Abstract
Previously we isolated a μ-calpain/PKCα 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 PKCα 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 PKCα 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 PKCα content in the particulate fraction and the appearance of PKMα fragment. These results suggest a possible calpain dependent down-regulation process of PKCαa in our model at the time of intense fusion.
In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of pkcα 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 PKCα, 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 μ-calpains).
Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCα.
Similar content being viewed by others
References
Huang Y, Wang KKW: The calpain family and human disease. Trends Mol Med 7: 355–361, 2001
Carafoli E, Molinari M: Calpain: A protease in search of a function? Biochem Biophys Res Commun 247: 193–203, 1998
Croall DE, Demartino GN: Calcium-activated neutral protease (calpain) system: Structure, function and regulation. Physiol Rev 71: 813–847, 1991
Mellgren RL: Evidence for participation of a calpain-like cysteine protease in cell cycle progression through late G1 phase. Biochem Biophys Res Comm 236: 555–558, 1997
Suzuki K, Sorimachi H: A novel aspect of calpain activation. FEBS Lett 433: 1–4, 1998
Richard I, Broux O, Allamand V, Fougerousse F, Chiannikulchai N, Bourg N, Brenguier L, Devaud C, Pasturaud P, Roudaut C, Hillaire D, Passos-Bueno MR, Zatz M, Tischfield JA, Fardeau M, Jackson CE, Cohen D, Beckman JS: Mutation in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27–40, 1995
Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI: Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet 26:163–175, 2000
Nishizuka Y: Intracellular signalling by hydrolysis phospholipids and activation of protein kinase C. Science 258: 607–614, 1992
Liu J-P: Protein kinase C and its substrates. Mol Cell Endocrinol 116: 1–29, 1996
Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y: The structure expression and properties of additional members of the protein kinase C family. J Biol Chem 263: 6927–6932, 1988
Al Z, Cohen CM: Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: Possible role of protein kinase M. Biochem J 296: 675–683, 1993
Young S, Parker PJ, Ullrich A, Stabel S: Down-regulation of protein kinase C is due to an increased rate of degradation. Biochem J 244: 775–779, 1987
Jaken S: Protein kinase C isozymes and substrates. Curr Opin Cell Biol 8: 168–173, 1996
Jaken S, Parker PJ: Protein kinase C binding partners. BioEssays 22: 245–254, 2000
Meacci E, Vasta V, Donati C, Farnararo M, Bruni P: Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C. FEBS Lett 457: 184–188, 1999
Osada S, Mizuno K, Saido TC, Akita Y, Suzuki K, Kuroki T, Ohno S: A new member of the protein kinase C family, nPKCθ, predominantly expressed in skeletal muscle. Mol Cell Biol 12: 3930–3938, 1992
Schmitz-Peiffer C, Browne CL, Biden T: Characterization of two forms of protein kinase C, with different substrate specificities, from skeletal muscle. Biochem J 320: 207–214, 1996
Dos Santos Mermelstein C, Costa ML, Filho CC, Netho VM: Intermediate filament protein in TPA-treated skeletal muscle cells in culture. J Muscle Res Cell Motil 17: 199–206, 1996
DeWindt LJ, Lim HW, Haq S, Force T, Molkentin JD: Calcineurin promotes protein kinase C and C-jun NH2-terminal kinase activation in the heart - cross talk between cardiac hypertrophic signaling pathways. J Biol Chem 275: 13571–13579, 2000
Cortright RN, Azevedo JL, Qian Zhou JR, Sinha M, Pories WJ, Itani SI, Dohm LG: Protein kinase C modulates insulin action in human skeletal muscle. Am J Physiol Endocrinol Metab 278: E553–E562, 2000
Caruso M, Miele C, Oriente F, Maitan A, Bifulco G, Andreozzi F, Condorelli G, Formisano P, Beguinot F: In L6 skeletal muscle cells, glucose induces cytosolic translocation of protein kinase C-alpha and trans-activates the insulin receptor kinase. J Biol Chem 274: 28637–28644, 1999
Capiati DA, Vazquez G, Tellez Inon MT, Boland RL: Role of protein kinase C in 1,25(OH)(2)-vitamin D(3) modulation of intracellular calcium during development of skeletal muscle cells in culture. J Cell Biochem 77: 200–212, 2000
Capiati DA, Limbozzi F, Tellez-Inon MT, Boland RL: Evidence on the participation of protein kinase C alpha in the proliferation of cultured myoblasts. J Cell Biochem 74: 292–300, 1999
