Title: Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR
Abstract: Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) can activate multiple signaling pathways in parallel, including those involving Janus kinase (JAK)–signal transducers and activators of transcription (STATs) [1Heinrich P.C. Behrmann I. Muller N.G. Schaper F. Graeve L. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway.Biochem J. 1998; 334: 297-314Crossref PubMed Scopus (1658) Google Scholar], mitogen-activated protein kinase (MAPK) [2Servidei T. Aoki Y. Lewis S.E. Symes A. Fink J.S. Reeves S.A. Coordinate regulation of STAT signaling and c-fos expression by the tyrosine phosphatase SHP-2.J Biol Chem. 1998; 273: 6233-6241Crossref PubMed Scopus (49) Google Scholar], phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamycin (mTOR)–p70 S6 kinase [3Oh H. Fujio Y. Kunisada K. Hirota H. Matsui H. Kishimoto T. et al.Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes.J Biol Chem. 1998; 273: 9703-9710Crossref PubMed Scopus (183) Google Scholar]. Crosstalk occurs between these pathways, because studies have shown that STAT3 requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription [4Wen Z. Zhong Z. Darnell J.J. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation.Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1680) Google Scholar]. Members of the JAK/Tyk family of tyrosine kinases mediate phosphorylation of STAT3 at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STAT3 Tyr727 appears to depend on both the extracellular stimulus and the cellular context [5Chung J. Uchida E. Grammer T.C. Blenis J. Stat3 serine phosphorylation by ERK-dependent and-independent pathways negatively modulates its tyrosine phosphorylation.Mol Cell Biol. 1997; 17: 6508-6516Crossref PubMed Scopus (532) Google Scholar, 6Ng J. Cantrell D. Stat3 is a serine kinase target in T lymphocytes. Interleukin 2 and T cell antigen receptor signals converge upon serine 727.J Biol Chem. 1997; 272: 24542-24549Crossref PubMed Scopus (126) Google Scholar, 7Sengupta T.K. Talbot E.S. Scherle P.A. Ivashkiv L.B. Rapid inhibition of interleukin-6 signaling and stat3 activation mediated by mitogen-activated protein kinases [In Process Citation].Proc Natl Acad Sci USA. 1998; 95: 11107-11112Crossref PubMed Scopus (199) Google Scholar, 8Ihle J.N. STATs and MAPKs: obligate or opportunistic partners in signaling.BioEssays. 1996; 18: 95-98Crossref PubMed Scopus (70) Google Scholar]. Here we investigate the kinase activity responsible for phosphorylation of STAT3 on Ser727 in CNTF-stimulated neuroblastoma cells. We found that CNTF-induced phosphorylation of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by inhibitors of MAPK and protein kinase C (PKC) activation. A STAT3 peptide was efficiently phosphorylated on Ser727 in a CNTF-dependent manner by mTOR, but not by a kinase-inactive mTOR mutant or by p70 S6 kinase. In agreement with these biochemical studies, rapamycin treatment of cells transfected with a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STAT3 Ser727Ala mutant. The ability of mTOR to contribute to activation of STAT3 extends the function of mTOR [9Hall M.N. The TOR signalling pathway and growth control in yeast.Biochem Soc Trans. 1996; 24: 234-239Crossref PubMed Scopus (45) Google Scholar] in mammalian cells to include transcriptional regulation.