Activation of STAT3 by G alpha(s) distinctively requires protein kinase A, JNK, and phosphatidylinositol 3-kinase.

Signal transducer and activator of transcription 3 (STAT3) can be stimulated by several G(s)-coupled receptors, but the precise mechanism of action has not yet been elucidated. We therefore examined the ability of Galpha(s)Q226L (Galpha(s)QL), a constitutively active mutant of Galpha(s), to stimulate STAT3 Tyr705 and Ser727 phosphorylations in human embryonic kidney 293 cells. Apart from Galpha(s)QL, the stimulation of Galpha(s) by cholera toxin or beta2-adrenergic receptor and the activation of adenylyl cyclase by forskolin, (Sp)-cAMP, or dibutyryl-cAMP all promoted both STAT3 Tyr705 and Ser727 phosphorylations. Moreover, the removal of Galpha(s) by RNA interference significantly reduced the beta2-adrenergic receptor-mediated STAT3 phosphorylations, denoting its capacity to regulate STAT3 activation by a G protein-coupled receptor. The possible downstream signaling molecules involved were assessed by using specific inhibitors and dominant negative mutants. Induction of STAT3 Tyr705 and Ser727 phosphorylations by Galpha(s)QL was suppressed by inhibition of protein kinase A, Janus kinase 2/3, Rac1, c-Jun N-terminal kinase (JNK), or phosphatidylinositol 3-kinase, and a similar profile was observed in response to beta2-adrenergic receptor stimulation. In contrast to the Galpha16-mediated regulation of STAT3 in HEK 293 cells (Lo, R. K., Cheung, H., and Wong, Y. H. (2003) J. Biol. Chem. 278, 52154-52165), the Galpha(s)-mediated responses, including STAT3-driven luciferase activation, were resistant to inhibition of phospholipase Cbeta. Surprisingly, Galpha(s)-mediated phosphorylation at Tyr705, but not at Ser727, was resistant to inhibition of c-Src, Raf-1, and MEK1/2 as well as to the expression of dominant negative Ras. Therefore, as with other Galpha-mediated activations of STAT3, the stimulatory signal arising from Galpha(s) is transduced via multiple signaling pathways. However, unlike the mechanisms employed by Galpha(i) and Galpha(14/16), Galpha(s) distinctively requires protein kinase A, JNK, and phosphatidylinositol 3-kinase for STAT3 activation.