Harris M B, Ju H, Venema V J, Blackstone M, Venema R C
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500, USA.
Gen Pharmacol. 2000 Sep;35(3):165-70. doi: 10.1016/s0306-3623(01)00104-5.
Previously we described ENAP-1, a 90-kDa protein that is tyrosine-phosphorylated in endothelial cells in response to bradykinin (BK) stimulation and is associated with endothelial nitric oxide synthase (eNOS). Subsequently, other investigators demonstrated that eNOS interacts with heat shock protein 90 (Hsp90) following stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, or fluid shear stress. Therefore, we tested the hypotheses that ENAP-1 and Hsp90 are the same protein and that BK activation of eNOS is dependent on Hsp90. Immunoblotting of immunoprecipitated Hsp90 with anti-phosphotyrosine antibody shows that Hsp90 is tyrosine-phosphorylated in response to BK stimulation of bovine aortic endothelial cells (BAECs). Coimmunoprecipitation of Hsp90 with anti-eNOS antibody reveals a Hsp90-eNOS complex in endothelial cells under basal conditions that is increased following BK stimulation. Taken together with the tyrosine phosphorylation data, these data suggest that ENAP-1 is Hsp90. BK-stimulated nitric oxide (NO) release is completely blocked by pretreatment with geldanamycin, a specific inhibitor of Hsp90, illustrating the importance of the Hsp90-eNOS interaction. In vitro binding assays with Hsp90-glutathione-S-transferase fusion proteins show direct binding of eNOS with the middle domain (residues 259-615) of Hsp90.
此前我们描述了ENAP-1，一种90 kDa的蛋白质，它在缓激肽（BK）刺激下在内皮细胞中发生酪氨酸磷酸化，并与内皮型一氧化氮合酶（eNOS）相关。随后，其他研究人员表明，在用血管内皮生长因子（VEGF）、组胺或流体剪切应力刺激内皮细胞后，eNOS与热休克蛋白90（Hsp90）相互作用。因此，我们检验了以下假设：ENAP-1和Hsp90是同一蛋白质，并且eNOS的BK激活依赖于Hsp90。用抗磷酸酪氨酸抗体对免疫沉淀的Hsp90进行免疫印迹分析表明，在BK刺激牛主动脉内皮细胞（BAECs）后，Hsp90发生酪氨酸磷酸化。用抗eNOS抗体对Hsp90进行共免疫沉淀，发现在基础条件下内皮细胞中存在Hsp90-eNOS复合物，在BK刺激后该复合物增加。结合酪氨酸磷酸化数据，这些数据表明ENAP-1就是Hsp90。用Hsp90的特异性抑制剂格尔德霉素预处理可完全阻断BK刺激引起的一氧化氮（NO）释放，这说明了Hsp90-eNOS相互作用的重要性。用Hsp90-谷胱甘肽-S-转移酶融合蛋白进行的体外结合试验表明，eNOS与Hsp90的中间结构域（第259 - 615位氨基酸残基）直接结合。
