Phorbol ester-stimulated phosphatidylcholine synthesis in human SK-N-MC neuroblastoma cells requires both protein kinase C and marcks

SK-N-MC human neuroblastoma cells, unlike some other neural cells in culture, do not increase phosphatidylcholine (PtdCho) synthesis in response to 43-12-0-tetradecanoylphorbol-13-acetate (TPA). This has been correlated with low levels of protein kinase C-a and Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) in SK-N-MC cells. To directly test for a requirement for PKC-a and MARCKS, we generated clones of SK-N-MC cells stably overexpressing human PKC-a (30-40 fold) or rat MARCKS (4-5 fold). The synthesis of PtdCho from [3H]choline was stimulated 1.8-2.3 fold by 100 nM TPA in four clones overexpressing MARCKS but only 1.1-1.4 fold in cells overexpressîng PKC-a or in vector controls. Stimulation was abolished by the PKC inhibitor, 6is-indolylmaleimide, or by down-regulation of PKC. Treatment with 100 M oleic acid stimulated PtdCho synthesis 2-4 fold in all clones. Clones overexpressing MARCKS showed 2-8 fold higher levels of membrane-associated PKC-a protein compared to vector controls in both basal and TPA-stimulated conditions, suggesting that MARCKS over-expression may regulate PKC-a expression. No increases in PKC-/3 or PKC-£ , the only other isoforms in these cells, were observed. Thus, both MARCKS and PKC are required for TPA-stimulated synthesis of PtdCho in SK-N-MC cells and there may be a coordinated interaction between MARCKS and PKC-α expression.