Protein Kinase Cε Actin-binding Site Is Important for Neurite Outgrowth during Neuronal Differentiation

We have previously shown that protein kinase Cepsilon (PKCepsilon) induces neurite outgrowth via its regulatory domain and independently of its kinase activity. This study aimed at identifying mechanisms regulating PKCepsilon-mediated neurite induction. We show an increased association of PKCepsilon to the cytoskeleton during neuronal differentiation. Furthermore, neurite induction by overexpression of full-length PKCepsilon is suppressed if serum is removed from the cultures or if an actin-binding site is deleted from the protein. A peptide corresponding to the PKCepsilon actin-binding site suppresses neurite outgrowth during neuronal differentiation and outgrowth elicited by PKCepsilon overexpression. Neither serum removal, deletion of the actin-binding site, nor introduction of the peptide affects neurite induction by the isolated regulatory domain. Membrane targeting by myristoylation renders full-length PKCepsilon independent of both serum and the actin-binding site, and PKCepsilon colocalized with F-actin at the cortical cytoskeleton during neurite outgrowth. These results demonstrate that the actin-binding site is of importance for signals acting on PKCepsilon in a pathway leading to neurite outgrowth. Localization of PKCepsilon to the plasma membrane and/or the cortical cytoskeleton is conceivably important for its effect on neurite outgrowth.     

Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms

We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.     

Control of neurite outgrowth by RhoA inactivation

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.     

p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation

The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Characterization of a brain-specific Rho GTPase-activating protein,p200RhoGAP

The Rho GTPase-activating proteins (RhoGAPs) are a family of multifunctional molecules that transduce diverse intracellular signals by regulating Rho GTPase activities. A novel RhoGAP family member, p200RhoGAP, is cloned in human and mouse. The murine p200RhoGAP shares 86% sequence identity with the human homolog. In addition to a conserved RhoGAP domain at the N terminus, multiple proline-rich motifs are found in the C-terminal region of the molecules. Northern blot analysis revealed a brain-specific expression pattern of p200RhoGAP. The RhoGAP domain of p200RhoGAP stimulated the GTPase activities of Rac1 and RhoA in vitro and in vivo, and the conserved catalytic arginine residue (Arg-58) contributed to the GAP activity. Expression of the RhoGAP domain of p200RhoGAP in Swiss 3T3 fibroblasts inhibited actin stress fiber formation stimulated by lysophosphatidic acid and platelet-derived growth factor-induced membrane ruffling but not Bradykinin-induced filopodia formation. Endogenous p200RhoGAP was localized to cortical actin in naive N1E-115 neuroblastoma cells and to the edges of extended neurites of differentiated N1E-115 cells. Transient expression of the RhoGAP domain and the full-length molecule, but not the catalytic arginine mutants, readily induced a differentiation phenotype in N1E-115 cells. Finally, p200RhoGAP was capable of binding to the Src homology 3 domains of Src, Crk, and phospholipase Cgamma in vitro and became tyrosine-phosphorylated upon association with activated Src in cells. These results suggest that p200RhoGAP is involved in the regulation of neurite outgrowth by exerting its RhoGAP activity and that its cellular activity may be regulated through interaction with Src-like tyrosine kinases.     

Neurite outgrowth from PC12 cells by basic fibroblast growth factor (bFGF) is mediated by RhoA inactivation through p190RhoGAP and ARAP3

The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)‐RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat‐C3 exoenzyme and the expression of dominant negative (DN)‐RhoA (RhoA N19) stimulated neurite outgrowth. GTP‐bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap‐dependent RhoGAP (ARAP3). DN‐p190RhoGAP prevented neurite outgrowth, while WT‐p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN‐Rap1 and DN‐ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF. J. Cell. Physiol. 224: 786–794, 2010. © 2010 Wiley‐Liss, Inc.     

PKCε, Via its Regulatory Domain and Independently of its Catalytic Domain, Induces Neurite-like Processes in Neuroblastoma Cells

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCalpha, betaII, delta, and epsilon fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCepsilon-EGFP induced cell processes whereas the other isoforms did not. The effect of PKCepsilon-EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCepsilon regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCepsilon and neurites induced during retinoic acid- and growth factor-induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCepsilon- PSC1V3 protein contained alpha-tubulin, neurofilament-160, and F-actin, but the PKCepsilon-PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y. These data suggest that PKCepsilon, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.     

Adhesion-dependent regulation of p190RhoGAP in the developing brain by the Abl-related gene tyrosine kinase

Abl family kinases, which include the mammalian Abl and Arg (Abl-related gene) kinases, regulate neuronal morphogenesis in developing metazoa (for review, see [1]). Activation of Abl kinase activity directs changes in actin-dependent processes such as membrane ruffling, filopodial protrusion, and cell motility. However, the mechanisms by which increased Abl or Arg kinase activity promote cytoskeletal rearrangements are unclear. We provide evidence that the Rho inhibitor p190RhoGAP (GTPase-activating protein) is an Arg substrate in the postnatal mouse brain. We show that p190RhoGAP has reduced phosphotyrosine content in postnatal arg(-/-) mouse brain extracts relative to wild-type extracts. In addition, the adhesion-dependent stimulation of p190RhoGAP phosphorylation observed in wild-type cells is not observed in arg(-/-) fibroblasts and neurons. Arg can phosphorylate p190RhoGAP in vitro and in vivo on tyrosine (Y) 1105. We find that Arg can stimulate p190RhoGAP to inhibit Rho and that Arg-mediated phosphorylation is required for this stimulation. Phosphorylation by Arg also promotes p190RhoGAP's association with p120RasGAP and stimulates p190RhoGAP's ability to induce neuritogenesis in neuroblastoma cells. Our results demonstrate that p190RhoGAP is an Arg substrate in the developing brain and suggest that Arg mediates the adhesion-dependent regulation of neuronal morphogenesis in the postnatal brain by phosphorylating p190RhoGAP.     

Comparison of the PKCalpha and the PKCepsilon C1b domains: identification of residues critical for PKCepsilon-mediated neurite induction

We showed earlier that over-expression of protein kinase C (PKC) epsilon induces neurite outgrowth. The effect is mediated by a region (PKCepsilonPSC1V3) encompassing the pseudosubstrate, the two C1 domains and part of the V3 region, and is independent of the catalytic activity of the enzyme. In this region, residues immediately N-terminal of the C1b domain are crucial for neurite outgrowth. However, in this study we show that the PKCepsilon C1b domain itself is necessary for neurite induction, since a mutant in which the PKCepsilon C1b domain has been replaced with the C1b domain from PKCalpha, PKCepsilonPSC1a(alphaC1b)V3 lacks neurite-inducing capacity. The molecular basis for the importance of the PKCepsilon C1b domain was investigated by mutation studies of the PKCalpha C1b domain. Point mutations were done in the PKCalpha C1b domain of the PKCepsilonPSC1a(alphaC1b)V3 construct, in which the PKCalpha residues were mutated into the corresponding residues in PKCepsilon. This highlighted residues in the C-terminal part of the primary sequence of the C1b domain, located in the base of the C1b domain, as important for neurite outgrowth. The mutations S48P, D32K and L49N all influenced neurite induction positively. Furthermore, the mutation of L49N alone was sufficient to make PKCepsilonPSC1a(alphaC1b)V3 neuritogenic in phorbol ester-stimulated cells, and mutation of this residue in full-length PKCepsilon into the corresponding residue in PKCalpha, N291L reduced the neurite-inducing effect of PKCepsilon. In conclusion, we have identified residues in the PKCepsilon C1b domain, in particular Asn49, that are essential for neurite outgrowth.     

p190A RhoGAP is a glycogen synthase kinase-3-beta substrate required for polarized cell migration

The Rho GTPases are critical regulators of the actin cytoskeleton and are required for cell adhesion, migration, and polarity. Among the key Rho regulatory proteins in the context of cell migration are the p190 RhoGAPs (p190A and p190B), which function to modulate Rho signaling in response to integrin engagement. The p190 RhoGAPs undergo complex regulation, including phosphorylation by several identified kinases, interactions with phospholipids, and association with a variety of cellular proteins. Here, we have identified an additional regulatory mechanism unique to p190A RhoGAP that involves priming-dependent phosphorylation by glycogen synthase-3-beta (GSK-3beta), a kinase previously implicated in establishing cell polarity. We found that p190A-deficient fibroblasts exhibit a defect in directional cell migration reflecting a requirement for GSK-3beta-mediated phosphorylation of amino acids in the C-terminal "tail" of p190A. This phosphorylation leads to inhibition of p190A RhoGAP activity in vitro and in vivo. These studies identify p190A as a novel GSK-3beta substrate and reveal a mechanism by which GSK-3beta contributes to cellular polarization in directionally migrating cells via effects on Rho GTPase activity.     

Distinct roles of PKC isoforms in NCAM-mediated neurite outgrowth

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.     

Tks5 recruits AFAP-110, p190RhoGAP, and cortactin for podosome formation

Podosome formation in vascular smooth muscle cells is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the molecular mechanism that underlies their specific recruitment to podosomes remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. We show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5 mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. By expressing a catalytically inactive point mutant and by siRNA-mediated expression knock-down we also provide evidence that p190RhoGAP is required for podosome formation. Together our findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation.     

Identification of an amino acid residue in the protein kinase C C1b domain crucial for its localization to the Golgi network

Protein kinase C (PKC) isoforms have been reported to be targeted to the Golgi complex via their C1 domains. We have shown recently that the regulatory domain of PKC induces apoptosis in neuroblastoma cells and that this effect is correlated to Golgi localization via the C1b domain. This study was designed to identify specific residues in the C1 domains that mediate Golgi localization. We demonstrate that the isolated C1b domains from PKCalpha, -delta, -epsilon, -eta, and - are targeted to the Golgi complex, whereas the corresponding C1a domains localize throughout the cell. Sequence alignment showed that amino acid residues corresponding to Glu-246 and Met-267 in PKC are conserved among C1b but absent from C1a domains. Mutation of Met-267, but not of Glu-246, to glycine abolished the Golgi localization of the isolated C1b domain and the regulatory domain of PKC. The mutated PKC regulatory domain constructs lacking Golgi localization were unable to induce apoptosis, suggesting a direct correlation between Golgi localization and apoptotic activity of PKC regulatory domain. Mutation of analogous residues in the C1b domain of PKCepsilon abrogated its Golgi localization, demonstrating that this effect is not restricted to one PKC isoform. The abolished Golgi localization did not affect neurite induction by PKCepsilon. However, the PKCepsilon mutant did not relocate to the Golgi network in response to ceramide and ceramide did not suppress the neurite-inducing capacity of the protein. Thus, the specific mutations in the C1b domain influence both the localization and function of full-length PKCepsilon.     

