Gα and Gβγ require distinct Src-dependent pathways to activate Rap1 and Ras

Abstract

The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the β-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gβγ subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, β-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the β2-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Gαs to Rap1 and ERKs and the other through Gβγ to Ras and AKT.