HSP70 binding modulates detachment of Na-K-ATPase following energy deprivation in renal epithelial cells.
HSP70 binding modulates detachment of Na-K-ATPase following energy deprivation in renal epithelial cells.
复制标题
DOI：
10.1152/ajprenal.00438.2004
复制
发表时间：
2005-06
期刊：
影响因子：
--
通讯作者：
M. Riordan;Rajasree Sreedharan;Shirley Wang;G. Thulin;A. Mann;Michael C. Stankewich;S. V. Van Why;M. Kashgarian;N. Siegel
中科院分区：
文献类型：
--
作者：
M. Riordan;Rajasree Sreedharan;Shirley Wang;G. Thulin;A. Mann;Michael C. Stankewich;S. V. Van Why;M. Kashgarian;N. Siegel
损伤后与肾上皮极性重建相关的分子机制仍未完全阐明。应激蛋白可以充当分子伴侣，潜在地调节损伤或增强恢复。我们测试了在 ATP 耗尽的条件下，热休克蛋白 70 (HSP70) 的过表达是否会稳定 Na-K-ATPase 与细胞骨架的附着，以及培养的肾上皮细胞中 Na-K-ATPase 和 HSP70 之间是否存在直接关联。 LLC-PK1 细胞用标记的 HSP70 (70FLAG) 或单独的载体 (VA) 转染。 ATP 耗尽后，在 Triton 可溶性裂解物中检测到 Na-K-ATP 酶的分离。 70FLAG 细胞在 ATP 耗尽 2 或 4 小时后，Na-K-ATP 酶的脱离显着减少 (P < 0.01)。 HSP70 和 Na-K-ATP 酶之间的相互作用通过 70FLAG 和 Na-K-ATP 酶的免疫共沉淀、直接和竞争性结合测定以及免疫细胞化学定位来确定。损伤后 HSP70 和 Na-K-ATP 酶的结合急剧增加。相互作用是：1）可逆； 2) 与 HSP70 结合蛋白网格蛋白的变化相反； 3) 仅当细胞裂解物中的 ATP 周转受到抑制时才存在，这是 HSP 结合的既定特征。这些研究表明：1) HSP70 的过度表达与损伤后 Na-K-ATP 酶从细胞骨架上的脱离减少有关； 2) HSP70 与 Na-K-ATPase 结合； 3) HSP70 与 Na-K-ATP 酶的结合是动态且特异性的，在损伤时增加，在恢复过程中减少。分子伴侣 HSP70 与受损或移位的 Na-K-ATP 酶之间的相互作用可能代表了能量剥夺后肾小管极性维持和恢复的基本细胞机制。
The molecular mechanisms associated with reestablishment of renal epithelial polarity after injury remain incompletely delineated. Stress proteins may act as molecular chaperones, potentially modulating injury or enhancing recovery. We tested whether overexpression of heat shock protein 70 (HSP70) would stabilize Na-K-ATPase attachment to the cytoskeleton, under conditions of ATP depletion, and whether a direct association existed between Na-K-ATPase and HSP70 in cultured renal epithelial cells. LLC-PK1 cells were transfected with a tagged HSP70 (70FLAG) or vector alone (VA). Detachment of Na-K-ATPase was detected in Triton soluble lysate after ATP depletion. 70FLAG cells demonstrated a significant (P < 0.01) decrease in detachment of Na-K-ATPase after either 2 or 4 h of ATP depletion. Interactions between HSP70 and Na-K-ATPase were determined by coimmunoprecipitation of 70FLAG and Na-K-ATPase, by direct and competitive binding assays and by immunocytochemical localization. Binding of HSP70 and Na-K-ATPase increased dramatically following injury. Interactions were: 1) reversible; 2) reciprocal to changes in the HSP70 binding protein clathrin; and 3) present only when ATP turnover was inhibited in cell lysate, an established characteristic of HSP binding. These studies indicate that 1) overexpression of HSP70 is associated with decreased detachment of Na-K-ATPase from the cytoskeleton following injury; 2) HSP70 binds to Na-K-ATPase; and 3) binding of HSP70 to Na-K-ATPase is dynamic and specific, increasing in response to injury and decreasing during recovery. Interaction between the molecular chaperone HSP70 and damaged or displaced Na-K-ATPase may represent a fundamental cellular mechanism underlying maintenance and recovery of renal tubule polarity following energy deprivation.