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Neuropoletic 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), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI 3-kinase) and mammalian target of rapamydn (mTOR)-p70 S6 kinase . 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. 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. 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 in mammalian cells to include transcriptional regulation.

Background: The small GTPase R-Ras displays a less potent transforming activity than the closely related Ras oncogene products. Although R-Ras has been reported to interact with c-Raf1 and Ral-GDS in vitro, the pathways by which it exerts its effects on cellular proliferation are not known., Results: Both Ras and R-Ras interact with phosphoinositide (PI) 3-kinase in vitro, and induce elevation of the levels of PI 3-kinase lipid products in intact cells. Unlike Ras, R-Ras does not activate Raf or mitogen-activated protein (MAP) kinase in cells. In co-transfection assays, the serine/threonine protein kinase PKB (or Akt) is effectively stimulated by R-Ras, Ras, mutants of Ras that activate PI 3-kinase but not other effectors, and activated forms of PI 3-kinase. Ras and R-Ras stimulate PKB/Akt through a non-autocrine mechanism that involves PI 3-kinase. The constitutive activation of PI 3-kinase alone is sufficient to activate PKB/Akt, but not the MAP kinase ERK or the stress-activated protein kinase, Jun N-terminal kinase. Transformation assays in fibroblasts suggest that PKB/Akt and Raf are part of distinct oncogenic signalling pathways., Conclusions: Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.