EphA2 receptor internalization and degradation, a process that has been explored as a means to reduce tumor malignancy. However, the mechanism and regulation of ligand-induced Eph receptor internalization are not well understood. Here we show that SHIP2 (Src homology 2 domain-containing phosphoinositide 5-phosphatase 2) is recruited to activated EphA2 via a heterotypic sterile alpha motif (SAM)-SAM domain interaction, leading to regulation of EphA2 internalization. Overexpression of SHIP2 inhibits EphA2 receptor endocytosis, whereas suppression of SHIP2 expression by small interfering RNA-mediated gene silencing promotes ligand-induced EphA2 internalization and degradation. SHIP2 regulates EphA2 endocytosis via phosphatidylinositol 3-kinase-dependent Rac1 activation. Phosphatidylinositol 3,4,5-trisphosphate levels are significantly elevated in SHIP2 knockdown cells, phosphatidylinositol 3-kinase inhibitor decreases phosphatidylinositol 3,4,5-trisphosphate levels and suppresses increased EphA2 endocytosis. Ephrin-A1 stimulation activates Rac1 GTPase, and the Rac1-GTP levels are further increased in SHIP2 knockdown cells. A dominant negative Rac1 GTPase effectively inhibited ephrin-A1-induced EphA2 endocytosis. Together, our findings provide evidence that recruitment of SHIP2 to EphA2 attenuates a positive signal to receptor endocytosis mediated by phosphatidylinositol 3-kinase and Rac1 GTPase.
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PMID:Regulation of EphA2 receptor endocytosis by SHIP2 lipid phosphatase via phosphatidylinositol 3-Kinase-dependent Rac1 activation. 1713 40

Gem is a protein of the Ras superfamily that plays a role in regulating voltage-gated Ca2+ channels and cytoskeletal reorganization. We now report that GTP-bound Gem interacts with the membrane-cytoskeleton linker protein Ezrin in its active state, and that Gem binds to active Ezrin in cells. The coexpression of Gem and Ezrin induces cell elongation accompanied by the disappearance of actin stress fibers and collapse of most focal adhesions. The same morphological effect is elicited when cells expressing Gem alone are stimulated with serum and requires the expression of ERM proteins. We show that endogenous Gem down-regulates the level of active RhoA and actin stress fibers. The effects of Gem downstream of Rho, i.e., ERM phosphorylation as well as disappearance of actin stress fibers and most focal adhesions, require the Rho-GAP partner of Gem, Gmip, a protein that is enriched in membranes under conditions in which Gem induced cell elongation. Our results suggest that Gem binds active Ezrin at the plasma membrane-cytoskeleton interface and acts via the Rho-GAP protein Gmip to down-regulate the processes dependent on the Rho pathway.
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PMID:Gem associates with Ezrin and acts via the Rho-GAP protein Gmip to down-regulate the Rho pathway. 1726 93

This chapter addresses, from a molecular structural perspective gained from examination of x-ray crystallographic and biochemical data, the mechanisms by which GTP-bound Galpha subunits of heterotrimeric G proteins recognize and regulate effectors. The mechanism of GTP hydrolysis by Galpha and rate acceleration by GAPs are also considered. The effector recognition site in all Galpha homologues is formed almost entirely of the residues extending from the C-terminal half of alpha2 (Switch II) together with the alpha3 helix and its junction with the beta5 strand. Effector binding does not induce substantial changes in the structure of Galpha*GTP. Effectors are structurally diverse. Different effectors may recognize distinct subsets of effector-binding residues of the same Galpha protein. Specificity may also be conferred by differences in the main chain conformation of effector-binding regions of Galpha subunits. Several Galpha regulatory mechanisms are operative. In the regulation of GMP phospodiesterase, Galphat sequesters an inhibitory subunit. Galphas is an allosteric activator and inhibitor of adenylyl cyclase, and Galphai is an allosteric inhibitor. Galphaq does not appear to regulate GRK, but is rather sequestered by it. GTP hydrolysis terminates the signaling state of Galpha. The binding energy of GTP that is used to stabilize the Galpha:effector complex is dissipated in this reaction. Chemical steps of GTP hydrolysis, specifically, formation of a dissociative transition state, is rate limiting in Ras, a model G protein GTPase, even in the presence of a GAP; however, the energy of enzyme reorganization to produce a catalytically active conformation appears to be substantial. It is possible that the collapse of the switch regions, associated with Galpha deactivation, also encounters a kinetic barrier, and is coupled to product (Pi) release or an event preceding formation of the GDP*Pi complex. Evidence for a catalytic intermediate, possibly metaphosphate, is discussed. Galpha GAPs, whether exogenous proteins or effector-linked domains, bind to a discrete locus of Galpha that is composed of Switch I and the N-terminus of Switch II. This site is immediately adjacent to, but does not substantially overlap, the Galpha effector binding site. Interactions of effectors and exogenous GAPs with Galpha proteins can be synergistic or antagonistic, mediated by allosteric interactions among the three molecules. Unlike GAPs for small GTPases, Galpha GAPs supply no catalytic residues, but rather appear to reduce the activation energy for catalytic activation of the Galpha catalytic site.
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PMID:Structural basis of effector regulation and signal termination in heterotrimeric Galpha proteins. 1785 54

