REGULATION OF NONCLASSICAL PROTEIN-KINASE-C ISOENZYMES IN A HUMAN T-CELL LINE

We have examined the expression and responses to activation, of novel/ atypical protein kinase C (PKC) isoforms epsilon,zeta, and delta in th e T cell lymphoma cell line K-4. The effects of 1-h phorbol 12-myrista te 13-acetate (PMA) and OKT3 activation of K-4 cells on PKC isoform di stribution were examined. In addition, the effects of PMA-mediated dow n-regulation on the expression of PKC epsilon and zeta were determined using high concentrations of PMA over 24- and 48-h time periods in th ese cells. PKC zeta expression was not altered by incubation of K-4 ce lls with up to 200 ng/ml PMA over a 24- or 48-h period. PKC epsilon wa s down-regulated in a concentration-dependent manner by PMA after both 24- and 48-h of activation. Expression of PKC epsilon was not complet ely depressed, however, even at the highest concentration of the phorb ol ester after 48-h incubation with PMA. The presence of PKC epsilon,z eta, and delta was confirmed by immunohistochemistry with distinct pat terns of expression observed. PMA-induced PKC activation for a l-h per iod resulted in a translocation of PKC delta from resting cytoplasmic/ nuclear staining to a cytoplasmic aggregate. Following 1-h activation through the T cell receptor-associated complex CD3, PKC delta transloc ated from a peri-nuclear/cytoplasmic compartment to a putative cytoske letal location in K-4 cells. This translocation was time dependent and redistributed to a cytoplasmic aggregate prior to the cytoskeleton. S imilarily, following l-h activation through the T cell receptor, PKC z eta redistributed directly to what is possibly a cytoskeletal cell com partment. The cytoplasmic distribution of PKC zeta was unaltered follo wing activation with PMA over a 1-h time period. There was no apparent redistribution of PKC epsilon cytoplasmic staining pattern following a 1-h direct or indirect activation. These results underline the diffe rences in individual PKC isoform distribution, and responses to differ ent stimuli, thereby providing additional evidence for the use of disc rete PKC isoform signaling pathways in T cells. Furthermore, this data underlines the differences in PMA-mediated PKC activation and activat ion through the T cell receptor.