Phosphorylation at Ser26Ser^{26} in the ATP-Binding Site of Ca2+Ca^{2+}/Calmodulin-Dependent Kinase II as a Mechanism for Switching off the Kinase Activity

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