A direct interaction between G-protein βγ subunits and the Raf-1 protein kinase

K. M. Pumiglia, H. LeVine, T. Haske, T. Habib, R. Jove, S. J. Decker

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76 Scopus citations


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(β2) subunit of heterotrimeric G-proteins. In vitro, purified G(βγ) 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(βγ). In competition experiments, the carboxyl terminus of β-adrenergic receptor kinase (βARK) blocked the binding of G(βγ) 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(βγ) revealed an affinity of interaction (K(d) = 163 ± 36 nM), similar to that seen between G(βγ) and βARK (K(d) = 87 ± 24 nM). The formation of native heterotrimeric G(αβγ) complexes, as measured by pertussis toxin ADP-ribosylation of G(α), could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G(βγ) were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G(β2). 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.

Original languageEnglish
Pages (from-to)14251-14254
Number of pages4
JournalJournal of Biological Chemistry
Issue number24
StatePublished - Jun 16 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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