Abstract
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.
Highlights
The regulatory mechanism of the deleted in liver cancer 1 (DLC1) tumor suppressor protein is unclear
We demonstrate that only the Src homology 3 (SH3) domain of p120 selectively inhibits the Rho-specific GTPase-activating proteins (RhoGAPs) activity of all three DLC isoforms as compared with a large set of other representative SH3 or RhoGAP proteins
We have shown that p120SH3, by utilizing a novel binding mode, selectively undergoes a high affinity interaction with the RhoGAP domain of DLC1 and effectively inhibits its GTPase-activating proteins (GAPs) activity by targeting its catalytic arginine finger
Summary
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. 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). 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. P120 associates with the DLC1 RhoGAP domain by targeting the catalytic argi-
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