Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR

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) [1], mitogen activated protein kinase (MAPK) [2], phosphatidylinositol 3 kinase (PI 3 kinase) and mammalian target of rapamycin (mTOR)-p70 S6 kinase [3], Crosstalk occurs between these pathways, because studies have shown that STATE requires phosphorylation on tyrosine and serine residues by independent protein kinase activities for maximal activation of target gene transcription [4]. Members of the JAk/Tyk family of tyrosine kinases mediate phosphorylation of STATE at Tyr705 during CNTF signaling; however, the kinase responsible for phosphorylation at STATE Tyr727 appears to depend on both the extracellular stimulus and the cellular context [5-8], Here we investigate the kinase activity responsible for phosphorylation of STATE 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 STATE 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 STATE Ser727Ala mutant. The ability of mTOR to contribute to activation of STATE extends the function of mTOR [9] in mammalian cells to include transcriptional regulation.