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

Abstract

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 Rasmediated 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.

Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells

“…In MCF-7 cells, the association requires p120 RasGAP tyrosine phosphorylation while in MCF-7ras cells, the association is Nck tyrosine phosphorylationdependent. A similar mechanism mediating the interaction of p120 RasGAP and p190 RhoGAP has also been recently described (Roof et al, 1998).…”

Sodium fluoride induces podosome formation in endothelial cells

“…At the GTPase level, Src inhibition alters NaF-mediated events in further increasing RhoA activation mediated by NaF, suggesting that Src may operate upstream RhoGTPases. As Src is a positive regulator of p190RhoGAP (Roof et al, 1998), the PP1 inhibitory effect on Src is expected to suppress a negative regulation on RhoA and therefore promote the GTP-bound form of the GTPase. Since Rho protein inhibition increased the number of cells showing podosomes, the PP1 inhibitory effect on NaF-induced podosome assembly could be a direct consequence of alteration in RhoA activity.…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…p190RhoGAP localizes to structures relatively poor in stress fibers [108] and arc-like structures in EGF stimulated cells with concomitant reduction in actin stress fibers [114], suggesting a functional link between the RhoGAP and actin dynamics during cell spreading and growth factor signaling [114,115]. However, while activation of p190RhoGAP requires tyrosine phosphorylation of Y1105 [116][117][118], tyrosine phosphorylation alone appears insufficient to activate p190 RhoGAP activity, at least in response to growth factor stimulation [119,120]. It is currently not known whether p190RhoGAP is regulated by ERK signaling.…”