Abstract
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.
Highlights
From the Parke-Davis Pharmaceutical Research Division, Departments of :j:8ignal Transduction and IlNeurosciences, Ann Arbor Michigan, 48106 and the §Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109
In vitro, purified GIl'Y subunits bound to a GST fusion protein encoding amino acids 1-330 of Raf-l (Raf/330)
Binding assays with truncation mutants of GST-Raf indicate that the region located be· tween amino acids 136 and 239 is a primary determinant for interaction with Gllr In competition experiments, the carboxyl terminus of p-adrenergic receptor kinase
Summary
Using a yeast two-hybr-id strategy to Identify other proteins that interact with and potentially regulate Raf-l, we isolated a clone encoding the carboxyl. In vitro, purified GIl'Y subunits bound to a GST fusion protein encoding amino acids 1-330 of Raf-l (Raf/330). Raf-1 is a serine-threonine protein kinase critically positioned in a kinase cascade linking activated growth factor receptors with the nucleus and regulating the mitogenic response. Regions contained in the amino-terminal portion of Raf-l are presumed to be critical for regulating the biological activity of this kinase. 1 In this report, we have characterized this interaction, raising the possibility that the binding of G13y to Raf-l could playa role in regulation of the mitogen-activated protein kinase pathway by G-proteincoupled receptors and/or potentially provide a junction for integration of signals generated by tyrosine kinase growth factor receptors and G-protein-coupled receptors An interacting clone was identified which encodes the carboxyl-terminal half ofthe G132 subunit ofheterotrimeric G-proteins. 1 In this report, we have characterized this interaction, raising the possibility that the binding of G13y to Raf-l could playa role in regulation of the mitogen-activated protein kinase pathway by G-proteincoupled receptors and/or potentially provide a junction for integration of signals generated by tyrosine kinase growth factor receptors and G-protein-coupled receptors
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