Reversible generation of a Ca²⁺-independent form of Ca²⁺(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism

The Ca²⁺(calmodulin (CaM))-dependent protein kinase II, purified from either rabbit liver or rat brain, was preincubated under conditions that are known to promote its autophosphorylation. When kinase activity was assayed after this preincubation, it was observed that excess EGTA could block no more than 40-60% of the total Ca²⁺- and CaM-dependent activity compared to 95% inhibition by EGTA prior to preincubation. In the EGTA assay, free Ca²⁺ was calculated to be <1 nM; therefore, this activity was designated Ca²⁺-independent activity. Formation of this Ca²⁺-independent form of the kinase was shown to be associated with autophosphorylation based on the following observations: (a) it required the presence of Ca²⁺, CaM, and ATP; (b) the ATP analogs adenylyl imidodiphosphate and adenylyl methylenediphosphate could not substitute for ATP; (c) generation of the independent form was associated with incorporation of phosphate into the kinase; and (d) addition of protein phosphatase partially dephosphorylated the kinase and restored its Ca²⁺ dependence. This phenomenon may be of physiological importance because it would prolong the effects of extracellular signals that only transiently increase the intracellular Ca²⁺ level.