A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus
A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus
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DOI：
10.1074/jbc.m314155200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Sedor, JR
中科院分区：
文献类型：
--
作者：
Srichai, MB;Konieczkowski, M;Sedor, JR
肾小球足细胞分化状态对滤过屏障功能至关重要，并受锌指转录因子WT1的调节。酵母双杂交试验发现了一种新的WT1相互作用蛋白(WTIP)，该蛋白定位于人类染色体19q13.1，该区域含有与家族性局灶性节段性肾小球硬化相关的基因。WTIP的结构域结构类似于胞质LIM结构域的Zysin亚家族，它包含三个羧基末端的LIM蛋白-蛋白质相互作用结构域和一个富含脯氨酸的、具有核输出信号的前LIM区域。在双杂交实验中，其他含有LIM结构域的蛋白(Zysin和小鼠肌肉LIM蛋白)不与WT1相互作用，WTIP也不与无关的转录因子Lmx1b相互作用。WTIP mRNA在培养的足细胞中检测到，并受发育调节，在小鼠肾脏中的表达在胚胎15-16天(E15-E16)达到高峰，但持续到成年。原位杂交显示WTIP在发育中的E15肾小球和培养的足细胞中有表达。在双杂交实验中与WTIP相互作用的部分WTIP克隆与WT1共定位于细胞核，与WT1共沉淀，并抑制WT1依赖的双调节蛋白启动子的转录激活。相反，全长WTIP被排除在细胞核之外，但在加入CRM1介导的核输出抑制剂软霉素B后，它在细胞核内积聚，并在整个细胞裂解产物中与WT1共沉淀。表位标记的WTIP与适配蛋白CD2AP(CMS)共同定位于足细胞肌动蛋白斑点，并与MENA位于细胞-细胞连接处。我们认为WTIP作为多蛋白质复合体的一部分，将这种特殊的黏附连接连接到肌动蛋白细胞骨架上，并在足细胞损伤后穿梭到细胞核中，提供了一种缝隙横隔膜结构变化调节基因表达的机制。
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.