Phosphorylation plays positive roles in regulating the inhibitory ability of calpastatin to calpain

Abstract

The objective of this study was to determine the regulatory mechanism of phosphorylation of calpastatin on the activity of l-calpain. Crude calpastatin extracted from the longissimus lumborum (LL) muscles of three Fat Tail Han Sheep were treated with protein kinase A (PKA) or alkaline phosphatase (AP) to increase or decrease their phosphorylation levels, respectively, and then, both phosphorylated/dephosphorylated solutions were incubated with different amounts of purified l-calpain (25, 50, 100 and 150 units). In the AP group, the rate of l-calpain degradation was higher than the control and PKA groups. Calpastatin with higher phosphorylation levels presented much higher inhibitory abilities. Five phosphorylation sites from two phosphopeptides were identified. Protein kinase A-catalysed calpastatin phosphorylation at Ser 649 positively regulates the inhibitory abilities of calpastatin by promoting the interaction between calpastatin and calpain. These results contribute to enrich the regulatory mechanism of l-calpain activity during post-mortem meat tenderisation.

Natural aging and ovariectomy induces parallel phosphoproteomic alterations in skeletal muscle of female mice

“…Significant upregulation of CAST (a calpain inhibitor) Ser-82 phosphorylation combined with altered phosphorylation of calpain substrates - downregulation of desmin and troponin T in both datasets and downregulation of myosin regulatory light chain phosphorylation in OA/YA dataset with upregulation in the Ovx/Sham dataset - imply abrogation of calpain activities in contracted muscle. Hyperphosphorylation of CAST positively regulates calpain inhibition [ 42 ], and phospho-modifications on calpain substrates has been shown to regulate the susceptibility of the substrate to calpain degradation [ 43 ]. Calpain-mediated proteolytic activities in skeletal muscle are crucial for myofibrillar protein turnover and aid in muscle plasticity through disassembly of the myofibril [ 44 ].…”

Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain

“…The differences in the degradation degree of calpain and calpastatin by calpain were caused by different phosphorylation levels of calpastatin. The inhibitory ability of dephosphorylated calpastatin on calpain activity was smaller than that of phosphorylated calpastatin catalyzed by PKA, and phosphorylation may play a positive role in regulating the inhibitory ability of calpastatin on calpain activity [ 33 ].…”