| Autor: |
Pumiglia, Kevin M., LeVine, Harry, Haske, Taraneh, Habib, Tania, Jove, Richard, Decker, Stuart J. |
| Zdroj: |
Journal of Biological Chemistry; June 1995, Vol. 270 Issue: 24 p14251-14254, 4p |
| Abstrakt: |
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 Gβ2subunit of heterotrimeric G-proteins. In vitro, purified Gβγsubunits 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 Gβγ. In competition experiments, the carboxyl terminus of β-adrenergic receptor kinase (βARK) blocked the binding of Gβγto Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitrobinding between Raf/330 and Gβγrevealed an affinity of interaction (Kd= 163 ± 36 nM), similar to that seen between Gβγand βARK (Kd= 87 ± 24 nM). The formation of native heterotrimeric Gαβγcomplexes, as measured by pertussis toxin ADP-ribosylation of Gα, could be disrupted by increasing amounts of Raf/330, with an EC50of approximately 200 nM, in close agreement with the estimated binding affinity. In vivocomplexes of Raf-1 and Gβγwere isolated from human embryonic kidney 293-T cells transfected with epitope-tagged Gβ2The 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. |
| Databáze: |
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