Abstract
Calcineurin, a Ca(2+)/calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. Calcineurin is the target of the immunosuppressive drugs cyclosporin A and FK506 complexed with their cytoplasmic receptors cyclophilin and FKBP12, respectively. In this study we report that calcineurin is also the target of a recently identified Ca(2+)-binding protein, CHP (for calcineurin homologous protein), which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin. In Jurkat and HeLa cells, overexpression of CHP specifically impaired the nuclear translocation and transcriptional activity of NFAT but had no effect on AP-1 transcriptional activity and only a small (<25%) inhibitory effect on the transcriptional activity of NFkappaB. Further study indicated that CHP inhibits calcineurin activity. In cells overexpressing CHP, the phosphatase activity of immunoprecipitated calcineurin was inhibited by approximately 50%; and in a reconstituted assay, the activity of purified calcineurin was inhibited up to 97% by the addition of purified recombinant CHP in a dose-dependent manner. Moreover, prolonged activation of Jurkat cells was associated with a decreased abundance of CHP, suggesting a possible regulatory mechanism allowing activation of calcineurin. CHP, therefore, is a previously unrecognized endogenous inhibitor of calcineurin activity.
Highlights
Calcineurin, a Ca2؉/calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT, a family of transcription factors required for the synthesis of several cytokine genes
In this study we report that calcineurin is the target of a recently identified Ca2؉-binding protein, CHP, which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin
Calcineurin is a heterodimeric enzyme consisting of a catalytic A subunit (CnA) and a tightly associated Ca2ϩ-binding regulatory B subunit (CnB) [12, 13]
Summary
Calcineurin, a Ca2؉/calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. The ability of CHP to inhibit NFAT nuclear translocation and transcriptional activity suggested that CHP, like CaM, might regulate calcineurin activity. In CHP-transfected cells, basal [Ca2ϩ]i and the stimulus-induced elevation of [Ca2ϩ]i were similar to those in vector controls (Fig. 3), which suggested that inhibition of NFAT activity by CHP was not because of impaired intracellular Ca2ϩ dynamics.
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