title: Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.
abstract: 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.
authors.affiliation: *Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, U.S.A.
authors.forename: Mehtap
authors.initials: M
authors.lastname: Yilmaz
authors.forename: Samudra S
authors.initials: SS
authors.lastname: Gangopadhyay
authors.affiliation: §Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, U.S.A.
authors.forename: Paul
authors.initials: P
authors.lastname: Leavis
authors.affiliation: §Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, U.S.A.
authors.forename: Zenon
authors.initials: Z
authors.lastname: Grabarek
authors.affiliation: *Department of Health Sciences, Boston University, 635 Commonwealth Avenue, Boston, MA 02215, U.S.A.
authors.forename: Kathleen G
authors.initials: KG
authors.lastname: Morgan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D005289:Ferrets / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; D020033:Protein Isoforms; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium