Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction
Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction
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DOI：
10.1111/gtc.12181
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发表时间：
2014-11-01
期刊：
影响因子：
2.1
通讯作者：
Sorimachi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Ojima, Koichi;Ono, Yasuko;Sorimachi, Hiroyuki
CAPN3（也称为p94/calpain-3）是骨骼肌特异性钙蛋白酶，是一种细胞内半胱氨酸蛋白酶。CAPN3蛋白酶活性和/或结构功能的丧失可导致2A型肢体肌营养不良（LGMD2A）。然而，体内骨骼肌中CAPN3的确切作用机制仍不清楚。通过研究调节CAPN3活性的蛋白修饰，我们发现CAPN3被磷酸化。通过诱变和质谱分析，我们在人类CAPN3的629和636位点鉴定出两个丝氨酸残基被磷酸化，并表明S629是一个主要的磷酸化位点。有趣的是，由于S629的取代，CAPN3的快速和彻底的自溶作用略有减弱。在骨骼肌中，磷酸化的CAPN3在肌原纤维部分富集。这些结果表明磷酸化的CAPN3是肌原纤维结构成分和/或参与基于肌原纤维的信号通路，而不是作为蛋白酶发挥作用。我们评估了磷酸化的CAPN3与LGMD2A病理之间的关系。磷酸化的CAPN3水平在LGMD2A肌肉中显著降低。我们的研究结果表明，磷酸化的CAPN3通过肌原纤维完整性和/或信号通路的缺陷参与LGMD2A的病理。这是首次报道CAPN3的磷酸化可能参与其生理功能。
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.