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Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR
K Yokogami1, S Wakisaka, J Avruch
1Molecular Neuro-Oncology, Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston 02129, USA.
Abstract:
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.
Insights
Mammalian target of rapamycin (mTOR) directly phosphorylates Signal Transducer and Activator of Transcription 3 (STAT3) on Ser727, impacting gene transcription. This finding reveals a novel role for mTOR in regulating STAT3 activity and cellular responses.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Neuroscience
Background:
- Ciliary neurotrophic factor (CNTF) activates multiple parallel signaling pathways, including JAK-STAT, MAPK, PI3K, and mTOR-p70 S6 kinase.
- Signal Transducer and Activator of Transcription 3 (STAT3) requires phosphorylation on both tyrosine and serine residues for maximal transcriptional activation.
- While JAK/Tyk kinases mediate STAT3 Tyr705 phosphorylation, the kinase for STAT3 Tyr727 phosphorylation is stimulus and context-dependent.
Purpose of the Study:
- To investigate the specific kinase activity responsible for STAT3 Ser727 phosphorylation in CNTF-stimulated neuroblastoma cells.
- To elucidate the role of mammalian target of rapamycin (mTOR) in STAT3 activation.
Main Methods:
- Utilized CNTF stimulation in neuroblastoma cells.
- Employed inhibitors for mTOR, MAPK, and protein kinase C (PKC).
- Performed in vitro kinase assays using a STAT3 peptide and mTOR/p70 S6 kinase.
- Assessed STAT3-responsive reporter gene activation in rapamycin-treated cells.
Main Results:
- CNTF-induced STAT3 Ser727 phosphorylation was significantly inhibited by the mTOR inhibitor rapamycin.
- MAPK and PKC inhibitors did not affect CNTF-induced STAT3 Ser727 phosphorylation.
- In vitro, mTOR directly phosphorylated a STAT3 peptide on Ser727 in a CNTF-dependent manner, unlike p70 S6 kinase or a kinase-inactive mTOR mutant.
- Rapamycin treatment reduced STAT3-responsive reporter gene activation similarly to a STAT3 Ser727Ala mutant.
Conclusions:
- Mammalian target of rapamycin (mTOR) is a key kinase responsible for STAT3 Ser727 phosphorylation in response to CNTF.
- This finding establishes a direct link between mTOR activity and STAT3-mediated transcriptional regulation.
- The study expands the known functions of mTOR to include direct control over STAT3's role in gene transcription.