Functional Cross-talk between Ras and Rho Pathways A Ras-SPECIFIC GTPase-ACTIVATING PROTEIN (p120RasGAP) COMPETITIVELY INHIBITS THE RhoGAP ACTIVITY OF DELETED IN LIVER CANCER (DLC) TUMOR SUPPRESSOR BY MASKING THE CATALYTIC ARGININE FINGER

Background: The regulatory mechanism of the DLC1 tumor suppressor protein is unclear. Results: Structure-function analysis revealed determinants for the selectivity, activity, and inhibition of DLC1 RhoGAP function. Conclusion: p120RasGAP competitively and selectively inhibits DLC1 by targeting its catalytic arginine finger. Significance: This mechanistic study emphasizes the importance of the functional inter-relationships of GTPase-activating proteins mediating cross-talk between the Ras and Rho pathways. The three deleted in liver cancer genes (DLC1-3) encode Rho-specific GTPase-activating proteins (RhoGAPs). Their expression is frequently silenced in a variety of cancers. The RhoGAP activity, which is required for full DLC-dependent tumor suppressor activity, can be inhibited by the Src homology 3 (SH3) domain of a Ras-specific GAP (p120RasGAP). Here, we comprehensively investigated the molecular mechanism underlying cross-talk between two distinct regulators of small GTP-binding proteins using structural and biochemical methods. We demonstrate that only the SH3 domain of p120 selectively inhibits the RhoGAP activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins. Structural and mutational analyses provide new insights into a putative interaction mode of the p120 SH3 domain with the DLC1 RhoGAP domain that is atypical and does not follow the classical PXXP-directed interaction. Hence, p120 associates with the DLC1 RhoGAP domain by targeting the catalytic arginine finger and thus by competitively and very potently inhibiting RhoGAP activity. The novel findings of this study shed light on the molecular mechanisms underlying the DLC inhibitory effects of p120 and suggest a functional cross-talk between Ras and Rho proteins at the level of regulatory proteins.