A DIRECT INTERACTION BETWEEN G-PROTEIN BETA-GAMMA-SUBUNITS AND THE, RAF-1 PROTEIN-KINASE

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G(beta 2) subunit of heterotrimeric G-proteins. In vitro, purified G(beta gamma) subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located. between amino acids 136 and 239 is a primary determinant for interaction with G(beta gamma). In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G(beta gamma) to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G(beta gamma) revealed an affinity of interaction (K-d = 163 +/- 36 nM), similar to that seen between G(beta gamma) and beta ARK (K-d = 87 +/- 24 nM), The formation of native heterotrimeric G(alpha beta gamma) complexes, as measured by pertussis toxin ADP-ribosylation of G(alpha), could be disrupted by increasing amounts of Raf/330, with an EC(50) of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G(beta gamma) were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G(beta 2). The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.