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-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat alpha CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM 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 intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.
Wang
Z
Z
Department of Biology and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Palmer
G
G
Griffith
L C
LC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D019556:COS Cells / Q000201:enzymology; D002118:Calcium / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D004330:Drosophila; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009420:Nervous System / Q000201:enzymology; D009473:Neuronal Plasticity / Q000502:physiology; D010766:Phosphorylation; D013379:Substrate Specificity
D002147:Calmodulin; D003067:Coenzymes; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium