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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 (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 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 EC50 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.

Pubmed ID: 7782277


  • Pumiglia KM
  • LeVine H
  • Haske T
  • Habib T
  • Jove R
  • Decker SJ


The Journal of biological chemistry

Publication Data

June 16, 1995

Associated Grants

  • Agency: NCI NIH HHS, Id: CA55652

Mesh Terms

  • Amino Acid Sequence
  • Animals
  • Cyclic AMP-Dependent Protein Kinases
  • GTP-Binding Proteins
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Protein Binding
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-raf
  • Rats
  • beta-Adrenergic Receptor Kinases