A novel role for Gab1 and SHP2 in epidermal growth factor-induced ras activation
A novel role for Gab1 and SHP2 in epidermal growth factor-induced ras activation
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DOI：
10.1074/jbc.m410012200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Raynal, P
中科院分区：
文献类型：
--
作者：
Montagner, A;Yart, A;Raynal, P
最近发现SHP 2通过去磷酸化Gabl下调PI 3 K活化，但是解释Gabl/SHP 2途径在EGF诱导的Ras活化中的积极作用的机制仍然不清楚。底物捕获实验现在表明，SHP 2去磷酸化其他Gabl磷酸酪氨酸位于显示四个YXXP基序的中心区域内。因为这些位点是RasGAP的潜在对接基序，我们测试了SHP 2是否使它们去磷酸化以促进Ras激活。我们观察到阻止SHP 2募集的Gabl构建体促进了RasGAP的膜重新定位。此外，在转染了SHP 2失活Gabl突变体的细胞或SHP 2缺陷型成纤维细胞中，RasGAP失活突变体恢复了Ras的活化，支持了RasGAP是SHP 2下游靶点的假设。为了确定Gabl是否是RasGAP结合配偶体，产生了缺失四个YAXP基序的Gabl突变体。该缺失抑制了RasGAP的再分布，并恢复了由SHP 2失活突变引起的有缺陷的Ras激活。此外，发现Gabl仅在SHP 2未被激活的条件下与RasGAP SH 2结构域相互作用。为了鉴定RasGAP结合位点，产生Gabl YXXP基序的Tyr至Phe突变体。在Tyr（317）上突变的Gabl构建体在RasGAP结合中受到严重影响，并且在补偿Ras缺陷的活化和阻断由SHP 2失活诱导的RasGAP再分布中最活跃。因此，我们已经定位在Gabl上的Ras负调控酪氨酸磷酸化位点参与RasGAP结合，并表明一个重要的SHP 2功能是下调其磷酸化以脱离RasGAP并维持Ras激活。
SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gabl but the mechanisms explaining the positive role of the Gabl/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gabl phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for RasGAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gabl construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gabl mutant or in SHP2deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gabl is a RasGAP-binding partner, a Gabl mutant deleted of four YAXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gabl was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify RasGAP-binding sites, Tyr to Phe mutants of Gabl YXXP motifs were produced. Gabl constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gabl a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.