Reduction of tyrosine kinase activity and protein tyrosine dephosphorylation by anoxic stimulation in vitro.
Tyrosine-specific protein phosphorylation has been recently implicated in mediating pathological changes associated with cerebral ischemia. In the present study, acute hypoxia/ischemia (anoxia) was simulated in vitro by incubating rat hippocampal slices in glucose-free artificial cerebrospinal fluid saturated with 95% N2/5% CO2. A marked decrease in the level of tyrosine phosphorylation of many protein bands compared with the control was observed. Immunoprecipitation and western blot confirmed that the NR2A/2B subunits of the N-methyl-D-aspartate receptors are among the dephosphorylated proteins. Maximal dephosphorylation of bands corresponding to NR2A/2B was reached after 10 min, and no recovery was observed even after 1 h in normal, oxygenated artificial cerebrospinal fluid. The effect was partially blocked by dephostatin, a membrane-permeable inhibitor of protein tyrosine phosphatases, but was not affected by the presence of glutamate receptor antagonists, or by removing extracellular Ca2+ or chelating intracellular Ca2+. Enzyme assay showed that anoxic stimulation resulted in a selective reduction in protein tyrosine kinase activity without affecting protein tyrosine phosphatase activity. Thus the present work suggests that anoxic stimulation produces a selective inhibition of protein tyrosine kinase activity leading to tyrosine-dephosphorylation of several proteins including the N-methyl-D-aspartate receptors. The underlying mechanism may involve a novel signal transduction pathway, which may protect neurons from degeneration during ischemic stress.
Braunton
J L
JL
Division of Pathology, Hospital for Sick Children, Toronto, ON, Canada.
Wong
V
V
Wang
W
W
Salter
M W
MW
Roder
J
J
Liu
M
M
Wang
Y T
YT
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D006624:Hippocampus / Q000201:enzymology / Q000378:metabolism; D002534:Hypoxia, Brain / Q000201:enzymology / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017470:Receptors, Glutamate / Q000378:metabolism; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D002118:Calcium