Tyrosine phosphorylation of the N-methyl-D-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases.
Phosphorylation of the NMDA receptor by Src-family tyrosine kinases has been implicated in the regulation of receptor function. We have investigated the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B by exogenous Src and Fyn and compared this to phosphorylation by tyrosine kinases associated with the postsynaptic density (PSD). Phosphorylation of the receptor by exogenous Src and Fyn was dependent upon initial binding of the kinases to PSDs via their SH2-domains. Src and Fyn phosphorylated similar sites in NR2A and NR2B, tryptic peptide mapping identifying seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B, respectively. All five tyrosine phosphorylation sites on NR2B were localized to the C-terminal, cytoplasmic domain. Phosphorylation of NR2B by endogenous PSD tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Phosphorylation-site specific antibodies identified NR2B Tyr1472 as a phosphorylation site for intrinsic PSD tyrosine kinases. Phosphorylation of this site was inhibited by the Src-family-specific inhibitor PP2. The results identify several potential phosphorylation sites for Src in the NMDA receptor, and indicate that not all of these sites are available for phosphorylation by kinases located within the structural framework of the PSD.
Cheung
H H
HH
Center for the Neurobiology of Stress, Division of Life Sciences, University of Toronto at Scarborough, Ontario, Canada.
Gurd
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000378:metabolism*; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D019000:Phosphotyrosine; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases