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 activ ate multiple signaling pathways in parallel, including those involving Janu s kinase (JAK)-signal transducers and activators of transcription (STATs) [ 1], mitogen activated protein kinase (MAPK) [2], phosphatidylinositol 3 kin ase (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 independ ent protein kinase activities for maximal activation of target gene transcr iption [4]. Members of the JAk/Tyk family of tyrosine kinases mediate phosp horylation of STATE at Tyr705 during CNTF signaling; however, the kinase re sponsible 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 phosphoryla tion of Ser727 was inhibited by the mTOR inhibitor rapamycin, but not by in hibitors of MAPK and protein kinase C (PKC) activation, A STATE peptide was efficiently phosphorylated on Ser727 in a CNTF dependent manner by mTOR, b ut not by a kinase-inactive mTOR mutant or by p70 S6 kinase, In agreement w ith these biochemical studies, rapamycin treatment of cells transfected wit h a STAT-responsive promoter reporter decreased activation of the reporter to the same degree as a STATE Ser727Ala mutant. The ability of mTOR to cont ribute to activation of STATE extends the function of mTOR [9] in mammalian cells to include transcriptional regulation.