Vol. 11, Issue 4, 1129-1142, April 2000

Activation of B-Raf and Regulation of the Mitogen-activated Protein Kinase Pathway by the G_o  chain

Valeria Antonelli,^* Francesca Bernasconi,^* Yung H. Wong, and Lucia Vallar^*

 ^*Department of Pharmacology, University of Milan, Consiglio Nazionale delle Ricerche, Cellular and Molecular Pharmacology Center, and Department for Biological and Technological Research, Scientific Institute San Raffaele, 20132 Milan, Italy; and  Department of Biology and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

Many receptors coupled to the pertussis toxin-sensitive G_i/o proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the  chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of G_o to regulate MAPK activity by transient expression of the activated mutant G_o-Q205L in Chinese hamster ovary cells. G_o-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. G_o-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated G_o stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by G_o. Moreover, G_o-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that G_o can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of G_o caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that G_o induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.