Kim SS, Kim JH, Kim HS, Park DE, Chung CH: Involvement of the θ-type protein kinase C in translocation of myristoylated alanine-rich C kinase substrate (MARCKS) during myogenesis of chick embryonic myoblasts. Biochem J 347: 139–146, 2000
Meacci E, Donati C, Cencetti F, Romiti E, Farnararo M, Bruni P: Receptor-activated phospholipase D is present in caveolin-3-enriched light membranes of C2C12 myotubes. FEBS Lett 473: 10–14, 2000
Meacci E, Donati C, Cencetti F, Romiti E, Bruni P: Permissive role of protein kinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-induced Rho A activation in C2C12 myoblasts. FEBS Lett 482: 97–101, 2000
Petit V, Thiery JP: Focal adhesions: Structure and dynamics. Biol Cell 92: 477–494, 2000
Song KS, Scherer PE, Tang Z, Okamoto T, Li S, Chafel M, Chu C, Kohtz DS, Lisanti MP: Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem 271: 15160–15165, 1996
Fox JE: On the role of calpain and Rho proteins in regulating integrininduced signaling. Thromb Haemost 82: 385–391, 1999
Disatnik MH, Rando TA: Integrin-mediated muscle cell spreading. The role of protein kinase c in outside-in and inside-out signaling and evidence of integrin cross-talk. J Biol Chem 274: 32486–32492, 1999
Kulkarni S, Saido TC, Suzuki K, Fox JE: Calpain mediates integrininduced signaling at a point upstream of Rho family members. J Biol Chem 274: 21265–21275, 1999
Jaken S, Leach K, Klauck T: Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts. J Cell Biol 109: 697–704, 1989
Beckerle MC, Burridge K, DeMartino GN, Croall DE: Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion. Cell 51: 569–577, 1987
Savart M, Verret C, Dutaud D, Touyarot K, Elamrani NA, Ducastaing A: Isolation and identification of a µ-calpain-protein kinase Cα complex in skeletal muscle. FEBS Lett 359: 60–64, 1995
Verret C, Poussard S, Touyarot K, Donger C, Savart M, Cottin P, Ducastaing A: Degradation of protein kinase Ma by µ-calpain in a µ-calpain-protein kinase Cα complex. Biochim Biophys Acta 1430: 141–148, 1999
Balcerzak D, Poussard S, Brustis JJ, Elamrami N, Soriano M, Cottin P, Ducastaing A: An antisense oligodeoxyribonucleotide to m-calpain mRNA inhibits myoblast fusion. J Cell Sci 108: 2077–2082, 1995
Poussard S, Duvert M, Balcerzak D, Ramassamy S, Brustis JJ, Cottin P, Ducastaing A: Evidence for implication of muscle-specific calpain (p94) in myofibrillar integrity. Cell Growth Diff 7: 1461–1469, 1996
Dourdin N, Balcerzak D, Brustis JJ, Poussard S, Cottin P, Ducastaing A: Potential m-calpain substrates during myoblast fusion. Exp Cell Res 246: 433–442, 1999
Bradford MM: Rapid and sensitive method for quantification of microgram quantity for protein utilising the principle of dye binding. Anal Biochem 72: 248–254, 1976
Cottin P, Poussard S, Dargelos E, Balcerzak D, Aragon B, Brustis JJ, Ducastaing A: Calpains and myogenesis. Meth Mol Biol 144: 173–180, 2000
Helene C, Toulme JJ: Specific regulation of gene expression by antisense, sense and antigene nucleic acids. Biochim Biophys Acta 1049: 99–125, 1990
Sorimachi H, Amano S, Ishiura S, Suzuki K: Primary sequences of rat µ-calpain large and small subunits are, respectively, moderately and highly similar to those of human. Biochim Biophys Acta 1309: 37–41, 1996
Delucas CI, Davies PL, Samis JA, Elce JS: Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit. Biochim Biophys Acta 1216: 81–93, 1993
Laemmli UK: Cleavage of structure protein during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970
Wolfe SH, Satle SK, Goll DE, Kleese WC, Edmunds T, Duperret SM: Chicken skeletal muscle has three Ca2+-dependent proteinase. Biochim Biophys Acta 998: 236–250, 1989
Hansra G, Garcia-Paramio P, Prevostel C, Whelan RD, Bornancin F, Parker PJ: Multisite dephosphorylation and desensitization of conventional protein kinase C isotypes. Biochem J 342: 337–344, 1999
Kishimoto A, Mikawa K, Hashimoto K, Yasuda S, Tanaka M, Tominaga M, Kuroda T, Nishizuka Y: Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain). J Biol Chem 264: 4088–4092, 1989
Pontremoli S, Melloni E, Salamino F, Patrone M, Michetti M, Horecker BL: Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or f-Met-Leu-Phe. Biochem Biophys Res Commun 160: 737–743, 1989
Dourdin N, Brustis JJ, Balcerzak D, Elamrani N, Poussard S, Cottin P, Ducastaing A: Myoblast fusion requires fibronectin degradation by exteriorized m-calpain. Exp Cell Res 235: 385–394, 1997
Eto A, Akita Y, Saido TC, Suzuki K, Kawashima S: The role of the calpain-calpastatin system in thyrotropin-releasing hormone-induced selective down-regulation of a protein kinase C isozyme, nPKCɛ, in rat pituitary GH4C1 cells. J Biol Chem 270: 25115–25120, 1995
Wang KKW: Developing selective inhibitors of calpain. Trends Pharm Sci 11: 139–142, 1990
Hilgenberg L, Yearwood S, Milstein S, Miles K: Neural influence on protein kinase C isoform expression in skeletal muscle. J Neurosci 16: 4994–5003, 1996
Yoshida Y, Huang FL, Nakabayashi H, Huang KP: Tissue distribution and developmental expression of protein kinase C isoenzymes. J Biol Chem 263: 9868–9873, 1988
Boczan J, Boros S, Mechler F, Kovacs L, Biro T: Differential expressions of protein kinase C isozymes during proliferation and differentiation of human skeletal muscle cells in vitro. Acta Neuropathol (Berl) 99: 96–104, 2000
Cottin P, Brustis JJ, Poussard S, Elamrani N, Broncard A, Ducastaing A: Ca2+-dependent proteinases (calpains) and muscle cell differentiation. Biochim Biophys Acta 1223: 170–178, 1994
Poussard S, Cottin P, Brustis JJ, Talmat S, Elamrani N, Ducastaing A: Quantitative measurement of calpain I and II mRNAs in differentiating rat muscle cells using a competitive polymerase chain reaction method. Biochimie 75: 885–890, 1993
Moraczewski J, Piekarska E, Bonavaud S, Wosinska K, Chazaud B, Barlovatz-Meimon G: Differential intracellular distribution and activities of µ-and m-calpains during differentiation of human myogenic cells in culture. C.R. Acad Sci Paris, Life Sci 319: 681–686, 1996
Stockholm D, Barbaud C, Marchand S, Ammarguellat F, Barritault D, Richard I, Beckmann J, Martelly I: Studies on calpain expression during differentiation of rat satellite cells in primary cultures in the presence of heparin or a mimic compound. Exp Cell Res 252: 392–400, 1999
Schollmeyer JE: Possible role of calpain I and II in differentiating muscle. Exp Cell Res 163: 413–422, 1986
Hong D, Huan JH, Ou B, Yeh J, Saido TC, Cheeke PR, Forsberg NE: Protein kinase C isoforms in muscle cells and their regulation by phorbol ester and calpain. Biochim Biophys Acta 1267: 45–54, 1995
Diaz-Guerra MJM, Sanchez Prieto J, Bosca L, Pocock J, Barrie A, Nicholls D: Phorbol-ester translocation of protein kinase C in guinea pig synaptosome and the potentiation of calcium dependent glutamate release. Biochim Biophys Acta 970: 157–165, 1988
Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y: Direct activation of calcium-activated phospholipid-dependent protein kinase C by tumor-promoting phorbol esters. J Biol Chem 257: 7847–7851, 1982
Figueiredo-Pereira ME, Banik N, Wilk S: Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: The multicatalytic proteinase complex and m-calpain. J Neurochem 62: 1989–1994, 1994
Lu Z, Liu D, Hornia A, Devonish W, Pagano M, Foster DA: Activation of protein kinase C triggers its ubiquitination and degradation. Mol Cell Biol 18: 839–845, 1998
Lee H-W, Smith L, Pettit GR, Smith JB: Bryostatin 1 and phorbol ester down-regulate protein kinase C-α and-ɛ via the ubiquitin/proteasom pathway in human fibroblasts. Mol Pharmacol 51: 439–447, 1997
Pongracz J, Webb P, Wang KQ, Deacon E, Lunn QJ, Lord JM: Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta. J Biol Chem 274: 37329–37334, 1999
Haussermann S, Kittstein W, Rincke G, Johannes FJ, Marks F, Gschwendt M: Proteolytic cleavage of protein kinase C mu upon induction of apoptosis in U937 cells - identification of the cleavage site and characterization of the fragment. FEBS Lett 462: 442–446, 1999
Li L, Zhou J, James G, Heller-Harrison R, Czech MP, Olson EN: FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 71: 1181–1194, 1992
James G, Olson E: Deletion of the regulatory domain of protein kinase Cα exposes regions in the hinge and catalytic domains that mediate nuclear targeting. J Cell Biol 116: 863–874, 1992
Poussard S, Dulong S, Aragon B, Brustis JJ, Veschambre P, Ducastaing A, Cottin P: Evidence for a MARCKS-PKCα complex in skeletal muscle. Int J Biochem Cell Biol 33: 711–721, 2001
Rights and permissions
About this article
Cite this article
Aragon, B., Poussard, S., Dulong, S. et al. Protein kinase Cα is a calpain target in cultured embryonic muscle cells. Mol Cell Biochem 231, 97–106 (2002). https://doi.org/10.1023/A:1014460730664
Issue Date:
DOI: https://doi.org/10.1023/A:1014460730664