Phosphorylation regulated by protein kinase A and alkaline phosphatase play positive roles in μ-calpain activity.

This study was aimed to determine the effect of phosphorylation/dephosphorylation regulated by protein kinase A (PKA) and alkaline phosphatase (AP) on μ-calpain activity at different Ca concentrations. μ-Calpain was treated with AP or PKA at 0.01, 0.05, 0.1 and 1 mM Ca. The pH value decreased in the AP group but remained stable in the control and PKA groups during incubation. Except samples incubated at 0.01 and 0.1 mM Ca for more than 20 min, μ-calpain incubated with PKA showed a higher degree of autolysis than control, but lower than the AP group. The content of α-helix structure of μ-calpain increased as phosphorylation level rose. Phosphorylation of μ-calpain at serine 255, 256, 476, 417 and 420 was identified. PKA catalyzed μ-calpain phosphorylation at serine 255, 256 and 476, located at domains II and III, positively regulated μ-calpain activity. These data demonstrated that dephosphorylation and PKA phosphorylation positively regulated μ-calpain activity, which was limited by increased Ca concentration.