Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities.
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 alpha-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca(2+)-calmodulin dependent or autonomous kinase activity of recombinant alpha-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased alpha-CaMKII binding to subcellular fractions enriched in post-synaptic 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.
Migues
Paola V
PV
School of Biomedical Sciences and Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia.
Lehmann
Ingo T
IT
Fluechter
Lisa
L
Cammarota
Martin
M
Gurd
James W
JW
Sim
Alistair T R
AT
Dickson
Phillip W
PW
Rostas
John A P
JA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000166:cytology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D011180:Postmortem Changes; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011869:Radioligand Assay / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000378:metabolism; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D013997:Time Factors
D050505:Mutant Proteins; D013912:Threonine; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases