Myogenic stage, sarcomere length, and protease activity modulate localization of muscle-specific calpain.
p94/calpain 3 is a Ca(2+)-binding intracellular protease predominantly expressed in skeletal muscles. p94 binds to the N2A and M-line regions of connectin/titin and localizes in the Z-bands. Genetic evidence showing that compromised p94 proteolytic activity leads to muscular dystrophy (limb-girdle muscular dystrophy type 2A) indicates the importance of p94 function in myofibrils. Here we show that a series of p94 splice variants is expressed immediately after muscle differentiation and differentially change localization during myofibrillogenesis. We found that the endogenous N-terminal (but not C-terminal) domain of p94 was not only localized in the Z-bands but also directly bound to sarcomeric alpha-actinin. These data suggest the incorporation of proteolytic N-terminal fragments of p94 into the Z-bands. In myofibrils localization of exogenously expressed p94 shifted from the M-line to N2A as the sarcomere lengthens beyond approximately 2.6 and 2.8 microm for wild-type and proteaseinactive p94, respectively. These data demonstrate for the first time that p94 proteolytic activity is involved in responses to muscle conditions, which may explain why p94 inactivation causes limb-girdle muscular dystrophy.
Ojima
Koichi
K
Department of Enzymatic Regulation for Cell Functions, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Ono
Yasuko
Y
Doi
Naoko
N
Yoshioka
Katsuhide
K
Kawabata
Yukiko
Y
Labeit
Siegfried
S
Sorimachi
Hiroyuki
H
D000185:Actinin / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D064211:Connectin; D017931:DNA Primers / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012518:Sarcomeres / Q000502:physiology*; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D064211:Connectin; D017931:DNA Primers; D009124:Muscle Proteins; D012333:RNA, Messenger; C577285:TTN protein, human; D000185:Actinin; D011494:Protein Kinases; D010447:Peptide Hydrolases; C105884:CAPN3 protein, human; D002154:Calpain; C491373:Capn3 protein, mouse