Inhibitors of actin polymerization and calmodulin binding enhance protein kinase C-induced translocation of MARCKS in C6 glioma cells.

MARCKS (myristoylated alanine-rich C-kinase substrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4beta-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC alpha to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC alpha in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.