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 int eract with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by pro tein kinase C (PKC). In the present study, cytochalasin D (CD) and cal modulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD tr eatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhance d the amount of MARCKS translocated to the cytosolic fraction in respo nse to the phorbol ester 4 beta-12-O-tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated ph osphorylation of MARCKS or on translocation of PKC ct to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation wa s noted and bis-indolylmaleimide (a more specific PKC inhibitor) did n ot alter MARCKS distribution. Staurosporine inhibited the phorbol este r-induced translocation of MARCKS but not of PKC alpha in both CD pret reated and untreated cells. Calmodulin antagonists (trifluoperazine, c almidazolium) had little effect on the cellular distribution or phosph orylation of MARCKS, but were synergistic with phorbol ester in transl ocating MARCKS from the membrane without a further increase in its pho sphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activat ed PKC, but that interaction with both F-actin and calmodulin might se rve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.