Continuous phosphorylation of GAP-43 and MARCKS by long-term TPA treatment in SK-N-SH human neuroblastoma cells.
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.
Goldsmith
A M
AM
Department of Pharmacology, University of Michigan Medical School, 2220E MSRB III, Ann Arbor, MI 48109-0632, USA.
Gnegy
M E
ME
D004789:Enzyme Activation / Q000187:drug effects; D019922:GAP-43 Protein / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009447:Neuroblastoma; D010766:Phosphorylation / Q000187:drug effects*; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D019922:GAP-43 Protein; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate