THE RAS-GTPASE-ACTIVATING PROTEIN SH3 DOMAIN IS REQUIRED FOR CDC2 ACTIVATION AND MOS INDUCTION BY ONCOGENIC RAS IN XENOPUS OOCYTES INDEPENDENTLY OF MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION

The Ras-GTPase-activating protein (RasGAP) is an important modulator o f p21(ras)-dependent signal transduction in Xenopus oocytes and in mam malian cells. We investigated the role of the RasGAP SH3 domain in sig nal transduction with a monoclonal antibody against the SH3 domain bf RasGAP. This antibody prevented the activation hf the maturation-promo ting factor complex (cyclin B-p34(cdc2)) by oncogenic Pas. The antibod y appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50%, whereas 100 ng of nonspe cific immunoglobulin G did not affect Cdc2 activation. The antibody bl ocked the CdcZ activation induced by oncogenic Pas but not that induce d by progesterone, which acts independently of Pas. A peptide correspo nding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to po sitions 273 to 305 of a sequence in the N-terminal moiety of the SH3 d omain of RasGAP had no effect. The antibody did not block the mitogen- activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90(rsk)). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncog enic Pas as well as blocking the activation of MAP kinase. Thus, MAP k inase is also implicated in the regulation of Cdc2 activity. In this s tudy, we further investigated the regulation of the synthesis of the c -mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of Mos protein induced by oncogenic Pas i s prevented by injecting the antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These r esults suggest that oncogenic Pas activates two signaling mechanisms: the MAP kinase cascade and a signaling path,vay implicating the SH3 do main of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.