ERK-mediated phosphorylation modulates the autolytic processing of the cysteine protease CAPN3

Calpain-3 (CAPN3) is a Ca²⁺-dependent cysteine protease predominantly expressed in skeletal muscles. Mutations in CAPN3 are associated with limb girdle muscular dystrophy R1 (LGMDR1), an autosomal recessive muscular dystrophy. CAPN3 is uniquely activated by rapid autolytic processing; however, the mechanisms regulating this activation are poorly understood. Here we show that phosphorylation regulates CAPN3 autolytic processing in living cells. We found that ouabain, a cardiac glycoside, activated ERK, which induced the phosphorylation of CAPN3 at S19 located within its N-terminal region. This phosphorylation promoted the N-terminal cleavage without altering the autolytic insertion sequence 1 (IS1) processing of CAPN3 or its subcellular localization in skeletal muscle cells. Notably, treatment of cultured skeletal muscle cells with insulin-like growth factor I (IGF-I), which can activate the ERK pathway, also induced S19 phosphorylation, suggesting that this modification can occur downstream of physiologically relevant extracellular signaling. These results demonstrate that ERK-mediated phosphorylation provides a regulatory mechanism for modulating N-terminal processing of CAPN3 without substantially affecting IS1 autolytic processing.