摘要
目的 :研究脑缺血损伤诱导非受体酪氨酸蛋白激酶Src之丝、苏、酪氨酸残基磷酸化及其与Pyk2结合的变化并探讨其可能的调节机制。方法 :四动脉结扎模型诱导大鼠前脑缺血损伤 ,免疫沉淀和免疫印迹法观察Src氨基酸残基磷酸化及其与Pyk2结合的变化。 结果 :缺血 15min后复灌 ,Src的Tyr 4 16和Ser而不是Thr的磷酸化有明显增加 ,且复灌 6hTyr 4 16磷酸化升至最高 ,而Ser磷酸化 1h升至顶峰 ,它们分别是正常组的 2 .8和 2 .3倍 ;此外 ,缺血 /复灌明显诱导Src与Pyk2的结合 ,相似于Tyr 4 16磷酸化变化 ,复灌 6h升至最高。 5 0mg/kg的氯胺酮能明显抑制缺血 /复灌诱导的SrcTyr 4 16磷酸化及其与Pyk2结合增加 ,与对照组比有显著性差异 (P <0 .0 5 )。结论 :脑缺血诱导了Src蛋白激酶Ser和Tyr 4 16磷酸化及其与Pyk2结合 ,它们参与了Src的激活和NMDA受体功能的自调。
Objective:To determine ischemia-induced phosphorylation of nonreceptor tyrosine kinase c-Src at tyrosine, serine and threonine residues and combination between c-Src and Pyk2, and to elucidate molecule mechanisms of c-Src activation underlying the brain ischemia lesion. Methods:Using immunoprecipitation and immunoblotting, phosphorylation of c-Src and its co-immunoprecipitation with Pyk2 were examined in the four-vessel occlusion (4-VO) model of Sprague-Dawley rats. Results:Ischemia/reperfusion (I/R) induced continuous tyrosine phosphorylation of c-Src in the kinase domain (Tyr-416) reached its peak at 6 h, and phosphorylation at serine residues reached its peak level at 1 h ( 2.6 and 2.3 folds vs sham control respectively), but I/R in 24 h lead to no alteration of its phosphorylation at Thr residues. Moreover, the binding level of Src to Pyk2 elicited by I/R was similar to its Tyr-416 phosphorylation and also reached a peak after 6 h. Ketamine at a 50mg/kg dose could suppress significantly the ischemia-induced Tyr-416 phosphorylation and combination between Src and Pyk2 ( P <0.05). Conclusion:I/R may increase the phosphorylation of c-Src at Tyr-416 and Ser and binding between c-Src and Pyk2 in the rat hippocampi, which is involved in the activation of c-Src and self-regulation of NMDA receptor function.
基金
国家自然科学基金 (3 0 170 2 2 0和 3 0 0 70 182 )
