Activation of STAT3 by Gαs 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 G alpha(s)Q226L (G alpha(s)QL), a constitutively active mutant of G alpha(s), to stimulate STAT3 Tyr(705) and Ser(727) phosphorylations in human embryonic kidney 293 cells. Apart from G alpha(s)QL, the stimulation of G alpha(s) by cholera toxin or beta(2)-adrenergic receptor and the activation of adenylyl cyclase by forskolin, (S-p)-cAMP, or dibutyryl-cAMP all promoted both STAT3 Tyr705 and Ser727 phosphorylations. Moreover, the removal of G alpha(s) by RNA interference significantly reduced the beta(2)-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 G alpha(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 beta 2-adrenergic receptor stimulation. In contrast to the G alpha(16)-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 G alpha(s)-mediated responses, including STAT3-driven luciferase activation, were resistant to inhibition of phospholipase C beta. Surprisingly, G alpha(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 G alpha-mediated activations of STAT3, the stimulatory signal arising from G alpha s is transduced via multiple signaling pathways. However, unlike the mechanisms employed by G alpha(i) and G alpha(14/16), G alpha(s) distinctively requires protein kinase A, JNK, and phosphatidylinositol 3-kinase for STAT3 activation.