OVEREXPRESSION OF MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE ENHANCES ACTIVATION OF PHOSPHOLIPASE-D BY PROTEIN-KINASE-C IN SK-N-MC HUMANNEUROBLASTOMA-CELLS

Signal transduction can involve the activation of protein kinase C (PK C) and the subsequent phosphorylation of protein substrates, including myristoylated alanine-rich C kinase substrate (MARCKS). Previously we showed that stimulation of phosphatidylcholine (PtdCho) synthesis by PMA in SK-N-MC human neuroblastoma cells required overexpression of MA RCKS, whereas PKC alpha alone was insufficient. We have now investigat ed the role of MARCKS in PMA-stimulated PtdCho hydrolysis by phospholi pase D (PLD). Overexpression of MARCKS enhanced PLD activity 1.3-2.5-f old compared with vector controls in unstimulated cells, and 3-4-fold in cells stimulated with 100 nM PMA. PMA-stimulated PLD activity was b locked by the PKC inhibitor bisindolylmaleimide. Activation of PLD by PMA was linear with time to 60 min, whereas stimulation of PtdCho synt hesis by PMA in clones overexpressing MARCKS was observed after a 15 m in time lag, suggesting that the hydrolysis of PtdCho by PLD preceded synthesis. The formation of phosphatidylbutanol by PLD was greatest wh en PtdCho was the predominantly labelled phospholipid, indicating that PtdCho was the preferred, but not the only, phospholipid substrate fo r PLD. Cells overexpressing MARCKS had 2-fold higher levels of PKC alp ha than in vector control cells analysed by Western blot analysis; lev els of PKC beta and PLD were similar in all clones. The loss of both M ARCKS and PKC alpha expression at higher subcultures of the clones was paralleled by the loss of stimulation of PLD activity and PtdCho synt hesis by PMA. Our results show that MARCKS is an essential link in the PKC-mediated activation of PtdCho-specific PLD in these cells and tha t the stimulation of PtdCho synthesis by PMA is a secondary response.