IDENTIFICATION OF A LATENT CA2-DEPENDENT PROTEIN KINASE-II PHOSPHORYLATION SITE IN VASCULAR CALPAIN-II( CALMODULIN)

The Ca2+-dependent protease, calpain II, isolated from vascular smooth muscle was found to be a substrate for Ca2+/calmodulin-dependent prot ein kinase II (CaM kinase II) in vitro. Phosphorylation was dependent upon prior autolysis of the regulatory subunit of calpain II. The stoi chiometry of phosphorylation of native, unautolyzed calpain II was 0.0 2 +/- 0.01 mol PO4/mol enzyme while for autolyzed calpain, the stoichi ometry was 1.04 +/- 0.15 mol PO4/mol enzyme. All phosphate was incorpo rated into the 76 kDa catalytic subunit of calpain II. A single serine residue in domain III of the catalytic subunit was identified as the phosphate acceptor: RGSTAGGCR. Phosphorylation doubled enzyme activit y measured both as proteolysis of an exogenous substrate (alpha-casein ) as well as by intermolecular catalytic subunit autolysis. The effect s of phosphorylation could be reversed by dephosphorylation using a ty pe IIA phosphoprotein phosphatase. These results demonstrate that calp ain II possesses a latent CaM kinase II phosphorylation site that is u nmasked by autolysis.