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.

Realization of phosphorylation hypothesis of sleep by mammalian CaMKIIβ

“…We also showed that other sleep-controlling residues (such as S26, S182, and T311) also undergo autophosphorylation ( Figure 7a ). S26 autophosphorylation occurs in CaMKIIγ 67 and suppresses the kinase activity 68 , which is consistent with our results in CaMKIIβ. The other phosphoproteomics study identified S25 autophosphorylation in CaMKIIα 30 .…”

Mechanisms of Vascular Smooth Muscle Contraction and the Basis for Pharmacologic Treatment of Smooth Muscle Disorders

“…CaMKinase II is another Ca/CaM-dependent kinase with the interesting property, when activated, of autophosphorylating itself on T287, which leads to a sustained activity after Ca is removed, giving it a chemical “memory” of having been activated ( Hudmon and Schulman, 2002 ; Lisman et al, 2002 ). Conversely, when S26 in the catalytic domain is autophosphorylated, it can terminate sustained kinase activity, making it “forget” prior activation ( Yilmaz et al, 2013 ).…”

Glyphosate pathways to modern diseases V: Amino acid analogue of glycine in diverse proteins

“…A possible mechanism by which CaMKII might be inhibited by glyphosate is through substitution of glyphosate for one of the highly conserved glycines near ser26. Ser26 is situated within a conserved stretch of nine residues (LGKGAFSVV) that constitute the upper lid of the ATP-binding site in the canonical kinase fold [325]. An intricate control mechanism for preventing excessive activity of this autophosphorylating enzyme involves phosphorylation of ser26, which then interferes with ATP binding and disrupts enzymatic activity in a feedback control mechanism.…”