Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity

• Published in: Bioscience reports Vol. 33; no. 2
• Main Authors: Yilmaz, Mehtap, Gangopadhyay, Samudra S, Leavis, Paul, Grabarek, Zenon, Morgan, Kathleen G
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 07.02.2013
• Subjects: Adenosine Triphosphate - metabolism; Animals; Binding Sites; Calcium - metabolism; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - genetics; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism; Calmodulin - genetics; Calmodulin - metabolism; Ferrets - genetics; Ferrets - metabolism; Mutation; Original Paper; Phosphorylation - genetics; Protein Isoforms - genetics; Protein Isoforms - metabolism; Serine - genetics; Serine - metabolism; Signal Transduction; Threonine - genetics; Threonine - metabolism
• ISSN: 0144-8463; 1573-4935
• DOI: 10.1042/bsr20120116

CaMKII (Ca²⁺/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 Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ 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 Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.