Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha.
Calpains are a family of calcium-dependent cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain 1 in the muscle contractile apparatus and specifically in the N1- and N2-lines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin--a specific muscle isoform of the filamin family--is a calpain 1 substrate and to characterise this interaction. Gamma-filamin is a major muscle architectural protein located in the Z-line and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain 1 in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain 1 are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C-terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain 1 in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.
Raynaud
Fabrice
F
UMR5539, EPHE-CNRS-UM2, cc107, Université Montpellier II, place E. Bataillon, 34095 Montpellier cedex 5, France. raynaudf@univ-montp2.fr
Fabrice
Raynaud
R
Jond-Necand
Carole
C
Carole
Jond-Necand
JN
Marcilhac
Anne
A
Anne
Marcilhac
M
Fürst
Dieter
D
Dieter
Fürst
F
Benyamin
Yves
Y
Yves
Benyamin
B
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D002522:Chlorocebus aethiops; D003285:Contractile Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064448:Filamins; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D051571:Protein Kinase C-alpha; D002154:Calpain; D002118:Calcium