AN EFFECTOR-LIKE FUNCTION OF RAS GTPASE-ACTIVATING PROTEIN PREDOMINATES IN CARDIAC-MUSCLE-CELLS

In contrast to familiar role for Ras in proliferation, we and others p reviously suggested that Ras also mediates hypertrophy, the increase i n cell mass characteristic of post-natal ventricular muscle. We showed that activated (G12R) and dominant-negative (S17N) Ha-Ras regulate '' constitutive'' and growth factor-responsive genes equivalently, in bot h cardiac myocytes and non-cardiac, Mv1Lu cells. Here, we attempt to d elineate pathways by which Ras exerts this global effect. The E63K mut ation, which impairs binding of guanine nucleotide releasing factor to Ras, alleviated suppression by S17N, consistent with sequestration of exchange factors as the mechanism for inhibition. To compare potentia l Ras effector proteins, we first engineered G12R/D38N, to abolish bin ding of Raf and phosphatidylinositol-3-kinase and established that thi s site was indispensable for augmenting gene expression. To distinguis h between inhibition of Ras by Ras GTPase-activating protein (GAP) ver sus a potential effector function of GAP, we tested the effector domai n substitution P34R: this mutation, which abolishes GAP binding, enhan ced Ras-dependent transcription in Mv1Lu cells, yet interfered with Ra s-dependent expression in ventricular myocytes. To examine the dichoto mous role of Ras-GAP predicted from these P34R results, we transfected both cell types with full-length GAP, the C-terminal catalytic domain (cGAP), or N-terminal Src homology domains (nGAP). In Mv1Lu cells, cG AP markedly inhibited both reporter genes, whereas GAP and nGAP had li ttle effect. Antithetically, in ventricular myocytes, GAP and nGAP act ivated gene expression, whereas cGAP was ineffective. Thus, Ras activa tes gene expression through differing effecters contingent on cell typ e, and an effector-like function of GAP predominates in ventricular mu scle.