Protein Kinase A Negatively Modulates the Nuclear Accumulation of NF-ATc1 by Priming for Subsequent Phosphorylation by Glycogen Synthase Kinase-3

Abstract

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 NFATc1 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.

A noncanonical sequence phosphorylated by casein kinase 1 in β-catenin may play a role in casein kinase 1 targeting of important signaling proteins

“…Through different mechanisms, masking a nuclear localization signal in the case of NF-AT4 or triggering proteolytic recognition for ␤-catenin, concerted phosphorylation involving CK1 and GSK-3 act to oppose the access to the nucleus of a transcription factor with the resulting block to proliferation. Pursuing the analogy further, the cAMP-dependent PKA also appears to participate in the priming phosphorylation of the NF-AT family of factors (30) and in the phosphorylation of Ser-45 of ␤-catenin bound to presenilin (54). In both cases, PKA acts to prime further phosphorylation by GSK-3.…”

“…Several kinases, including GSK-3␤, c-Jun N-terminal kinase (JNK), p38, extracellularly regulated kinase (ERK), protein kinase A (PKA), and CK2 have been reported to participate in the phosphorylation of the NF-AT protein family (26)(27)(28)(29)(30). In 1998, however, Zhu et al (20) showed that CK1␣ was bound to the regulatory region of NF-AT4, an interaction that enhanced its phosphorylation by this kinase.…”

PKCθ and PKA are antagonistic partners in the NF-AT transactivation pathway of primary mouse CD3+ T lymphocytes

“…Consistently, NF-AT mutants of the PKA phosphorylation sites lost 14-3-3 binding, translocated to the nucleus, and became transcriptionally active. 37 In contrast, upon TCR stimulation, NF-AT is dephosphorylated by calcineurin, which is established to functionally cooperate with PKC 38,39 in this pathway. This Ca 2ϩ /calcinerurin/PKC pathway leads to activation and nuclear entry of NF-AT and subsequent IL-2 production of the activated T cells.…”

Transcriptional Activation of hTERT, the Human Telomerase Reverse Transcriptase, by Nuclear Factor of Activated T Cells

“…The different hTERT-Luc reporter plasmids were transfected alone or cotransfected with HA-NFAT1 and/or CN expression vectors. HA-NFAT1 and CN expression vectors were transfected using a ratio of 5:1 based on the previous studies of Sheridan et al (45) and on the results of preliminary transfections (supplemental Figs. S3 and S4).…”