Regulation of Th17 Differentiation by IKKα-Dependent and - Independent Phosphorylation of RORγt

Transcription factor retinoid acid-related orphan receptor (ROR)gamma t transcriptionally regulates the genes required for differentiation of Th17 cells that mediate both protective and pathogenic immunity. However, little is known about the function of posttranslational modifications in the regulation of ROR gamma t activity. Mass spectrometric analysis of immunoprecipitated ROR gamma t from Th17 cells identified multiple phosphorylation sites. Systematic mutation analysis of the identified phosphorylation sites found that phosphorylation of S376 enhances whereas phosphorylation of S484 inhibits Th17 differentiation. I kappa B kinase (IKK) a binds and phosphorylates ROR gamma t at S376 but not S484. Knockdown of IKK alpha, dominant-negative IKKa, and ROR gamma t mutants incapable of interacting with IKK alpha all decrease Th17 differentiation. Furthermore, nonphosophorylatable ROR gamma t mutant (S376A) impairs whereas phosphomimetic mutant (S376E) stimulates Th17 differentiation independent of IKK alpha. Therefore, IKK alpha-dependent phosphorylation of S376 stimulated whereas IKK alpha-independent phosphorylation of S484 inhibited ROR gamma t function in Th17 differentiation.