Multiple kinase regulation of the nuclear factor of activated T cells

The nuclear localization and transcriptional activity of the NF-ATc family of transcription factors, essential to many developmental, differentiation, and adaptation processes, are determined by the opposing activities of the phosphatase calcineurin, which promotes nuclear accumulation of NF-ATc, and several kinases, which promote cytoplasmic accumulation. Here we show that PKA opposes calcineurin-mediated dephosphorylation and nuclear accumulation of NF-ATc1 by phosphorylating serines S245, S269, and S294 and that mutation of these serines blocks PKA's effect. Activation of endogenous PKA is similarly able to promote phosphorylation of these sites on NF-ATc1 in two lymphoid cell lines. We further show that complete inhibition of NF-ATc1 nuclear localization by PKA requires a second kinase activity that can be supplied by glycogen synthase kinase-3 (GSK-3), and that mutation of either the PKA phosphorylation sites or the upstream GSK-3 sites inhibits PKA's effect. Thus, we propose that PKA and GSK-3 function in an hierarchical phosphorylation mechanism to negatively regulate the nuclear accumulation of NF-ATc1.; The NF-ATc family members are regulated at multiple phosphorylation sites by many potential kinases. The implications for differential regulation of NF-ATc family members by PKA and of multisite phosphorylation on the regulation of the NF-ATc family members is discussed.