Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney
Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney
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DOI：
10.1093/ndt/gfp697
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发表时间：
2010-06-01
影响因子：
6.1
通讯作者：
Yamamoto, Tadashi
中科院分区：
文献类型：
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作者：
Zhang, Ying;Yoshida, Yutaka;Yamamoto, Tadashi
背景。酪氨酸蛋白的磷酸化在多种生物反应的调控中起着至关重要的作用，已成为广泛研究的焦点。为了了解酪氨酸磷酸化在肾脏功能中的作用，我们对正常大鼠肾脏中酪氨酸磷酸化蛋白进行了全面的蛋白质组学研究。采用双向凝胶电泳和抗磷酸酪氨酸抗体免疫沉淀法检测酪氨酸磷酸化蛋白。用质谱法对蛋白质进行了分析，并用特异性抗体进行了免疫学分析。大多数酪氨酸磷酸化蛋白局限于肾小球，主要定位于肾小球毛细血管壁，尤其是足细胞足突。我们的系统蛋白质组学分析发现，nephrin、SHPS-1（酪氨酸蛋白磷酸酶非受体型底物1）、FAK1和paxillin是主要的酪氨酸磷酸化蛋白，而Neph1、talin和vinculin是次要的酪氨酸磷酸化蛋白。在本研究中，shps - 1被确定为肾小球中一种新的酪氨酸磷酸化蛋白，也主要定位于足突。质谱分析确定了SHPS-1在Y460、Y477和y501上的磷酸化位点。本研究在正常大鼠肾脏中发现酪氨酸磷酸化蛋白，该蛋白在肾小球中显著丰富，并定位于足细胞足突。这些蛋白被归类为细胞-细胞或细胞-基质粘附复合物相关分子，提示它们在肾小球超滤中起关键作用。
Background. Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney.Methods. Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies.Results. Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-I was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501.Conclusion. This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.