Evidence for a MARCKS-PKCalpha complex in skeletal muscle.
MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a protein known to cross-link actin filament and consequently, is very important in the stabilization of the cytoskeletal structure. In addition, it has been recently demonstrated that the phosphorylation rate of this protein changes during myogenesis and that this protein is implicated in fusion events. For a better understanding of the biological function of MARCKS during myogenesis, we have undertaken to identify and purify this protein from rabbit skeletal muscle. 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 PKCalpha substrate and/or a regulator of PKC activity. These results are supported by previous studies showing preferential interactions and co-localization of PKC isozyme and MARCKS at focal adhesion sites. This is the first 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 PKalpha. Our results provide evidence for a tight and specific association of MARCKS and PKCalpha (a major conventional PKC isozyme in skeletal muscle) as indicated by the co-purification of the two proteins.
Poussard
S
S
Laboratoire de Biochimie et Technologie des Aliments, ISTAB USC-INRA 429, Université Bordeaux I, Avenue des Facultés, 33405, Cedex, Talence, France. s.poussard@istab.u-bordeaux.fr
Dulong
S
S
Aragon
B
B
Jacques Brustis
J
J
Veschambre
P
P
Ducastaing
A
A
Cottin
P
P
D000818:Animals; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008565:Membrane Proteins; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000201:enzymology; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011817:Rabbits
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha