Protein kinase Calpha is a calpain target in cultured embryonic muscle cells.
Previously we isolated a micro-calpain/PKCalpha complex from skeletal muscle which suggested tight interactions between the Ca2+-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKCalpha and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture. Our results show that calpains and PKCalpha are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKCalpha content in the particulate fraction and the appearance of PKMalpha fragment. These results suggest a possible calpain dependent down-regulation process of PKCalpha in our model at the time of intense fusion. In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of PKCalpha in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKCalpha, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and micro-calpains). Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCalpha.
Aragon
B
B
Laboratoire de Biochimie et Technologie des Aliments, ISTAB USC-INRA 429, Université Bordeaux I, Talence, France.
Poussard
S
S
Dulong
S
S
Touyarot
K
K
Dargelos
E
E
Brustis
J J
JJ
Levieux
D
D
Ducastaing
A
A
Cottin
P
P
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D024510:Muscle Development / Q000187:drug effects; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology* / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors
D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; C410302:PKM-alpha protein, Bos taurus; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate