Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.     

Formation of complexes involving RasGAP and p190 RhoGAP during morphogenetic events of the gastrulation in xenopus.

In relation to the activation of the Src-family of tyrosine kinases during early morphogenetic events of gastrulation in Xenopus, we have identified two multiprotein complexes. The first complex, including RasGAP, p190 RhoGAP and p62, was previously characterized in murine fibroblasts overexpressing c-Src or transformed by v-Src and has been correlated with cytoskeleton remodelling. A second complex, not identified in other models includes tyrosine-phosphorylated p66SHC, Grb2, RasGAP and p190 RhoGAP. The association with p66SHC, considered as a negative regulator of ERK (extracellular signal-regulated kinase), p120RasGAP and p190RhoGAP, suggests a possible mechanism for coupling Ras and Rho signalling pathways. The interaction of RasGAP and p190 RhoGAP in two multiprotein complexes could constitute an additional level of Rho regulation during morphogenetic events of gastrulation.     

Protein tyrosine phosphatase PTP20 induces actin cytoskeleton reorganization by dephosphorylating p190 RhoGAP in rat ovarian granulosa cells stimulated with follicle-stimulating hormone

We identified 25 protein tyrosine phosphatases (PTPs) expressed in rat ovarian granulosa cells. Of these PTPs, the expression levels of at least PTP20, PTP-MEG1, PTPepsilonM, and PTPepsilonC significantly changed during the estrous cycle. We examined the cellular functions of PTP20 in granulosa cells by expressing the wild type, a catalytically inactive CS mutant in which Cys229 of PTP20 was changed to Ser, or a substrate-trapping DA mutant in which Asp197 was mutated to Ala, using an adenovirus vector. Overexpression of the wild type, but not of the CS mutant, induced retraction of the cell body with the extension of long, dendritic-like processes after stimulation with FSH, a critical factor for the survival and differentiation of these cells. In addition, cell adhesion to the substratum decreased in an FSH-dependent manner. Inhibiting Rho GTPase activity with C3 botulinum toxin caused similar morphological changes. The FSH-enhanced phosphotyrosine (p-Tyr) level of p190 RhoGAP was selectively reduced by the overexpressed wild type, but not by mutated PTP20. Although p190 RhoGAP is tyrosine phosphorylated by c-Src via the tyrosine kinase Pyk2, wild-type PTP20 had little effect on p-Tyr418 of c-Src and no effect on p-Tyr402 of Pyk2, which are required for full c-Src activity and for interacting between Pyk2 and c-Src, respectively. The CS and DA mutants as well as the wild type reduced the formation of p190 RhoGAP-p120 RasGAP complexes. Confocal microscopy analysis revealed that PTP20 intracellularly colocalizes with p190 RhoGAP. These results demonstrate that PTP20 regulates the functions of granulosa cells in an FSH-dependent manner by dephosphorylating p190 RhoGAP and subsequently inducing reorganization of the actin cytoskeleton. Moreover, our data suggest that PTPs play significant roles in controlling the dynamics of ovarian functions.     

Modulation of epidermal growth factor-dependent protein phosphorylation in cell membrane preparations by receptor down regulation

We have reported previously [6] that epidermal growth factor (EGF)-induced down regulation of EGF receptors in normal rat kidney (NRK) cells results in a selective decrease in the in vitro EGF-dependent ³²P-phosphorylation of two membrane phosphoproteins of Mr I70K and Mr I50K. In this report, we further characterized the modulation of ³²P-phosphorylation of the 170K- and 150K-dalton proteins by down regulation with EGF in NRK cells. While EGF binding to its receptors was a necessary condition to induce loss of EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins, it was not sufficient. Thus, reduction in the temperature of the incubation of cells with EGF from 37°C to 4°C abolished the loss of EGF-dependent phosphorylation of the 170K- and 150K-dalton membrane proteins. When EGF was removed from the medium the EGF-dependent phosphorylation of the 170K- and l50K-dalton proteins was quickly replenished; by 3 hr one-half of the “down regulated” phosphorylation was restored. All EGF-dependent phosphorylating capacity of the 170K- and l50K-dalton protein bands returned by 6 hr after removal of the growth factor. The loss of EGF-dependent phosphorylation of the 170K- and I50K-dalton proteins occurred at physiological EGF concentrations (0.25–25 ng/ml) that span the concentration range which is mitogenic for NRK cells. Exposure of confluent nondividing NRK cells to 1 ng/ml EGF, followed by incubation for 5 hr at 37°C. led to a 50% reduction in the EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins. Maximal reduction (～95%) in the EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins was noted with 10 ng/ml EGF for 5 hr. The EGF-induced loss of EGF-dependent phosphorylation was specific: several other growth factors did not produce phosphorylation loss of the 170K-     

Cadherin engagement inhibits RhoA via p190RhoGAP

Cadherins are transmembrane receptors that mediate cell-cell adhesion in epithelial cells. A number of changes occur during cadherin-mediated junction formation, one of which is a rearrangement of the actin cytoskeleton. Key regulators of actin cytoskeletal dynamics in cells are the Rho family of GTPases. We have demonstrated in previous studies that cadherin signaling suppresses RhoA activity and activates Rac1. The signaling events downstream of cadherins that modulate the activity of Rho family proteins remain unknown. Here we have identified a pathway by which RhoA becomes inactivated by cadherins. To determine whether cadherins regulate RhoA through activation of a GTPase-activating protein (GAP) for RhoA, we used constitutively active RhoA to isolate activated GAPs. Using this assay, we have identified the RhoA-specific GAP, p190RhoGAP, downstream from engaged cadherins. We found that cadherin engagement induced tyrosine phosphorylation of p190RhoGAP and increased its binding to p120RasGAP. The increased precipitation of p190RhoGAP with 63LRhoA was blocked by addition of PP2 suggesting that Src family kinases are required downstream from cadherin signaling. The inhibition of RhoA activity by cadherins was antagonized by expression of a dominant negative p190RhoGAP. Taken together, these data demonstrate that p190RhoGAP activity is critical for RhoA inactivation by cadherins.     

Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane

The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.     

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.     

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.     

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.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell–cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.     

Stretch-induced actin remodeling requires targeting of zyxin to stress fibers and recruitment of actin regulators

Reinforcement of actin stress fibers in response to mechanical stimulation depends on a posttranslational mechanism that requires the LIM protein zyxin. The C-terminal LIM region of zyxin directs the force-sensitive accumulation of zyxin on actin stress fibers. The N-terminal region of zyxin promotes actin reinforcement even when Rho kinase is inhibited. The mechanosensitive integrin effector p130Cas binds zyxin but is not required for mitogen-activated protein kinase-dependent zyxin phosphorylation or stress fiber remodeling in cells exposed to uniaxial cyclic stretch. α-Actinin and Ena/VASP proteins bind to the stress fiber reinforcement domain of zyxin. Mutation of their docking sites reveals that zyxin is required for recruitment of both groups of proteins to regions of stress fiber remodeling. Zyxin-null cells reconstituted with zyxin variants that lack either α-actinin or Ena/VASP-binding capacity display compromised response to mechanical stimulation. Our findings define a bipartite mechanism for stretch-induced actin remodeling that involves mechanosensitive targeting of zyxin to actin stress fibers and localized recruitment of actin regulatory machinery.     

Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src,NADPH Oxidase,PI3K,and Akt in Human Colon Cancer Cells

Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47^phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.     

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.     

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.     

p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho

Integration of receptor tyrosine kinase, integrin, and cadherin activities is crucial for normal cell growth, motility, and adhesion. Here, we describe roles for p120-catenin (p120) and p190RhoGAP that coordinate crosstalk between these systems and regulate cadherin function. Surprisingly, PDGFR-induced actin remodeling in NIH3T3 cells is blocked in the absence of p120, and the cells are partially transformed via constitutive activation of Rho. We have traced the mechanism to unexpected codependent roles for p120 and p190RhoGAP in regulating Rac-dependent antagonism of Rho. Receptor-induced Rac activity causes translocation of p190RhoGAP to adherens junctions (AJs), where it couples to the cadherin complex via interaction with p120. AJ formation is dependent on this p120-p190RhoGAP interaction and fails altogether if either of these proteins are compromised. We propose that Rac activation links diverse signaling systems to AJ assembly by controlling transient p190RhoGAP interactions with p120 and localized inhibition of Rho.     

Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation

During development of the central nervous system, oligodendrocyte progenitor cells differentiate into mature myelinating cells. The molecular signals that promote this process, however, are not well defined. One molecule that has been implicated in oligodendrocyte differentiation is the Src family kinase Fyn. In order to probe the function of Fyn in this system, a yeast two hybrid screen was performed. Using Fyn as bait, p190 RhoGAP was isolated in the screen of an oligodendrocyte cDNA library. Coimmunoprecipitation and in vitro binding assays verified that p190 RhoGAP bound to the Fyn SH2 domain. Phosphorylation of p190 required active Fyn tyrosine kinase and was increased threefold upon differentiation of primary oligodendrocytes. Moreover, complex formation between p190 and p120 RasGAP occurred in differentiated oligodendrocytes. p190 RhoGAP activity is known to regulate the RhoGDP:RhoGTP ratio. Indeed, expression of dominant negative Rho in primary oligodendrocytes caused a hyperextension of processes. Conversely, constitutively activated Rho caused reduced process formation. These findings define a pathway in which Fyn activity regulates the phosphorylation of p190, leading to an increase in RhoGAP activity with a subsequent increase in RhoGDP, which in turn, regulates the morphological changes that accompany oligodendrocyte differentiation.     

Desensitization of the PDGFbeta receptor by modulation of the cytoskeleton: the role of p21(Ras) and Rho family GTPases

Ligand-induced PDGF-type beta receptor (PDGFbeta-R) autophosphorylation is profoundly suppressed in cells transformed by activated p21(Ras). We report here that the integrity of the actin cytoskeleton is a critical regulator of PDGFbeta-R function in the presence of p21(Ras). Morphological reversion of Balb cells expressing a constitutively activated p21(Ras), with re-formation of actin stress fibers and cytoskeletal architecture, rendering them phenotypically similar to untransformed fibroblasts, allowed recovery of ligand-dependent PDGFbeta-R autophosphorylation. Conversely, disruption of the actin cytoskeleton in Balb/c-3T3 cells obliterated the normal ligand-induced phosphorylation of the PDGFbeta-R. The Rho family GTPases Rac and Rho are activated by p21(Ras) and are critical mediators of cell motility and morphology via their influence on the actin cytoskeleton. Transient expression of wild-type or constitutively active mutant forms of RhoA suppressed ligand-dependent PDGFbeta-R autophosphorylation and downstream signal transduction. These studies demonstrate the necessary role of Rho in the inhibition of PDGFbeta-R autophosphorylation in cells containing activated p21(Ras) and also demonstrate the importance of cell context and the integrity of the actin cytoskeleton in the regulation of PDGFbeta-R ligand-induced autophosphorylation.