Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities

Abstract

Autophosphorylation of Ca 2+-calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG-tagged wild type and phospho-and dephospho-mimic mutants of a-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca 2+ -calmodulin dependent or autonomous kinase activity of recombinant a-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased a-CaMKII binding to subcellular fractions enriched in postsynaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho-Thr253-CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell.

Regulation of CaMKII In vivo: The Importance of Targeting and the Intracellular Microenvironment

“…This phosphorylation site, which is present in every subunit and conserved across species, has been shown to be phosphorylated in vivo [48]. As shown in Table 1, phosphorylation at Thr253 has no direct effect on the kinase activity of CaMKII in vitro although it has marked effects on CaMKII targeting [48].…”

“…When the stoichiometry of Thr253 phosphorylation is measured in the whole brain it appears to be low and therefore of questionable functional significance. However, the phosphorylation stoichiometry is high in a specific pool of CaMKII that is associated with the PSD [48] and therefore the functional consequences of Thr253 phosphorylation may be concentrated at this and other specialised cellular locations.…”

The Role of Molecular Regulation and Targeting in Regulating Calcium/Calmodulin Stimulated Protein Kinases

“…Phosphorylation of T305/306 decreases the amount of CaMKII bound to the PSD, stimulating translocation from the PSD to the cytosol [ 137 ] . By contrast, phosphorylation of either T286 or T253 enhances binding to the PSD, stimulating translocation from the cytosol to the PSD and the effects appear to be through independent binding proteins since phosphorylation at both sites results in an additive effect [ 121 ] .…”

“…T253 has previously been overlooked as a phosphorylation site of interest as it has no direct effect on the kinase activity of CaMKII in vitro . However, T253 phosphorylation has marked effects on CaMKII targeting [ 107,121 ] . Furthermore, the overall stoichiometry of T253 phosphorylation is relatively low in the cell as a whole because it occurs only in a subpopulation of CaMKII molecules at particular cellular locations [ 121 ] .…”

“…Once these sites are phosphorylated, CaM can no longer bind so CaMKII becomes insensitive to changes in Ca 2+ /CaM [ 120 ] . Recently, a new phosphorylation site on CaMKII at T253 was identifi ed in vivo [ 121 ] . T253 has previously been overlooked as a phosphorylation site of interest as it has no direct effect on the kinase activity of CaMKII in vitro .…”