Abstract:

CAPN3 (also called p94/calpain-3) is a skeletal muscle-specific calpain, an intracellular cysteine protease. Loss of CAPN3 protease activity and/or structural functions cause limb-girdle muscular dystrophy type 2A (LGMD2A). However, the precise mechanism of action of CAPN3 in skeletal muscles in vivo remains largely elusive. By studying the protein modifications that regulate CAPN3 activity, we found that CAPN3 was phosphorylated. By performing mutagenesis and mass spectrometry analyses, we identified two Ser residues at positions 629 and 636 in human CAPN3 that are phosphorylated and showed that S629 is a major phosphorylation site. Intriguingly, rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the substitution of S629. In skeletal muscles, phosphorylated CAPN3 was enriched in the myofibril fraction. These results imply that phosphorylated CAPN3 is a myofibril structural component and/or participates in myofibril-based signaling pathways, rather than functions as a protease. We evaluated the relationship between phosphorylated CAPN3 and the pathology of LGMD2A. The level of phosphorylated CAPN3 was greatly reduced in LGMD2A muscles. Our findings suggest that phosphorylated CAPN3 is involved in the pathology of LGMD2A through defects in myofibril integrity and/or signaling pathways. This is the first report that phosphorylation of CAPN3 may be involved in its physiological function.

日本語要旨:

肢帯型筋ジストロフィー2A型の原因タンパク質であるカルパイン3がリン酸化されていることを見いだした。このリン酸化によりカルパイン3の自己分解活性が若干抑えられること、リン酸化されたカルパイン3は主に筋原線維に含まれることがわかった。ミスセンス変異をもつ肢帯型筋ジストロフィー2A患者筋のカルパイン3では、リン酸化が低下していた。このことから、カルパイン3のリン酸化は病態に関与することが考えられた。