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 ␤-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 G␤␥ subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, ␤-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 ␤ 2 -adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through G␣ s to Rap1 and ERKs and the other through G␤␥ to Ras and AKT.

PKA phosphorylation of Src mediates Rap1 activation in NGF and cAMP signaling in PC12 cells

“…2E). These data demonstrate that Rap1 and ERKs can be activated by a PKA/SFK-dependent mechanism in PC12 cells, as has been shown for other cell types (Schmitt and Stork, 2002a).…”

“…SrcS17A has been shown to selectively block the ability of Journal of Cell Science 117 (25) cAMP to activate Src in other cell types (Schmitt and Stork, 2002a). Cells were transfected with Flag-tagged wild-type Src or SrcS17A along with Flag-Rap1 and assayed for Rap1 activation following forskolin/IBMX treatment.…”

G Protein regulation of MAPK networks

“…Further studies have indicated that in many cell types, Ga s -mediated activation of ERK1/2 signaling via Rap-1 involves novel Rap-1-GEF known as Crk SH3 domain-binding guanine nucleotide-releasing factor, or C3G (Tanaka et al, 1994;Gotoh et al, 1995). It should be noted here that unlike the direct activation of EPAC by cAMP (de Rooij et al, 1998;Quilliam et al, 2002;Bos, 2006), the activation of C3G requires the PKA-mediated phosphorylation of Ser-17 of Src (Schmitt and Stork, 2002a;Obara et al, 2004;Weissman et al, 2002;Wang et al, 2006b), subsequent Src-mediated activation of Cbl, Cbl-mediated recruitment of Crk-C3G complex and Crk-SH3 domain-mediated recruitment as well as activation of C3G (Ichiba et al, 1999;Stork, 2000, 2002a). Results from these studies establish a paradigm in which Ga s stimulates ERK1/2 via a pathway involving cAMP-PKA-Src-mediated activation of C3G, C3G stimulation of Rap-1 and, finally, Rap-1-mediated activation of B-Raf-MEK-ERK module (Schmitt and Stork, 2000;Weissman et al, 2002;Obara et al, 2004;Wang et al, 2006b).…”

Beyond the dogma: novel 2-adrenoceptor signalling in the airways

“…Again, this process appears to involve a PKA-dependent phosphorylation of Src, or a Src-like kinase, leading to the activation of the small GTPase, Rap1, and subsequent repression of the RafRMKK1/2RERK cascade [214]. However, it is important to note that, in a different cell line, this signalling cascade can also be activated via PKA-dependent phosphorylation of Src [215]. Clearly, these data demonstrate the potential diversity of b 2 -adrenoceptor signalling and highlight the importance of characterising physiologically relevant responses under physiological conditions in nontransformed cells of the tissue of interest.…”