Mechanism of the generation of autonomous activity of Ca ²⁺/calmodulin-dependent protein kinase IV

Ca²⁺/calmodulin-dependent protein kinase IV (CaM-KIV) is phosphorylated at Thr¹⁹⁶ by Ca²⁺/calmodulin-dependent protein kinase kinase (CaM-KK), resulting in induction of both autonomous activity and a high level of Ca²⁺/CaM-dependent activity. We have shown that the kinetics of Thr¹⁹⁶ phosphorylation of CaM-KIV by CaM-KK is well correlated with the generation of its autonomous activity, although Thr¹⁷⁷ phosphorylation of CaM-KI does not induce its autonomous activity. The activities of CaM-KI chimera mutants fused with C-terminal regions (residues 296-469 and 296-350) of CaM-KIV are completely dependent on Ca²⁺/CaM, which is also the case for CaM-KI. Unlike wild-type CaM-KI, however, phosphorylation of Thr¹⁷⁷ in the chimera mutants by CaM-KK resulted in generation of significant autonomous activities, indicating that the phosphorylation of Thr in the activation loop is sufficient to partially release the autoinhibitory region of CaM-KIV from the catalytic core. Indeed, the CaM-KIV peptide (residues 304-325) containing minimum autoinhibitory sequences (residues 314-321) suppressed the activity of non-phosphorylated CaM-KIV with an IC₅₀ of ∼50 μM, and this suppression was competitive with respect to the peptide substrate; however, the CaM-KIV peptide was not capable of inhibiting Thr¹⁹⁶-phosphorylated CaM-KIV. Taken together, these results indicated that the Thr¹⁹⁶ phosphorylation of CaM-KIV by CaM-KK reduced the interaction of the catalytic core with the autoinhibitory region, resulting in generation of the autonomous activity.