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 Ca2+ concentrations.  μ-Calpain was treated with AP or PKA at 0.01, 0.05, 0.1 and 1 mM Ca2+.  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 Ca2+ 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 Ca2+ concentration.