Srichai Manakan B, Konieczkowski Martha, Padiyar Aparna, Konieczkowski David J, Mukherjee Amitava, Hayden Patrick S, Kamat Sweta, El-Meanawy M Ashraf, Khan Shenaz, Mundel Peter, Lee Sean Bong, Bruggeman Leslie A, Schelling Jeffrey R, Sedor John R
Departments of Medicine and Physiology and Biophysics, School of Medicine, Case Western Reserve University and Rammelkamp Center for Research and Education, MetroHealth System Campus, Cleveland, Ohio 44109-1998, USA.
J Biol Chem. 2004 Apr 2;279(14):14398-408. doi: 10.1074/jbc.M314155200. Epub 2004 Jan 20.
Glomerular podocyte differentiation state is critical for filtration barrier function and is regulated by WT1, a zinc finger transcription factor. A yeast two-hybrid assay identified a novel, WT1-interacting protein (WTIP) that maps to human chromosome 19q13.1, a region with genes linked to familial focal segmental glomerulosclerosis. The domain structure of WTIP is similar to the zyxin subfamily of cytosolic LIM domain-containing proteins, which contain three carboxyl-terminal LIM protein-protein interaction domains and a proline-rich, pre-LIM region with a nuclear export signal. Other LIM domain-containing proteins (zyxin and mouse muscle LIM protein) did not interact with WT1 in two-hybrid assays, and WTIP did not interact with an unrelated transcription factor, LMX1B. WTIP mRNA was detected in cultured podocytes and was developmentally regulated, with expression peaking in mouse kidney at embryonic day 15-16 (E15-E16) in kidney but persisting into adulthood. In situ hybridization demonstrated WTIP expression in developing E15 glomeruli and in cultured podocytes. The partial WTIP clone, which interacted with WTIP in the two-hybrid assay, co-localized with WT1 in nuclei, co-precipitated with WT1, and inhibited WT1-dependent transcriptional activation of the amphiregulin promoter. In contrast, full-length WTIP was excluded from cell nuclei, but after the addition of leptomycin B, an inhibitor of Crm1-mediated nuclear export, it accumulated in the nucleus and co-precipitated with WT1 in whole cell lysates. Epitope-tagged WTIP co-localized with the adaptor protein CD2AP (CMS) in podocyte actin spots and with Mena at cell-cell junctions. We propose that WTIP monitors slit diaphragm protein assembly as part of a multiple protein complex, linking this specialized adhesion junction to the actin cytoskeleton, and shuttles into the nucleus after podocyte injury, providing a mechanism whereby changes in slit diaphragm structure modulate gene expression.
肾小球足细胞的分化状态对于滤过屏障功能至关重要，且受锌指转录因子WT1调控。酵母双杂交实验鉴定出一种新的与WT1相互作用的蛋白（WTIP），该蛋白定位于人类19号染色体q13.1区域，此区域的基因与家族性局灶节段性肾小球硬化相关。WTIP的结构域与含胞质LIM结构域蛋白的zyxin亚家族相似，后者含有三个羧基末端LIM蛋白-蛋白相互作用结构域以及一个富含脯氨酸的前LIM区域，该区域带有核输出信号。在双杂交实验中，其他含LIM结构域的蛋白（zyxin和小鼠肌肉LIM蛋白）不与WT1相互作用，并且WTIP也不与无关转录因子LMX1B相互作用。在培养的足细胞中检测到WTIP mRNA，其表达受发育调控，在小鼠胚胎期第15 - 16天（E15 - E16）时在肾脏中表达达到峰值，但在成年期仍持续存在。原位杂交显示WTIP在发育中的E15肾小球及培养的足细胞中表达。在双杂交实验中与WTIP相互作用的部分WTIP克隆，在细胞核中与WT1共定位，与WT1共沉淀，并抑制WT1依赖的双调蛋白启动子的转录激活。相反，全长WTIP被排除在细胞核外，但在添加Crm1介导的核输出抑制剂雷帕霉素B后，它在细胞核中积累并在全细胞裂解物中与WT1共沉淀。表位标记的WTIP在足细胞肌动蛋白斑点中与衔接蛋白CD2AP（CMS）共定位，并在细胞间连接处与Mena共定位。我们提出WTIP作为多蛋白复合物的一部分监测裂孔隔膜蛋白组装，将这种特殊的黏附连接与肌动蛋白细胞骨架相连，并在足细胞损伤后穿梭进入细胞核，提供了一种机制，通过该机制裂孔隔膜结构的变化调节基因表达。
