Evidence for a MARCKS-PKC alpha complex in skeletal muscle

MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a protein known t o cross-link actin filament and consequently. is very important in the stab ilization of the cytoskeletal structure. In addition, it has been recently demonstrated that the phosphorylation rate of this protein changes during m yogenesis and that this protein is implicated in fusion events. For a bette r understanding of the biological function of MARCKS during myogenesis, we have undertaken to identify and purify this protein from rabbit skeletal mu scle. Three chromatographic steps including an affinity calmodulin-agarose column were performed. The existence of a complex between the two proteins was confirmed by non-denaturing gel electrophoresis and immunoprecipitation . Two complexes were isolated which present an apparent molecular weight of about 600 kDa. Such interactions suggest that MARCKS is either a very good PKC alpha substrate and/or a regulator of PKC activity. These results are supported by previous studies showing preferential interactions and co-loca lization of PKC isozyme and MARCKS at focal adhesion sites. This is the fir st time that MARCKS has been purified from skeletal muscle and our data are consistent with a major role of this actin- and calmodulin-binding protein in cytoskeletal rearrangement or other functions mediated by PK alpha. Our results provide evidence for a tight and specific association of MARCKS an d PKC alpha (a major conventional PKC isozyme in skeletal muscle) as indica ted by the co-purification of the two proteins. (C) 2001 Elsevier Science L td. All rights reserved.