The Mental Retardation Associated Protein,srGAP3 Negatively Regulates VPA-Induced Neuronal Differentiation of Neuro2A Cells

The Slit-Robo GTPase-activating proteins (srGAPs) are important multifunctional adaptor proteins involved in various aspects of neuronal development, including axon guidance, neuronal migration, neurite outgrowth, dendritic morphology and synaptic plasticity. Among them, srGAP3, also named MEGAP (Mental disorder-associated GTPase-activating protein), plays a putative role in severe mental retardation. SrGAP3 expression in ventricular zones of neurogenesis indicates its involvement in early stage of neuronal development and differentiation. Here, we show that overexpression of srGAP3 inhibits VPA (valproic acid)-induced neurite initiation and neuronal differentiation in Neuro2A neuroblastoma cells, whereas knockdown of srGAP3 facilitates the neuronal differentiation in this cell line. In contrast to the wild type, overexpression of srGAP3 harboring an artificially mutation R542A within the functionally important RhoGAP domain does not exert a visible inhibitory effect on neuronal differentiation. The endogenous srGAP3 selectively binds to activated form of Rac1 in a RhoGAP pull-down assay. We also show that constitutively active (CA) Rac1 can rescue the effect of srGAP3 on attenuating neuronal differentiation. Furthermore, change in expression and localization of endogenous srGAP3 is observed in neuronal differentiated Neuro2A cells. Together, our data suggest that srGAP3 could regulate neuronal differentiation in a Rac1-dependent manner.     

RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity

The binding of extracellular matrix proteins to integrins triggers rearrangements in the actin cytoskeleton by regulating the Rho family of small GTPases. The signaling events that mediate changes in the activity of Rho proteins in response to the extracellular matrix remain largely unknown. We have demonstrated in previous studies that integrin signaling transiently suppresses RhoA activity through stimulation of p190RhoGAP. Here, we investigated the biological significance of adhesion-dependent RhoA inactivation by manipulating p190RhoGAP signaling in Rat1 fibroblasts. The inhibition of RhoA activity that is induced transiently by adhesion was antagonized by expression of dominant negative p190RhoGAP. This resulted in impaired cell spreading on a fibronectin substrate, reduced cell protrusion, and premature assembly of stress fibers. Conversely, overexpression of p190RhoGAP augmented cell spreading. Dominant negative p190RhoGAP elevated RhoA activity in cells on fibronectin and inhibited migration, whereas overexpression of the wild-type GAP decreased RhoA activity, promoted the formation of membrane protrusions, and enhanced motility. Cells expressing dominant negative p190RhoGAP, but not control cells or cells overexpressing the wild-type GAP, were unable to establish polarity in the direction of migration. Taken together, these data demonstrate that integrin-triggered RhoA inhibition by p190RhoGAP enhances spreading and migration by regulating cell protrusion and polarity.     

Tyrosine 807 of the v-Fms oncogene product controls cell morphology and association with p120RasGAP.

Expression of the v-fms oncogene of feline sarcoma virus in fibroblasts causes surface exposure of an activated receptor tyrosine kinase, v-Fms, that is autophosphorylated at multiple sites within its cytoplasmic domain. Cellular proteins interacting with this part of v-Fms modulate the mitogenic activity and morphology of the cells. We show here that the tyrosine residue in position 807 (Y-807) of the v-Fms molecule constitutes a major autophosphorylation site. The replacement of this residue by phenylalanine (Y807F mutation) allowed us to functionally dissect v-Fms-specific mitogenic and morphogenic cascades. Cells expressing the mutant v-Fms molecule resembled wild-type (wt) v-Fms-transformed (wt-v-Fms) cells in terms of [3H]thymidine uptake rates and activation of the Ras/Raf-1 mitogenic cascade. Such cells showed, however, a flat morphology and contained intact actin cables and fibronectin network. Our studies indicate that the v-Fms molecule controls cell morphology by a cascade that involves a direct interaction with p120RasGAP and p190RhoGAP: (i) in contrast to wt v-Fms molecules, the Y807F v-Fms protein failed to associate with and phosphorylate p120RasGAP; (ii) tight complexes between p120RasGAP and p190RhoGAP as well as detectable RhoGAP activity were present exclusively in wt-v-Fms cells; and (iii) p190RhoGAP was dispersed throughout the cytoplasm of wt-v-Fms cells, whereas its distribution was restricted to perinuclear regions of cells expressing the mutant v-Fms gene.     

p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.