Long-term treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulates select
protein kinase C (PKC)
isozymes and may differentially affect PKC substrates. We investigated the role of PKC down-regulation on phosphorylation of two PKC substrates, the 43 kDa growth-associated
protein (GAP-43) and the
myristoylated alanine-rich C-kinase substrate (MARCKS) in SK-N-SH human
neuroblastoma cells. Cells were treated with 70 nM TPA for 15 min, 17 or 72 h. Phosphorylation of MARCKS and
GAP-43 was elevated throughout 72 h of TPA. The magnitude and peptidic sites of phosphorylation in
GAP-43 and MARCKS were similar after all TPA treatments.
GAP-43, but not MARCKS, content was increased after 17 and 72 h of TPA. The ratio of
GAP-43 phosphorylation to content was elevated throughout 17 h but returned to control by 72 h as content increased.
PKC epsilon and alpha
isozyme content was greatly reduced after 72 h of TPA but membranes retained 23% of PKC activity. Only
PKC epsilon translocated to membranes after 15 min TPA.
GAP-43 content after 72 h of TPA was increased in subcellular fractions in which significant
PKC epsilon isozyme concentration remained. These results demonstrate that continuous TPA differentially affected phosphorylation of PKC substrate
proteins and regulation of PKC
isozyme content in SK-N-SH cells.