Calpastatin inhibits the activity of phosphorylated μ-calpain in vitro.

The objective of this study was to investigate the effect of phosphorylation on the sensitivity of μ-calpain to the inhibition induced by calpastatin. Purified μ-calpain was incubated with alkaline phosphatase (AP) or protein kinase A (PKA) to modulate the phosphorylation level of μ-calpain. Accurately 25, 50, 100 and 150 units of AP/PKA-treated μ-calpain were mixed with the same amounts of heat stable proteins and incubated at 4 °C. In the calpastatin-free system, AP and PKA-treated μ-calpain had higher proteolytic activity compared to the control. Intact AP-treated μ-calpain degraded fastest in the 50, 100 and 150 unit μ-calpain incubation systems. However, the degradation rate of μ-calpain in control and PKA group was non-significant in 100 and 150 unit μ-calpain systems. Our results demonstrated that, compared to dephosphorylated and control μ-calpain, calpastatin presents greater inhibition to PKA phosphorylated μ-calpain. This study increases understanding of the mechanism of μ-calpain activity regulated by phosphorylation.