MTORC1 Promotes T-bet Phosphorylation To Regulate Th1 Differentiation.

ABSTRACT: CD4+ T cells lacking the mTORC1 activator Rheb fail to secrete IFN-? under Th1 polarizing conditions. We hypothesized that this phenotype is due to defects in regulation of the canonical Th1 transcription factor T-bet at the level of protein phosphorylation downstream of mTORC1. To test this hypothesis, we employed targeted mass-spectrometry proteomic analysis-multiple reaction monitoring mass spectrometry. We used this method to detect and quantify predicted phosphopeptides derived from T-bet. By analyzing activated murine wild-type and Rheb-deficient CD4+ T cells, as well as murine CD4+ T cells activated in the presence of rapamycin, a pharmacologic inhibitor of mTORC1, we were able to identify six T-bet phosphorylation sites. Five of these are novel, and four sites are consistently dephosphorylated in both Rheb-deficient CD4+ T cells and T cells treated with rapamycin, suggesting mTORC1 signaling controls their phosphorylation. Alanine mutagenesis of each of the six phosphorylation sites was tested for the ability to impair IFN-? expression. Single phosphorylation site mutants still support induction of IFN-? expression; however, simultaneous mutation of three of the mTORC1-dependent sites results in significantly reduced IFN-? expression. The reduced activity of the triple mutant T-bet is associated with its failure to recruit chromatin remodeling complexes to the Ifng gene promoter. These results establish a novel mechanism by which mTORC1 regulates Th1 differentiation, through control of T-bet phosphorylation.

SUBMITTER: Chornoguz O 

PROVIDER: S-EPMC5458608 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature