Phosphorylation at \(Ser^{26}\) in the ATP-Binding Site of \(Ca^{2+}\)/Calmodulin-Dependent Kinase II as a Mechanism for Switching off the Kinase Activity

Abstract

CaMKII ((Ca^{2+})/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The (\gamma) isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII (\gamma) at (Ser^{26}), a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at (Ser^{26}), we generated a phosphospecific (Ser^{26}) antibody and demonstrated an increase in (Ser^{26}) phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of (Ser^{26}) affects the kinase activity, we mutated (Ser^{26}) to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in (Thr^{287}) autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at (Ser^{26}) of CaMKII (\gamma) inhibits the catalytic activity of the enzyme towards its autophosphorylation site at (Thr^{287}) most probably by blocking ATP binding. We propose that (Ser^{26}) phosphorylation constitutes an important mechanism for switching off CaMKII activity.