The Role of Protein Kinase C Signaling in Activated DRA Transcription

Expression of human MHC HLA-DRA class II gene can be up-regulated in B cells by Ig cross-linking as well as by phorbol esters such as 12- O -tetradecanoyl phorbol 13-acetate (TPA). Induced DRA expression involves activation of restricted protein kinase C (PKC) isoforms, resulting in activated activator protein-1-dependent transcription. In this report expression profiles and activation of PKC were analyzed in human Raji B lymphoblastoid cells. Transient transfection analysis with target plasmids containing either DRA promoter (wild-type or mutated) or TPA response elements demonstrated that pretreatment with the selective PKC inhibitor GF 109203X repressed TPA-mediated activation. Western analysis performed on cellular fractions of resting cells and of TPA-activated cells revealed abundant expression of classical PKC-α (cPKC-α), cPKC-βII, and atypical PKC-ζ isoforms and identified a sustained translocation of cPKC-α and cPKC-βII from the cytosolic compartment to membranes. As expected, the distribution of atypical PKC-ζ was unaffected by TPA treatment and displayed an even distribution between cytosol and membranes. This finding was confirmed by immunofluorescence microscopy. The TPA-mediated translocation of cPKC-α and cPKC-βII was not influenced by pretreatment with GF 109203X. Finally, functional activation and translocation of PKC were investigated with a selective in vitro kinase assay. Together, these results show that activated HLA-DRA expression in response to TPA treatment is strictly dependent on PKC activation acting on the X2 box of the DRA promoter and that selective inhibition of PKC enzymatic activity does not influence subcellular localization of expressed PKC isoenzymes. Thus, the translocation event per se occurs independently of PKC activation in these cells.