Regulatory interactions of the calmodulin-binding, inhibitory, and autophosphorylation domains of Ca²⁺/calmodulin-dependent protein kinase II

Two peptide analogs of Ca²⁺/calmodulin-dependent protein kinase II (CaMK-(peptides)) were synthesized and used to probe interactions of the various regulatory domains of the kinase. CaMK-(281-289) contained only Thr²⁸⁶, the major Ca²⁺-dependent autophosphorylation site of the kinase (Schworer, C.M., Colbran, R.J., Keefer, J.R. & Soderling, T.R. (1988) J. Biol. Chem. 263, 13486-13489), whereas CaMK-(281-309) contained Thr²⁸⁶ together with the previously identified calmodulin binding and inhibitory domains (Payne, M.E., Fong, Y.-L., Ono, T., Colbran, R.J., Kemp, B.E., Soderling, T.R. & Means, A.R. (1988) J. Biol. Chem. 263, 7190-7195). CaMK-(281-309), but not CaMK-(281-289), bound calmodulin and was a potent inhibitor (IC₅₀ = 0.88 ± 0.7 μM using 20 μM syntide-2) of exogenous substrate (syntide-2 or glycogen synthase) phosphorylation by a completely Ca²⁺/calmodulin-independent form of the kinase generated by limited proteolysis with chymotrypsin. This inhibition was completely relieved by the inclusion of Ca²⁺/calmodulin in excess of CaMK-(281-309) in the assays. CaMK-(281-289) was a good substrate (K(m) = 11 μM; V(max) = 3.15 μmol/min/mg) for the proteolyzed kinase whereas phosphorylation of CaMK-(281-309) showed nonlinear Michaelis-Menton kinetics, with maximal phosphorylation (0.1 μmol/min/mg) at 20 μM and decreased phosphorylation at higher concentrations. The addition of Ca²⁺/calmodulin to assays stimulated the phosphorylation of CaMK-(281-309) by the proteolyzed kinase approximately 10-fold but did not affect the phosphorylation of CaMK-(281-289). A model for the regulation of Ca²⁺/calmodulin-dependent protein kinase II is proposed based on the above observations and results from other laboratories.