Evidence that autophosphorylation at Thr-286/Thr-287 is required for full activation of calmodulin-dependent protein kinase II

Calmodulin-dependent protein kinase II (CaM-kinase II) undergoes a very rapid autophosphorylation at Thr-286/Thr-287 in the presence of Ca2+/calmodulin and ATP/Mg2+, and this has greatly hampered studies on the role of the autophosphorylation in the regulation of the enzyme activity, because it has been difficult to measure the activity of the non-autophosphorylated enzyme in the presence of Ca2+/calmodulin. In the present study, this difficulty was overcome by using adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) in place of ATP. When the enzyme was assayed with 2 mu M ATP gamma S and 200 mu M syntide-2 at 5 degrees C in the presence of Ca2+/calmodulin, the linear reaction rate of thiophosphorylation of syntide-2 was much slower than that of the enzyme which had previously undergone autothiophosphorylation. Under the limiting assay conditions, thiophosphorylation of the enzyme did not occur significantly during assay. Using this assay condition, activation by autothiophosphorylation was examined. When CaM-kinase II was autothiophosphorylated at 5 degrees C, the concomitant stimulation of both activities in the presence and absence of Ca2+/calmodulin was observed. The activity of the recombinant wild-type enzyme in the presence of Ca2+/calmodulin as well as in its absence was also markedly activated upon autothiophosphorylation, whereas those of the recombinant mutated enzyme, whose Thr-287 was replaced by Ala, was not activated at all. These results provide strong support for the contention that CaM-kinase II initially possesses a basal low level of the total activity and that the initial rapid autophosphorylation on Thr-286/Thr-287 results in full activation of the enzyme.