Cot/Tpl2 and PKCζ cooperate in the regulation of the transcriptional activity of NFATc2 through the phosphorylation of its amino-terminal domain

Nuclear factor of activated T cells (NFAT) plays a prominent role in gene transcription during the immune response. Growing evidence demonstrates the implication of inducible phosphorylation of the transactivation domain (TAD) of NFAT in transcriptional activation of genes. We have analyzed the regulation of NFATc2 activation by Cot/Tpl2 and protein kinase C ζ (PKCζ) in T cells. Our results show that PKCζ and Cot/Tpl2 cooperate in regulating the transactivation activity mediated by the amino-terminal domain of NFATc2. Neither Cot/Tpl2 kinase nor PKCζ-mediated induction of the transactivation activity of NFATc2 was affected by cyclosporin-A treatment, supporting a calcineurin independent route in the signaling pathways mediating NFATc2 activation. Co-precipitation experiments showed physical interaction among Cot/Tpl2, PKCζ and NFATc2. Analysis of the transactivation activity of deletions in the N-terminal region of NFATc2, suggested the involvement of amino acids 52–64 of NFATc2 in the induction of its transactivating function by PKCζ. This kinase in vitro phosphorylates NFATc2 and deletion and mutational studies identified Ser53 and Ser56 (of the SPPS motif) as substrates for PKCζ. Thus, our results suggest that PKCζ phosphorylation of Ser53 and Ser56 in the N-terminal TAD from NFATc2 potentiates its transactivating function in human T cells.