Protein kinase C-theta participates in the activation of cyclic AMP-responsive element-binding protein and its subsequent binding to the-180 site of the IL-2 promoter in normal human T lymphocytes

IL-2 gene expression is regulated by the cooperative binding of discrete tr anscription factors to the IL-2 promoter/enhancer and is predominantly cont rolled at the transcriptional level. In this study, we show that in normal T cells, the - 180 site (-164/-189) of the IL-2 promoter/enhancer is a p-cA MP-responsive element-binding protein (p-CREB) binding site. Following acti vation of the T cells through various membrane-initiated and membrane-indep endent pathways, protein kinase C (PKC)-theta phosphorylates CREB, which su bsequently binds to the -180 site and associates with the transcriptional c oactivator p300. Rottlerin, a specific PKC-theta inhibitor, diminished p-CR EB protein levels when normal T cells were treated with it. Rottlerin also prevented the formation of p-CREB/p300 complexes and the DNA-CREB protein b inding. Cotransfection of fresh normal T cells with luciferase reporter con struct driven by two tandem - 180 sites and a PKC-theta construct caused a significant increase in the transcription of the reporter gene, indicating that this site is functional and regulated by PKC-theta. Cotransfection of T cells with a luciferase construct driven by the -575/+57 region of the IL -2 promoter/enhancer and a PKC-theta construct caused a similar increase in the reporter gene transcription, which was significantly limited when two bases within the -180 site were mutated. These findings show that CREB play s a major role in the transcriptional regulation of IL-2 and that a major p athway for the activation of CREB and its subsequent binding to the IL-2 pr omoter/enhancer in normal T cells is mediated by PKC-theta.