Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylaton

Regulatory mechanisms of rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) were probed using a synthetic peptide (CaMK-(281-309)) corresponding to residues 281-309 (α-subunit) which contained the calmodulin (CaM)-binding and inhibitory domains and also the initial autophosphorylation site (Thr²⁸⁶). Kinetic analyses indicated that inhibition of a completely Ca²⁺/CaM-independent form of CaM-kinase II by CaMK-(281-309) was noncompetitive with respect to peptide substrate (syntide-2) but was competitive with respect to ATP. Interaction of CaMK-(281-309) with the ATP-binding site was independently confirmed since inactivation of proteolyzed CaM-kinase II by phenylglyoxal (t( 1/2 ) = 7 min) was blocked by ATP analog plus Mg²⁺ or by CaMK-(281-309). In the presence of Ca²⁺/CaM, CaMK-(281-309) no longer protected against phenylglyoxal inactivation, consistent with our previous observations (Colbran, R.J., Fong, Y.-L., Schworer, C.M., and Soderling, T.R. (1988) J. Biol. Chem. 263, 18145-18151) that binding of Ca²⁺/CaM to CaMK-(281-309) 1) blocks its inhibitory property, and 2) enhances its phosphorylation at Thr²⁸⁶. The present study also showed that phosphorylation of CaMK-(281-309) decreased its inhibitory potency at least 10-fold without affecting its Ca²⁺/CaM-binding ability. Thus, CaM-kinase II is inactive in the absence of Ca²⁺/CaM because an inhibitory domain within residues 281-309 interacts with the catalytic domain and blocks ATP binding. Autophosphorylation of Thr²⁸⁶ results in a Ca²⁺/CaM-independent form of the kinase by disrupting the inhibitory interaction with the catalytic domain.