Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.
The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.
Omkumar
R V
RV
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
Kiely
M J
MJ
Rosenstein
A J
AJ
Min
K T
KT
Kennedy
M B
MB
D000818:Animals; D001665:Binding Sites; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione; D007700:Kinetics; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*; D012694:Serine; D014357:Trypsin / Q000378:metabolism
D016194:Receptors, N-Methyl-D-Aspartate; D012694:Serine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D005978:Glutathione