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A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase
K M Pumiglia1, H LeVine, T Haske
1Parke-Davis Pharmaceutical Research Division, Department of Signal Transduction, Ann Arbor, Michigan 48106, USA.
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.
Insights
Researchers discovered that G beta gamma subunits directly bind to Raf-1, a key kinase in cell signaling. This novel interaction suggests cross-talk between growth factor and G-protein coupled receptors.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Raf-1 is a serine/threonine protein kinase downstream of Ras in the mitogen-activated protein kinase (MAPK) cascade.
- Heterotrimeric G-proteins are crucial signal transducers activated by various receptors.
Purpose of the Study:
- To identify novel regulators of Raf-1 using a yeast two-hybrid system.
- To characterize the interaction between Raf-1 and G-protein subunits.
Main Methods:
- Yeast two-hybrid screening to identify interacting proteins.
- In vitro binding assays using purified proteins and GST fusion proteins.
- Competition assays and Scatchard analysis to quantify binding affinity.
- In vivo co-immunoprecipitation from transfected cells.
Main Results:
- The carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins was identified as a Raf-1 interacting protein.
- G beta gamma subunits specifically bind to Raf-1, with the primary interaction domain located between amino acids 136-239.
- The binding affinity (Kd) was determined to be approximately 163 nM, comparable to G beta gamma binding to beta-adrenergic receptor kinase (beta ARK).
- Raf-1 disrupted the formation of native G alpha beta gamma complexes in vitro.
- In vivo complexes of Raf-1 and G beta gamma were isolated from transfected cells.
Conclusions:
- A novel interaction between Raf-1 and G beta gamma subunits was identified.
- This interaction provides a molecular link for potential signaling cross-talk between the MAPK pathway and G-protein coupled receptor pathways.
- The findings suggest a new mechanism for integrating signals from growth factor and G-protein coupled receptors.