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

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 γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ 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 γ 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.