The APol Site at - 150 bp , but Not the NF - KB Site , Is Likely to Represent the Major Target of Protein Kinase C in the Interleukin 2 Promoter

Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the c~ and/8 isozymes of PKC, and abrogates induction of Ib2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in At-5 cells transfected with a plasmid containing the Ib2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NFxB, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-KB site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at 150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-Jbs and c-jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at 150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter.