Activation of STAT3 by Gαs Distinctively Requires Protein Kinase A, JNK, and Phosphatidylinositol 3-Kinase

Andrew M.F. Liu,Helen Wise,Rico K.H. Lo,Yung H. Wong,Cecilia S.S. Wong,Christina Morris

doi:10.1074/jbc.m605288200

Abstract

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.

Highlights

Activation of G␣s Induces STAT3 Activations in HEK 293 Cells—We have previously demonstrated that hIP is coupled to both Gs and Gq for signal transduction [36, 37], and Gs may participate in mediating the hIP-induced STAT3 phosphorylations in human erythroleukemia (HEL) cells [29]
Numerical values shown above the immunoreactive bands represent relative intensities of G␣sQL-induced STAT3 phosphorylations expressed as a ratio of the basal level
Because the primary signal generated upon activation of G␣s is cAMP instead of inositol 1,4,5-trisphosphate/Ca2ϩ, it is hardly surprising that G␣s-mediated STAT3 phosphorylations employ components of the cAMP pathway (Fig. 1A) and are refractory to the inhibition of PLC␤ (Figs. 1D, 6A, and 10D)

Summary

Introduction

Activation can be attained via other pathways besides Ras. Inhibition of the Rac1/JNK pathway by overexpression of Rac1 negative mutant (Rac1DN) completely abolished G␣sQL-induced STAT3 phosphorylations at both Tyr705 and Ser727 (Fig. 2C) where wild type The G␣sQL-induced STAT3 Ser727 phosphorylation was independent of PLC␤ and appeared to be mediated via multiple signaling intermediates, including Ras/MEK1/2/ ERK, Rac1/JNK, c-Src, JAK2/3, and PI3K, whereas the G␣sQLinduced STAT3 Tyr705 phosphorylation did not require the participation of MEK1/2 or c-Src. Effects of Various Inhibitors on G␣sQL-induced STAT3-dependent Luciferase Activity—We have previously shown that G␣16QL is fully capable of inducing STAT3-driven luciferase expression and that this response is c-Src- and MEK1/2-dependent but does not require the participation of PI3K [26].

Results

Conclusion