Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor receptor/NF-κB activation through Gab1 in glioblastoma cells
Distinct domains in the SHP-2 phosphatase differentially regulate epidermal growth factor receptor/NF-κB activation through Gab1 in glioblastoma cells
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DOI：
10.1128/mcb.24.2.823-836.2004
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发表时间：
2004-01-01
影响因子：
5.3
通讯作者：
O'Rourke, DM
中科院分区：
文献类型：
--
作者：
Kapoor, GS;Zhan, Y;O'Rourke, DM
核转录因子kappaB在炎症和癌症中起重要作用，被多种刺激激活，包括肿瘤坏死因子α、白介素1、紫外线照射、病毒，以及受体酪氨酸激酶，如表皮生长因子受体(EGFR)。虽然先前的研究表明，EGFR可以诱导核因子-kappaB的表达，但这种激活的机制仍不清楚。在这项研究中，我们确定了EGFR诱导的人脑胶质母细胞瘤细胞中调节核因子-kappaB激活所需的信号小体的成分。ErbB调节细胞的免疫沉淀分析表明，SHP-2与Grb2相关结合蛋白1(GAB1)之间的结合是连接EGFR和NF-kappaB激活的信号体形成的关键步骤。我们还表明，EGFR诱导的NF-kappaB激活是由PI3-Kinase/Akt激活环介导的。SHP-2、GAB1和肉豆蔻酰化Akt的过表达显著上调了胶质母细胞瘤细胞中NF-kappaB的转录活性和DNA结合活性。有趣的是，过表达SHP-2的两个SH2结构域中的任何一个，R32E或R138E，与野生型SHP-2相比，NF-kappaB的活性略有降低，表明SHP-2的SH2结构域是EGFR诱导的NF-kappaB激活所必需的。另一方面，无论是不能与SHP-2结合的GAB1突变体(Y627F)还是磷酸酶失活的SHP-2突变体(C459S)的异位过表达导致了NF-kappaB活性的显著增加。此外，表达SHP-2C459S的细胞表现出较高的GAB1磷酸化酪氨酸含量，提示SHP-2通过其磷酸酶结构域调节GAB1的磷酸化，从而对NF-kappaB的活性产生负调控作用。这些结果表明，在胶质母细胞瘤细胞中，SHP-2/GAB1结合是通过PI3-K/Akt信号轴将EGFR与NF-kappaB转录活性联系起来的关键，SHP-2是NF-kappaB激活的双重调节因子。
The transcription factor nuclear factor kappaB (NF-kappaB) plays an important role in inflammation and cancer, is activated by a variety of stimuli including tumor necrosis factor alpha, interleukin-1, UV irradiation, and viruses, as well as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Although previous studies suggest that EGFR can induce NF-kappaB, the mechanism of this activation remains unknown. In this study, we identify the components of the EGFR-induced signalosome in human glioblastoma cells required to regulate NF-kappaB activation. Immunoprecipitation analyses with ErbB-modulated cells indicate that association between SHP-2 and Grb2-associated binder 1 (Gab1) is the critical step in the formation of the signalosome linking EGFR to NF-kappaB activation. We also show that EGFR-induced NF-kappaB activation is mediated by the PI3-kinase/Akt activation loop. Overexpression of SHP-2, Gab1, and myristoylated Akt significantly upregulated NF-kappaB transcriptional activity and DNA binding activity in glioblastoma cells. Interestingly, overexpression of either one of the two SH2 domain mutants of SHP-2, R32E or R138E, slightly reduced NF-kappaB activity relative to that of wild-type SHP-2, indicating that the SH2 domains of SHP-2 are required for EGFR-induced NF-kappaB activation. On the other hand, ectopic overexpression of either a Gab1 mutant incapable of binding to SHP-2 (Y627F) or a phosphatase-inactive SHP-2 mutant (C459S) caused a significant increase in NF-kappaB activity. Moreover, SHP-2 C459S-expressing cells displayed higher Gab1 phosphotyrosine content, suggesting that SHP-2 regulates Gab1 phosphoryllation through its phosphatase domain, which confers a negative regulatory effect on NF-kappaB activity. These results indicate that SHP-2/Gab1 association is critical for linking EGFR to NF-kappaB transcriptional activity via the PI3-kinase/Akt signaling axis in glioblastoma cells and that SHP-2 acts as a dual regulator of NF-kappaB activation.