PHORBOL ESTER-INDUCED AND RETINOIC ACID-INDUCED REGULATION OF THE PROTEIN-KINASE-C SUBSTRATE MARCKS IN IMMORTALIZED HIPPOCAMPAL CELLS

The expression of MARCKS, a major protein kinase C (PKC) substrate, wa s examined in the immortalized hippocampal cell line HN33, following d ifferentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an appa rent PKC-dependent mechanism. Exposure to 1 mu M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity i n the soluble fraction with a concurrent increase in membrane-associat ed PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Si gnificant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h , respectively. The reduction in MARCKS protein levels was maximal fol lowing 24 h of PMA exposure. MARCKS protein expression was also down-r egulated in a dose-dependent manner on exposure of HN33 cells to retin oic acid. In cells exposed to 10 mu M retinoic acid, the MARCKS protei n level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF 109203X blocked the down-regulation of MARCKS protein in PMA-treated c ultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.