| The N-methyl-D-aspartate (NMDA) receptor is a Ca2+/Na+ channel that is regulated by phosphorylation. In this thesis, it was demonstrated that Src-family protein tyrosine kinases (Src) phosphorylate the rodent NMDA receptor subunits NR2A and NR2B. Tryptic peptide mapping identified seven and five major tyrosine-phosphorylated peptides derived from NR2A and NR2B respectively, but phosphorylation of NR2B by endogenous postsynaptic density (PSD) tyrosine kinases yielded only three tyrosine-phosphorylated tryptic peptides, two of which corresponded to Src phosphorylation sites, and one of which was novel. Y1472 of NR2B was identified as a phosphorylation site for intrinsic PSD tyrosine kinases, including Src. Following a transient (15 minutes) global ischemic episode produced by the four-vessel occlusion procedure, phosphorylation of several PSD proteins, including NR2B on Y1472, NR2A, and NR1 on S890, S896 and S897, was enhanced relative to shams. The ability of intrinsic PSD tyrosine kinases to phosphorylate the NMDA receptor increased following ischemia. The levels of PSD-associated protein kinases, including gp145TrkB, activated PKC and PYK2, were elevated at early times [0 minute (TrkB) and 15 minutes (PKC and PYK2)] after the ischemic event. Although an increase in PSD-associated Src was delayed for between 20 minutes and 6 hours, activated Src was present in the PSD by as early as 15 minutes. Phosphorylation of NR2A and NR2B by exogenous Src was dependent upon initial binding of the kinase to PSDs via its SH2-domain. The in vitro binding of NR2A and NR2B to the SH2-domain of Src increased after ischemia and 6 hours of reperfusion. Inhibition of PKC reduced the ischemia-induced activation of PKC, PYK2 and Src in PSDs, and diminished the increase in tyrosine phosphorylation of NR2A and NR2B. Together, these results contribute to our understanding of the basic mechanisms that underlie ischemia-induced increases in tyrosine phosphorylation of the NMDA receptor. |
