Premium
Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin‐dependent protein kinase II
Author(s) -
Raveendran Rajeevkumar,
Devi Suma Priya Sudarsana,
Mayadevi Madhavan,
Steephan Mathew,
Santhoshkumar Thankayyan R.,
Cheriyan John,
Sanalkumar Rajendran,
Pradeep Kurup K.,
James Jackson,
Omkumar Ramakrishnapillai V.
Publication year - 2009
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2009.06108.x
Subject(s) - autophosphorylation , calmodulin , phosphorylation , phosphatase , dephosphorylation , protein kinase a , biochemistry , protein subunit , biology , protein phosphatase 1 , chemistry , microbiology and biotechnology , kinase , enzyme , gene
Ca 2+ influx through NMDA‐type glutamate receptor at excitatory synapses causes activation of post‐synaptic Ca 2+ /calmodulin‐dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B‐Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca 2+ /calmodulin activated form and the autonomously active Thr286‐autophosphorylated form of CaMKII. Green fluorescent protein–α‐CaMKII co‐expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione‐ S ‐transferase pull‐down assay. Thr286‐autophosphorylated α‐CaMKII or the autophosphorylation mimicking mutant, T286D‐α‐CaMKII, binds NR2B sequence independent of Ca 2+ /calmodulin unlike native wild‐type α‐CaMKII. We show enhancement of this binding by Ca 2+ /calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B‐S1303D) abolishes the Ca 2+ /calmodulin‐independent binding whereas it allows the Ca 2+ /calmodulin‐dependent binding of α‐CaMKII in vitro . Similarly, the autonomously active mutants, T286D‐α‐CaMKII and F293E/N294D‐α‐CaMKII, exhibited Ca 2+ ‐independent binding to non‐phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho‐Ser1303 on NR2B.