Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein
Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein
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DOI：
10.1074/jbc.m609061200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Nakajima, Kazunori
中科院分区：
文献类型：
--
作者：
Honda, Takao;Nakajima, Kazunori
Disabled1 （DAB1）是reelin信号通路的细胞内介质，对大脑发育过程中正确的神经元定位至关重要。到目前为止，DAB1被认为是一种细胞质蛋白。在这里，我们发现DAB1受核细胞质穿梭的影响。在稳定状态下，DAB1主要位于细胞质中。然而，使用leptomycine B（一种CRM1（染色体区域维持1）- rangtp依赖的核输出的特异性抑制剂）治疗可导致DAB1的核积累。通过将DAB1的缺失或替换突变体与增强的绿色荧光蛋白融合，我们绘制了一个二部核定位信号和两个依赖crm1的核输出信号。这些靶向信号在Neuro2a细胞和原代大脑皮层神经元中都有功能。使用纯化的重组蛋白，我们已经证明CRM1以依赖于rangtp的方式直接与DAB1结合。我们还发现，Fyn酪氨酸激酶或Reelin刺激对DAB1的酪氨酸磷酸化（这是脑层形成所必需的）并不影响DAB1在体外的亚细胞定位。这些结果表明DAB1是一种核细胞质穿梭蛋白，并提出了DAB1在细胞核和细胞质中发挥作用的可能性。
Disabled1 (DAB1) is an intracellular mediator of the Reelinsignaling pathway and essential for correct neuronal positioning during brain development. So far, DAB1 has been considered a cytoplasmic protein. Here, we show that DAB1 is subject to nucleocytoplasmic shuttling. In its steady state, DAB1 is mainly located in the cytoplasm. However, treatment with leptomycine B, a specific inhibitor of the CRM1 (chromosomal region maintenance 1)-RanGTP-dependent nuclear export, resulted in nuclear accumulation of DAB1. By using deletion or substitutional mutants of DAB1 fused with enhanced green fluorescent protein, we have mapped a bipartite nuclear localization signal and two CRM1-dependent nuclear export signals. These targeting signals were functional in both Neuro2a cells and primary cerebral cortical neurons. Using purified recombinant proteins, we have shown that CRM1 binds to DAB1 directly in a RanGTP-dependent manner. We also show that tyrosine phosphorylation of DAB1, which is indispensable for the layer formation of the brain, by Fyn tyrosine kinase or Reelin stimulation did not affect the subcellular localization of DAB1 in vitro. These results suggest that DAB1 is a nucleocytoplasmic shuttling protein and raise the possibility that DAB1 plays a role in the nucleus as well as in the cytoplasm.