Honda Takao, Nakajima Kazunori
Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
J Biol Chem. 2006 Dec 15;281(50):38951-65. doi: 10.1074/jbc.M609061200. Epub 2006 Oct 23.
Disabled1 (DAB1) is an intracellular mediator of the Reelin-signaling 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.
Disabled1（DAB1）是Reelin信号通路的细胞内介质，对大脑发育过程中神经元的正确定位至关重要。到目前为止，DAB1一直被认为是一种细胞质蛋白。在此，我们表明DAB1存在核质穿梭现象。在其稳态下，DAB1主要位于细胞质中。然而，用 leptomycine B（一种CRM1（染色体区域维持蛋白1）-RanGTP依赖性核输出的特异性抑制剂）处理后，DAB1会在细胞核中积累。通过使用与增强型绿色荧光蛋白融合的DAB1缺失或替代突变体，我们确定了一个双分型核定位信号和两个CRM1依赖性核输出信号。这些靶向信号在Neuro2a细胞和原代大脑皮层神经元中均具有功能。使用纯化的重组蛋白，我们表明CRM1以RanGTP依赖性方式直接与DAB1结合。我们还表明，Fyn酪氨酸激酶或Reelin刺激导致的DAB1酪氨酸磷酸化（这对大脑层形成不可或缺）在体外并不影响DAB1的亚细胞定位。这些结果表明DAB1是一种核质穿梭蛋白，并增加了DAB1在细胞核和细胞质中均发挥作用的可能性。
