The AP-1 site at -150 bp, but not the NF-kappa B 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 alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, 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-kappa B 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-fos 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.
Jain
J
J
Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Valge-Archer
V E
VE
Sinskey
A J
AJ
Rao
A
A
D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000737:chemistry* / Q000235:genetics; D010703:Phorbol Esters / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D011493:Protein Kinase C / Q000235:genetics*; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry*; D013601:T-Lymphocytes / Q000502:physiology; D014162:Transfection
D007376:Interleukin-2; D016328:NF-kappa B; D010703:Phorbol Esters; D016755:Proto-Oncogene Proteins c-jun; D011493:Protein Kinase C