Phosphorylation at Ser²6 in the ATP-binding site of Ca²âº/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

CaMKII (Ca²âº/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.

The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions.

Gene duplication followed by functional specialization is a potent force in the evolution of biological diversity. A comparative study of two highly conserved duplicated genes, ARABIDOPSIS TRITHORAX-LIKE PROTEIN1 (ATX1) and ATX2, revealed features of both partial redundancy and of functional divergence. Although structurally similar, their regulatory sequences have diverged, resulting in distinct temporal and spatial patterns of expression of the ATX1 and ATX2 genes. We found that ATX2 methylates only a limited fraction of nucleosomes and that ATX1 and ATX2 influence the expression of largely nonoverlapping gene sets. Even when coregulating shared targets, ATX1 and ATX2 may employ different mechanisms. Most remarkable is the divergence of their biochemical activities: both proteins methylate K4 of histone H3, but while ATX1 trimethylates it, ATX2 dimethylates it. ATX2 and ATX1 provide an example of separated K4 di from K4 trimethyltransferase activity.