TI  - A direct interaction between  <prot>G-protein beta gamma</prot>  subunits and the  <prot>Raf-1 protein kinase</prot> .
PG  - 14251-4
AB  - <prot>Raf-1</prot> is a serine/threonine protein kinase positioned downstream of <prot>Ras</prot> in
      the <prot>mitogen-activated protein kinase</prot> cascade. Using a yeast two-hybrid
      strategy to identify other proteins that interact with and potentially
      regulate  <prot>Raf-1</prot> , we isolated a clone encoding the carboxyl-terminal half of
      the <prot>G beta 2 subunit</prot> of heterotrimeric  G-proteins . In vitro, purified  <prot>G
      beta gamma</prot>  subunits specifically bound to a GST fusion protein encoding
      amino acids 1-330 of  <prot>Raf-1</prot>  (<prot>Raf/330</prot>). Binding assays with truncation
      mutants of  GST-<prot>Raf</prot>  indicate that the region located between amino acids
      136 and 239 is a primary determinant for interaction with  <prot>G beta gamma</prot> . In
      competition experiments, the carboxyl terminus of <prot>beta-adrenergic receptor
      kinase</prot> (<prot>beta ARK</prot>) blocked the binding of  <prot>G beta gamma</prot>  to  <prot>Raf/330</prot>; however,
      the   <prot>Raf-1</prot>  -binding proteins,  <prot>Ras</prot>  and  14-3-3 , had no effect. Scatchard
      analysis of in vitro binding between  <prot>Raf/330</prot> and  <prot>G beta gamma</prot>  revealed an
      affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between
       <prot>G beta gamma</prot>  and  <prot>beta ARK</prot>  (Kd = 87 +/- 24 nM). The formation of native
      heterotrimeric <prot>G alpha beta gamma</prot> complexes, as measured by pertussis
      toxin ADP-ribosylation of G alpha, could be disrupted by increasing
      amounts of <prot>Raf/330</prot>, with an EC50 of approximately 200 nM, in close
      agreement with the estimated binding affinity. In vivo complexes of  <prot>Raf-1</prot> 
      and  <prot>G beta gamma</prot>  were isolated from human embryonic kidney 293-T cells
      transfected with epitope-tagged <prot>G beta 2</prot>. 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.
AD  - Parke-Davis Pharmaceutical Research Division, Department of Signal
