REGULATION OF DROSOPHILA CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE-II BY AUTOPHOSPHORYLATION ANALYZED BY SITE-DIRECTED MUTAGENESIS/

In this study we demonstrate that Drosophila calcium/calmodulin-depend ent protein kinase II (CaMKII) is capable of complex regulation by aut ophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Dr osophila CaMKII is equivalent to phosphorylation of threonine-286 in r at alpha CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphoryl ated. Autophosphorylation of this residue occurs only within the holoe nzyme structure and requires calmodulin (CaM) to be bound to the subst rate subunit. Phosphorylation of threonine-306 and threonine-307 in th e CaM binding domain of the Drosophila kinase occurs only in the absen ce of CaM, and this phosphorylation is capable of inhibiting further C aM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermol ecular threonine-287 phosphorylation. These results demonstrate that t he mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.