Studies on the regulatory domain of Ca²⁺/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the α subunit

The regulatory role of Arg²⁸³ in the autoinhibitory domain of Ca²⁺/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca²⁺/calmodulin-dependent protein kinase II, substitution of Arg²⁸³ by other residues increased the IC₅₀ values of the peptides in the following order: Arg << Lys << Gln << Glu. Site-directed mutations of Arg²⁸³ to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca²⁺/CaM, but the Glu²⁸³ mutant had a slightly higher Ca²⁺-independent kinase activity (5.46 ± 0.88%) compared to the wild-type Arg²⁸³ (1.86 ± 0.71%) and the Gln²⁸³ mutant (2.15 ± 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca²⁺-independent activity, the Arg²⁸³ kinase attained maximal Ca²⁺-independent activity (about 20%) within 30 s, whereas the Gln²⁸³ and Glu²⁸³ mutants attained maximal Ca²⁺-independence only after about 40 min of autophosphorylation. The results indicate that Arg²⁸³ is a very important determinant for the regulatory autophosphorylation of Thr²⁸⁶ that generates the Ca²⁺-independent activity but is not essential for the other multiple autophosphorylations within Ca²⁺/calmodulin-dependent protein kinase II, and that Arg²⁸³ is only one of several important residues for the inhibitory potency of the autoinhibitory domain.