A Direct Interaction between G-Protein Subunits and the Raf-1 Protein Kinase

• Published in: The Journal of biological chemistry 1995-06, Vol.270 (24), p.14251
• Main Authors: Kevin M. Pumiglia, Harry LeVine, Taraneh Haske, Tania Habib, Richard Jove, Stuart J. Decker
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.24.14251

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 subunit of heterotrimeric G-proteins. In vitro , purified G 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 . In competition experiments, the carboxyl terminus of β-adrenergic receptor kinase (βARK) blocked the binding of G 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 revealed an affinity of interaction ( K = 163 ± 36 nM), similar to that seen between G and βARK ( K = 87 ± 24 nM). The formation of native heterotrimeric G complexes, as measured by pertussis toxin ADP-ribosylation of G , could be disrupted by increasing amounts of Raf/330, with an EC of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G 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.