MARCKS is phosphorylated and translocated by a calcium and phorbol ester-insensitive protein kinase c in c6 glioma cell membranes

MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a prominent substrate for conventional protein kinase C (PKC) isoforms and is believed to be involved in the regulation of membrane-cytoskeletal interactions. We have observed that MARCKS is released in an ATP-stimulated manner from the membrane fraction of digitonin-permeabilized C6 glioma cells. Addition of [732P|-ATP to the membrane fraction resulted in translocation and phosphorylation of MARCKS. thus indicating involvement of an active membrane-bound kinase. Pretreatment of cells with 2 μM 4/312-o-tetradecanoyl-phorbol- 13-acetate (/3-TPA). conditions shown to downregulate conventional and novel isoforms of PKC, did not inhibit the ATPdependent transiocation and phosphorylation of MARCKS. Under both normal and down-regulated conditions, the ATP-dependent translocation and phosphorylation of MARCKS was unaffected by EGTA, inhibited by staurosporinp and fris-indolylmaleimide, and inhibited by a calmodulin-binding domain MARCKS peptide, implicating a membrane-bound PKC isoform which does not have a requirement for calcium and is insensitive to down-regulation by 3-TPA. These findings support a novel mechanism by which MARCKS may be phosphorylated and translocated by an atypical PKC isoform m vivo.