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.

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.

SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins.

Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.