Protein kinase A negatively modulates the nuclear accumulation of NF-ATc1 by priming for subsequent phosphorylation by glycogen synthase kinase-3.
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. Many reports suggest that protein kinase A (PKA) negatively modulates calcineurin-mediated NF-ATc activation. Here we show that overexpression of PKA causes phosphorylation and cytoplasmic accumulation of NF-ATc1 in direct opposition to calcineurin by phosphorylating Ser-245, Ser-269, and Ser-294 in the conserved serine-proline repeat domain, and that mutation of these serines blocks the effect of PKA. 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 a complete block 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 prevents the effect of PKA. Thus, we propose that PKA functions cooperatively as a priming kinase for further phosphorylation by GSK-3 to oppose calcineurin-mediated nuclear accumulation and transcriptional activity of NF-ATc1 and that, through this mechanism, PKA may be an important modulator of many NF-ATc-dependent processes.
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D012694:Serine; D015759:Ionomycin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D002118:Calcium