Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1.

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.

Tandem engagement of phosphotyrosines by the dual SH2 domains of p120RasGAP.

p120RasGAP is a multidomain GTPase-activating protein for Ras. The presence of two Src homology 2 domains in an SH2-SH3-SH2 module raises the possibility that p120RasGAP simultaneously binds dual phosphotyrosine residues in target proteins. One known binding partner with two proximal phosphotyrosines is p190RhoGAP, a GTPase-activating protein for Rho GTPases. Here, we present the crystal structure of the p120RasGAP SH2-SH3-SH2 module bound to a doubly tyrosine-phosphorylated p190RhoGAP peptide, revealing simultaneous phosphotyrosine recognition by the SH2 domains. The compact arrangement places the SH2 domains in close proximity resembling an SH2 domain tandem and exposed SH3 domain. Affinity measurements support synergistic binding, while solution scattering reveals that dual phosphotyrosine binding induces compaction of this region. Our studies reflect a binding mode that limits conformational flexibility within the SH2-SH3-SH2 cassette and relies on the spacing and sequence surrounding the two phosphotyrosines, potentially representing a selectivity mechanism for downstream signaling events.

SH3 domain regulation of RhoGAP activity: Crosstalk between p120RasGAP and DLC1 RhoGAP.

RhoGAP proteins are key regulators of Rho family GTPases and influence a variety of cellular processes, including cell migration, adhesion, and cytokinesis. These GTPase activating proteins (GAPs) downregulate Rho signaling by binding and enhancing the intrinsic GTPase activity of Rho proteins. Deleted in liver cancer 1 (DLC1) is a tumor suppressor and ubiquitously expressed RhoGAP protein; its activity is regulated in part by binding p120RasGAP, a GAP protein for the Ras GTPases. In this study, we report the co-crystal structure of the p120RasGAP SH3 domain bound directly to DLC1 RhoGAP, at a site partially overlapping the RhoA binding site and impinging on the catalytic arginine finger. We demonstrate biochemically that mutation of this interface relieves inhibition of RhoGAP activity by the SH3 domain. These results reveal the mechanism for inhibition of DLC1 RhoGAP activity by p120RasGAP and demonstrate the molecular basis for direct SH3 domain modulation of GAP activity.