Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF
Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF
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DOI：
10.1242/jcs.028647
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发表时间：
2008-08-15
影响因子：
4
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
--
作者：
Itoh, Reina E.;Kiyokawa, Etsuko;Matsuda, Michiyuki
在表皮生长因子(EGF)诱导的膜褶皱、膜脂突起和细胞迁移中，rac1起着至关重要的作用。包括Sos1、Sos2、Tiam1和Vav2在内的几个鸟嘌呤核苷酸交换因子(GEF)已被证明可以将生长信号从EGF受体转导到rac1。为了阐明每一种gef的作用，我们在靶向每个rac1gee的siRNA转染的A431细胞中，对EGF诱导的rac1活性变化进行了时间推移成像。由于这些GEF的敲除只部分抑制了EGF诱导的rac1激活，我们寻找EGF受体下游的另一种rac1 gef，发现与肿瘤抑制因子APC结合的rac1-CDc42 gef也参与了EGF诱导的rac1激活。有趣的是，EGF刺激以一种依赖于Src家族酪氨酸激酶的方式在ASEF的APC结合区诱导Tyr94的磷酸化。野生型ASEF可使siRNA处理细胞中EGF诱导的rac1活性受到抑制，但突变体ASEF的Tyr94Phe不能恢复该抑制作用。这一观察结果有力地证明了Tyr94磷酸化在EGF诱导的ASEF激活中的积极作用，该激活伴随着rac1的激活。
Rac1 has a crucial role in epidermal growth factor (EGF)induced membrane ruffling, lamellipodial protrusion, and cell migration. Several guanine nucleotide exchange factors (GEFs) including Sos1, Sos2, Tiam1 and Vav2 have been shown to transduce the growth signal from the EGF receptor to Rac1. To clarify the role of each GEF, we time-lapse imaged the EGF-induced activity change of Rac1 in A431 cells transfected with siRNA targeting each Rac1 GEE Because knockdown of these GEFs suppressed EGF-induced Rac1 activation only partially, we looked for another Rac1 GEF downstream of the EGF receptor and found that Asef, a Rac1-Cdc42 GEF bound to the tumor suppressor APC, also contributed to EGF-induced Rac1 activation. Intriguingly, EGF stimulation induced phosphorylation of Tyr94 within the APC-binding region of Asef in a manner dependent on Src-family tyrosine kinases. The suppression of EGF-induced Rac1 activation in siRNA-treated cells was restored by wild-type Asef, but not by the Tyr94Phe mutant of Asef. This observation strongly argues for the positive role of Tyr94 phosphorylation in EGF-induced Asef activation following the activation of Rac1.