Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.
The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.
Ikeda
A
A
Department of Biochemistry, Asahikawa Medical College, Japan.
Okuno
S
S
Fujisawa
H
H
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D007700:Kinetics; D008297:Male; D009185:Myelin Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism; D014357:Trypsin
D002147:Calmodulin; D009185:Myelin Proteins; D013912:Threonine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D002118:Calcium