Regulatory interactions of calmodulin-binding proteins: Phosphorylation of calcineurin by autophosphorylated Ca²⁺/calmodulin-dependent protein kinase II

The Ca²⁺/calmodulin (CaM)-dependent protein phosphatase calcineurin is rapidly phosphorylated (0.8 mol of ³²PO₄ per mol of 60-kDa subunit of calcineurin) by brain Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II). This reaction requires the autophosphorylated, Ca²⁺-independent form of CaM-kinase II since Ca²⁺/CaM binding to calcineurin inhibits phosphorylation. However, the phosphorylation reaction does require Ca²⁺, presumably acting through the 19-kDa subunit of calcineurin. Calcineurin is a good substrate for CaM-kinase II, with a K(m) of 19 μM and V(max) of 2.4 μmol/min per mg. Phosphorylation of calcineurin changed its phosphatase activity with either a 2-fold increase in K(m) (³²P-labeled myosin light chain as substrate) or a 50% decrease in V(max) (p-nitrophenyl phosphate as substrate). The phosphorylated calcineurin exhibited very slow autodephosphorylation (0.09 nmol/min per mg) but was effectively dephosphorylated by brain protein phosphatase IIA. Dephosphorylation, like phosphorylation, was blocked by high concentrations of Ca²⁺/CaM and stimulated by Ca²⁺ alone. Thus calcineurin has a regulatory phosphorylation site that is phosphorylated by the Ca²⁺-independent form of CaM-kinase II and blocked by high concentrations of Ca²⁺/CaM.