N-TERMINAL SEQUENCES CONTAINED IN THE SRC-HOMOLOGY-2 AND SRC-HOMOLOGY-3 DOMAINS OF P120 GTPASE-ACTIVATING PROTEIN ARE REQUIRED FOR FULL CATALYTIC ACTIVITY TOWARD RAS

The p120 GTPase-activating protein (GAP) is a negative regulator of Ra s, which has a central role in signal transduction pathways that contr ol cell proliferation. p120 GAP accelerates the conversion of activate d Ras-GTP to its inactive form, Ras-GDP, thereby inhibiting mitogenic signaling. To examine potential contributions of p120 N-terminal seque nces to regulation of its C-terminal catalytic domain, we constructed deletion mutants lacking defined regions, including the variable hydro phobic region as well as the Src homology 2 (SH2) and 3 (SH3) domains. These mutant proteins were expressed in infected Sf9 insect cells fro m recombinant baculoviruses and assayed in vitro for their ability to stimulate the intrinsic GTPase activity of purified Ras. While deletio n of the variable hydrophobic region had no effect on p120 GAP activit y, deletion of the entire SH2/SH3/SH2 region severely impaired catalyt ic activity toward Ras. Deletion of individual SH2 and SH3 domains wit hin this region partially inhibited p120 GAP activity. Moreover, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of t he neurofibromin GAP-related domain. These results demonstrate that se quences in the SH2/SH3/SH2 region of p120 GAP are required for full ca talytic activity toward Ras. Together with earlier findings that the p 120 GAP SH2 domains mediate interactions with several GAP-associated p roteins, our results suggest multiple roles for the N-terminal sequenc es in regulating p120 GAP catalytic activity and mitogenic signaling p athways. In addition, our results raise the possibility that SH2 domai n point mutations in p120 GAP detected in some basal cell carcinomas r educe catalytic activity toward Ras and thereby contribute to oncogene sis.