Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS).
We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.
Goudenege
Sébastien
S
Laboratoire Biosciences de 1'Aliment, USC-INRA 2009 Université Bordeaux I, Talence, France.
Poussard
Sylvie
S
Dulong
Sandrine
S
Cottin
Patrick
P
D000818:Animals; D002154:Calpain / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D021941:Caveolae / Q000378:metabolism*; D002451:Cell Compartmentation; D002459:Cell Fusion; D003600:Cytosol / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate