Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C

We have previously demonstrated that 12-O-tetradecanoylphorbol-18-acetate ( TPA) activates human T-cell leukemia virus type-I long terminal repeat (LTR ) in Jurkat cells by a protein kinase C (PKC)-independent mechanism involvi ng a posttranslational activation of Sp1 binding to an Sp1 site located wit hin the Ets responsive region-1 (ERR-1). By employing the PKC inhibitor, bi sindolylmaleimide I and cotransfecting the reporter LTR construct with a ve ctor expressing PKC-alpha, we demonstrated, in the present study, that this effect of TPA was not only independent of, but actually antagonized by, PK C. Electrophoretic mobility shift assays together with antibody-mediated su pershift and immuno-coprecipitation analyses, revealed that the posttransla tional activation of Sp1 was exerted by inducing the formation of Sp1-p53 h eterocomplex capable of binding to the Sp1 site in ERR-1. Furthermore, we d emonstrated that Jurkat cells contain both wild-type (w.t) and mutant forms of p53 and we detected both of them in this complex at variable combinatio ns; some molecules of the complex contained either the wt. or the mutant p5 3 separately, whereas others contained the two of them together. Finally, w e showed that the Sp1-p53 complexes could bind also to an Sp1 site present in the promoter of another gene such as the cyclin-dependent kinase inhibit or p21(WAF-1), but not to consensus recognition sequences of the wt. p53. T herefore, we speculate that there might be several other PKC-independent bi ological effects of TPA which result from interaction of such Sp1-p53 compl exes with Sp1 recognition sites residing in the promoters of a wide variety of cellular and viral genes, (C) 2001 Academic Press.