肌动蛋白磷酸化及其作用的研究

【作者】 李迎春；

【导师】 顾洛； 戈应滨；

【作者基本信息】 南京医科大学 ， 生理学， 2005， 硕士

【摘要】 肾脏的重吸收功能主要是通过肾脏上皮细胞内的微绒毛这一重要结构而实现的。肌动蛋白聚合形成了肾脏上皮细胞内对微绒毛起支撑作用的细胞骨架,因此肌动蛋白细胞骨架的破坏势必导致肾小管上皮细胞结构和功能的改变,从而引起肾脏的重吸收功能障碍。 肾脏近端小管上皮细胞ATP缺乏是研究肾脏缺血时多种细胞骨架成分及其功能改变的良好模型。我们以往的研究表明,肾脏近端小管上皮细胞ATP缺乏时,从微绒毛脱落的肌动蛋白是和非肌动蛋白结合在一起的。然而,有关其捕获的分子机制还不清楚。 就蛋白质的功能调节而言,磷酸化是最为常见的方式。但是有关肾脏上皮细胞内肌动蛋白磷酸化以及肌动蛋白磷酸化改变对肾脏上皮细胞内细胞骨架的影响报道很少。 基于此考虑,本研究用二维电泳和免疫印迹法分离并鉴定细胞内磷酸化和非磷酸化的肌动蛋白;然后用特异性的单克隆抗磷酸化丝氨酸(或抗磷酸化苏氨酸或抗磷酸化酪氨酸)抗体确定肌动蛋白磷酸化氨基酸残基的种类,从而探讨ATP缺乏时兔肾脏近端小管上皮细胞内肌动蛋白磷酸化与肌动蛋白捕获间的关系。 本研究结果发现,兔肾脏近端小管上皮细胞在ATP缺乏时:(1) 几乎有一半被捕获的肌动蛋白处于磷酸化状态;(2) 进一步的研究发现肌动蛋白的磷酸化是发生在丝氨酸残基上的;(3) 磷酸更多还原

【Abstract】 Renal proximal tubular (PT) ceils organize into highly specialized apical membrane domains, constituting microvilli and allowing them to perform the reabsorption of bulk of nutrients and ions in kidney. The establishment and maintenance of microvilli are dependent on the integrity of the actin cytoskeleton because actin polymerizes to form the actin cytoskeleton in the corn of microvilli in renal epithelial cells. Therefore, the disruption of the actin cytoskeleton initiates a cascade of structural and functional alteration in renal epithelial cells and which will lead to the dysfunction of reabsorption in kidney.ATP-depletion is a ideal model for studying various cytoskeletal and functional alternations induced by renal ischemia in renal proximal tubules (PT). Our previous study showed that the actin broken down from the microvillar F-actin filaments was bound to non-actin protein(s) in pellet of renal PT during ATP-depletion. However, the molecular details regarding actin sequestration remained elusive.Phosphorylation is the most common way of regulating protein functions. There is little known about the regulation of actin cytoskeletonal structure and function by actin phosphorylation inrenal epithelial cells.In this report, by using two-dimensional (2D) Western blotting, we separated phosphorylated actin from unphosphorylated actin in ATP-depleted PT, and identified phosphorylation actin by monoclonal anti-actin and which residues of amino acid were phosphorylated by using monoclonal anti-phosphoserine (or anti-phosphotyrosine or anti-phosphothreonine). The purpose of this study was to attempt to demonstrate the correlation between actin sequestration and actin phosphorylation in ATP-depleted PT.The analysis of the sequestered actin indicated that in ATP-depleted PT nearly half of the sequestered actin was phosphorylated on serine residue(s). The further study demonstrated that phosphorylated actin was only found in cytoskletonal fraction rather than in cytoplasmic fractions.In conclusion, the present studies demonstrated that there may be a close correlation between actin sequestration and actin phosphorylation in ATP-depleted PT.更多还原