Cooperation of DEF6 with activated rac in regulating cell morphology
Cooperation of DEF6 with activated rac in regulating cell morphology
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DOI：
10.1074/jbc.m605153200
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发表时间：
2007-01-19
影响因子：
4.8
通讯作者：
Fukui, Yasuhisa
中科院分区：
文献类型：
--
作者：
Oka, Tsutomu;Ihara, Sayoko;Fukui, Yasuhisa
rho家族gtpase与各种细胞的肌动蛋白重塑和随后的形态学改变有关。DEF6是一种含有pleckstrin同源结构域的蛋白，已被报道作为鸟嘌呤核苷酸交换因子调节Rho-家族GTPases。在这里，我们证明了DEF6也具有与活化的Rac1协同的特性。DEF6选择性地与加载GTP的Rac1结合。这种相互作用由Rac1的效应域介导。GFP-DEF6和组成型活性Rac1在COS-7细胞中的过表达显著改变了其细胞形状；这在缺乏活化的Rac1时未见。DEF6对细胞形态的影响与鸟嘌呤核苷酸交换活性无关。由于DEF6不包含任何先前已知的与Rac相互作用的序列，因此我们探索了这种结合所必需的结构域。结合需要DEF6的氨基末端和中心部分。最后，我们成功地创建了DEF6的突变体，其氨基末端部分发生了点突变，从而取消了与活化的Rac1的结合。当这些突变体与活化的Rac1共表达时，在COS-7细胞中没有表现出形态变化。这些结果表明，DEF6不仅激活Rho-家族gtpase，而且还与活化的Rac1协同发挥其细胞功能。
Rho-family GTPases have been implicated in actin remodeling and subsequent morphologic changes in various cells. DEF6, a pleckstrin homology domain-containing protein, has been reported to regulate Rho- family GTPases as a guanine nucleotide exchange factor. Here, we demonstrate that DEF6 also has the property of cooperating with activated Rac1. DEF6 bound selectively to Rac1 loaded with GTP. The interaction is mediated by the effector domain of Rac1. Overexpression of GFP-DEF6 together with constitutively active Rac1 in COS-7 cells significantly changed their cell shape; this was not seen in the absence of activated Rac1. This effect of DEF6 on cellular morphology was shown to be independent of its guanine nucleotide exchange activity. Because DEF6 does not contain any sequences previously known to interact with Rac, we explored the domain necessary for the binding. The amino-terminal portion and central parts of DEF6 were required for the binding. Finally, we succeeded in creating mutants of DEF6 with point mutations in the amino-terminal portion, which abrogate the binding to activated Rac1. These mutants did not exhibit the morphologic change in COS-7 cells when they were co-expressed with activated Rac1. These results suggest that DEF6 not only activates Rho- family GTPases but also cooperates with activated Rac1 to exert its cellular function.