Mitotic Aurora-A kinase was found to be required for formation of bipolar spindle, ensuring accurate chromosome segregation in mitosis. Recently, Aurora-A was shown to promote Ran-GTP-induced spindle formation and astral microtubule development. Here, by selective immunodepletion, we showed that Aurora-A was required for centrosome- but not Ran-GTP-induced astral microtubule formation in Xenopus egg extracts. Aurora-A enhanced microtubule polymerization in both centrosome- and Ran-GTP-induced aster assemblies: shortening the timing of aster assembly and increasing the aster size. Indeed, adding of Aurora-A protein alone induced microtubule clustering, which was abrogated by Aurora kinase inhibitory small molecule ZM447439. In addition, we showed that Aurora-A was indispensable for Ran-GTP-induced bipolar spindle formation. Inhibition of Aurora-A activity by adding of kinase inactive dominant mutant led to spindle collapse and formation of monopolar spindle whereas minus-end motor protein dynein/dynactin inhibitor p50/dynamitin rescued the bipolar structure. Lastly, we revealed that Aurora-A was necessary for microtubule poleward flux and this requirement depended on kinase activity. Thus, we showed that Aurora-A promoted microtubule polymerization and maintained microtubule flux in ensuring proper bipolar spindle assembly.
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PMID:Requirement of aurora-A kinase in astral microtubule polymerization and spindle microtubule flux. 1841 60

Class three semaphorins (SEMAs) were originally shown to be mediators of axon guidance that repelled axons and collapsed growth cones, but it is now evident that SEMA3F, for example, has similar effects on tumor cells and endothelial cells (EC). In both human U87MG glioma cells and human umbilical vein EC, SEMA3F induced rapid cytoskeletal collapse, suppressed cell contractility, decreased phosphorylation of cofilin, and inhibited cell migration in culture. Analysis of the signaling pathways showed that SEMA3F formed a complex with NRP2 (neuropilin-2) and plexin A1. These interactions eventually led to inactivation of the small GTPase, RhoA, which is necessary for stress fiber formation and cytoskeleton integrity. A novel upstream RhoA mediator was shown to be ABL2, also known as ARG, a membrane-anchored nonreceptor tyrosine kinase. Within minutes after the addition of SEMA3F, ABL2 directly bound plexin A1 but not to a plexin A1 mutant lacking the cytoplasmic domain. In addition, ABL2 phosphorylated and thereby activated p190RhoGAP, which inactivated RhoA (GTP to GDP), resulting in cytoskeleton collapse and inhibition of cell migration. On the other hand, cells overexpressing an ABL2 inactive kinase mutant or treated with ABL2 small interfering RNA did not inactivate RhoA. Cells treated with p190RhoGAP small interfering RNA also did not inactivate RhoA. Together, these results suggested that ABL2/ARG is a novel mediator of SEMA3F-induced RhoA inactivation and collapsing activity.
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PMID:ABL2/ARG tyrosine kinase mediates SEMA3F-induced RhoA inactivation and cytoskeleton collapse in human glioma cells. 1866 May 2

The Rac GTPase regulates Rho signaling in a broad range of physiological settings and in oncogenic transformation [1-3]. Here, we report a novel mechanism by which crosstalk between Rac and Rho GTPases is achieved. Activated Rac1 binds directly to p190B Rho GTPase-activating protein (RhoGAP), a major modulator of Rho signaling. p190B colocalizes with constitutively active Rac1 in membrane ruffles. Moreover, activated Rac1 is sufficient to recruit p190B into a detergent-insoluble membrane fraction, a process that is accompanied by a decrease in GTP-bound RhoA from membranes. p190B is recruited to the plasma membrane in response to integrin engagement [4]. We demonstrate that collagen type I, a potent inducer of Rac1-dependent cell motility in HeLa cells, counteracts cytoskeletal collapse resulting from overexpression of wild-type p190B, but not that resulting from overexpression of a p190B mutant specifically lacking the Rac1-binding sequence. Furthermore, this p190B mutant exhibits dramatically enhanced RhoGAP activity, consistent with a model whereby binding of Rac1 relieves autoinhibition of p190B RhoGAP function. Collectively, these observations establish that activated Rac1, through direct interaction with p190B, modulates subcellular RhoGAP localization and activity, thereby providing a novel mechanism for Rac control of Rho signaling in a broad range of physiological processes.
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PMID:Coordination of Rho and Rac GTPase function via p190B RhoGAP. 1894 7


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