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.     

c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190- RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF- dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.     

The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development

Rho GTPases direct actin rearrangements in response to a variety of extracellular signals. P190 RhoGAP (GTPase activating protein) is a potent Rho regulator that mediates integrin-dependent adhesion signaling in cultured cells. We have determined that p190 RhoGAP is specifically expressed at high levels throughout the developing nervous system. Mice lacking functional p190 RhoGAP exhibit several defects in neural development that are reminiscent of those described in mice lacking certain mediators of neural cell adhesion. The defects reflect aberrant tissue morphogenesis and include abnormalities in forebrain hemisphere fusion, ventricle shape, optic cup formation, neural tube closure, and layering of the cerebral cortex. In cells of the neural tube floor plate of p190 RhoGAP mutant mice, polymerized actin accumulates excessively, suggesting a role for p190 RhoGAP in the regulation of +Rho-mediated actin assembly within the neuroepithelium. Significantly, several of the observed tissue fusion defects seen in the mutant mice are also found in mice lacking MARCKS, the major substrate of protein kinase C (PKC), and we have found that p190 RhoGAP is also a PKC substrate in vivo. Upon either direct activation of PKC or in response to integrin engagement, p190 RhoGAP is rapidly translocated to regions of membrane ruffling, where it colocalizes with polymerized actin. Together, these results suggest that upon activation of neural adhesion molecules, the action of PKC and p190 RhoGAP leads to a modulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normal neural development.     

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.     

Coordination of Rho and Rac GTPase function via p190B RhoGAP

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.     

Opposing roles of p190RhoGAP and Ect2 RhoGEF in regulating cytokinesis

Evidence suggests that p190RhoGAP (p190), a GTPase activating protein (GAP) specific for Rho, plays a role in cytokinesis. First, ectopic expression of p190 induces a multinucleated cellular phenotype. Second, endogenous p190 localizes to the cleavage furrow of dividing cells. Lastly, its levels are reduced in late mitosis by ubiquitin-mediated proteasomal degradation, consistent with the idea that low levels of p190 and high levels of active Rho are required for completion of cytokinesis. As with p190, RhoA and the RhoGEF, ECT2, have been localized to the cleavage furrow. These findings raise the question of whether p190 and ECT2 cooperate antagonistically to regulate the activity of Rho and contraction of the actomyosin ring during cytokinesis. Here we demonstrate ECT2 can, in a dose-dependent manner, reduce multinucleation induced by p190. Furthermore, endogenous p190 and ECT2 colocalize at the cleavage furrow of dividing cells and stably associate with one another in co-immunoprecipitation assays. Functional and physical interactions between p190 and ECT2 are reflected in the levels of Rho activity, as assessed by Rho pull-down assays. Together, these results suggest that co-regulation of Rho activity by p190RhoGAP and ECT2 in the cleavage furrow determines whether cells properly complete cytokinesis.     

Mitotic Down-regulation of p190RhoGAP Is Required for the Successful Completion of Cytokinesis

p190RhoGAP-A (p190) is a GTPase-activating protein known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of Rho intrinsic GTPase activity. We have previously shown that p190 protein levels are cell cycle-regulated, decreasing in mitosis, and that this decrease is mediated by the ubiquitin-proteasome pathway. In addition, overexpression of p190 results in decreased RhoGTP levels at the cleavage furrow during cytokinesis, p190 and the RhoGEF Ect2 play opposing roles in cytokinesis, and sustained levels of p190 in mitosis are associated with cytokinesis failure, all findings that suggest but do not directly demonstrate that completion of cytokinesis is dependent on reduced levels of p190. Here we report, using an RNAi reconstitution approach with a degradation-resistant mutant, that decreased p190 levels are required for successful cytokinesis. We also show that the multinucleation phenotype is dependent on p190 RhoGAP activity, determine that the N-terminal GBDS1 region is necessary and sufficient for p190 mitotic ubiquitination and degradation, and identify four N-terminal residues as necessary for the degradation of p190 in mitosis. Our data indicate that in addition to activation of RhoGEF(s), reduction of RhoGAP (p190) is a critical mechanism by which increased RhoGTP levels are achieved in late mitosis, thereby ensuring proper cell division.     

Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability

p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.     

Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway.

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. p190 inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by p190. Local regulation of p190 could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of p190 by integrin and Src, both implicated in neural plasticity.     

p190RhoGAP negatively regulates Rho activity at the cleavage furrow of mitotic cells

Previous studies demonstrated that p190RhoGAP (p190) negatively affects cytokinesis in a RhoGAP-dependent manner, suggesting that regulation of Rho may be a critical mechanism of p190 action during cytokinesis. P190 localizes to the cleavage furrow (CF) of dividing cells, and its levels decrease during late mitosis by an ubiquitin-mediated mechanism, consistent with the hypothesis that high RhoGTP levels are required for completion of cytokinesis. To determine whether RhoGTP levels in the CF are affected by p190 and to define the phase(s) of cytokinesis in which p190 is involved, we used FRET analysis alone or in combination with time-lapse microscopy. In normal cell division activated Rho accumulated at the cell equator in early anaphase and in the contractile ring, where it co-localized with p190. Real-time movies revealed that cells expressing elevated levels of p190 exhibited multiple cycles of abnormal CF site selection and ingression/regression, which resulted in failed or prolonged cytokinesis. This was accompanied by mislocalization of active Rho at the aberrant CF sites. Quantified data revealed that in contrast to ECT2 and dominate negative p190 (Y1283Ap190), which resulted in hyper-activated Rho, Rho activity in the CF was reduced by wild type p190 in a dose-dependent manner. These results suggest that p190 regulates cytokinesis through modulation of RhoGTP levels, thereby affecting CF specification site selection and subsequent ring contraction.     

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.     

p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.     

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.     

Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils

We found that engagement of beta(2) integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta(2) integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta(2) integrin-induced activation of p190RHOGAP: The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of beta(2) integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RHOGAP: Instead, the beta(2) integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta(2) integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta(2) integrin-stimulated cells. Thus, in neutrophils, beta(2) integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RHOA:     

A Point Mutation in p190A RhoGAP Affects Ciliogenesis and Leads to Glomerulocystic Kidney Defects

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.     

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.     

The PAR-6 polarity protein regulates dendritic spine morphogenesis through p190 RhoGAP and the Rho GTPase

The majority of excitatory synaptic transmission in the brain occurs at dendritic spines, which are actin-rich protrusions on the dendrites. The asymmetric nature of these structures suggests that proteins regulating cell polarity might be involved in their formation. Indeed, the polarity protein PAR-3 is required for normal spine morphogenesis. However, this function is independent of association with atypical protein kinase C (aPKC) and PAR-6. Here we show that PAR-6 together with aPKC plays a distinct but essential role in spine morphogenesis. Knockdown of PAR-6 inhibits spine morphogenesis, whereas overexpression of PAR-6 increases spine density, and these effects are mediated by aPKC. Using a FRET biosensor, we further show that p190 RhoGAP and RhoA act downstream of the PAR-6/aPKC complex. These results define a role for PAR-6 and aPKC in dendritic spine biogenesis and maintenance, and reveal an unexpected link between the PAR-6/aPKC complex and RhoA activity.     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.     

Phosphorylation of the p190 RhoGAP N-terminal domain by c-Src results in a loss of GTP binding activity

p190 RhoGAP is a multi-domain protein that is thought to regulate actin cytoskeleton dynamics. It can be phosphorylated both in vitro and in vivo at multiple sites by the Src tyrosine kinase and one or more of these sites is postulated to modulate p190 function. One of the regions which is multiply phosphorylated by Src in vitro is the N-terminal GTP binding domain. Using a partially purified, bacterially expressed recombinant protein that includes the GTP binding domain (residues 1-389), we show that GTP binds to this fragment in a specific and saturable manner that is both time- and dose-dependent and that tyrosine phosphorylation of this fragment by c-Src results in a loss of GTP binding activity. These findings suggest that tyrosine phosphorylation of the p190 N-terminal domain can alter its ability to bind GTP.