Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II.
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-alpha-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 alpha-CaMKII or the autophosphorylation mimicking mutant, T286D-alpha-CaMKII, binds NR2B sequence independent of Ca(2+)/calmodulin unlike native wild-type alpha-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 alpha-CaMKII in vitro. Similarly, the autonomously active mutants, T286D-alpha-CaMKII and F293E/N294D-alpha-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.
Raveendran
Rajeevkumar
R
Rajiv Gandhi Centre for Biotechnology, Thycaud, P. O., Thiruvananthapuram-695014, Kerala, India.
Devi Suma Priya
Sudarsana
S
Mayadevi
Madhavan
M
Steephan
Mathew
M
Santhoshkumar
Thankayyan R
TR
Cheriyan
John
J
Sanalkumar
Rajendran
R
Pradeep
Kurup K
KK
James
Jackson
J
Omkumar
Ramakrishnapillai V
RV
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002490:Central Nervous System / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007313:Insecta; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology
C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D012694:Serine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D010744:Phosphoric Monoester Hydrolases