Identification of IκBα as a substrate of Fas-associated phosphatase-1
Identification of IκBα as a substrate of Fas-associated phosphatase-1
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DOI：
10.1046/j.1432-1327.2000.01818.x
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发表时间：
2000-12-01
期刊：
影响因子：
--
通讯作者：
Sato, TA
中科院分区：
文献类型：
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作者：
Nakai, Y;Irie, S;Sato, TA
Fas（APO-1/CD 95）是肿瘤坏死因子受体（TNFR）/神经生长因子受体（NGFR）超家族成员，是一种细胞表面分子，激活后可诱导细胞凋亡。Fas相关磷酸酶-1（FAP-1）是一种250 kDa的蛋白酪氨酸磷酸酶（PTP），与Fas的负调控结构域（C-末端15个氨基酸）相关。当用FAP-1转染时，人肿瘤细胞系变得对Fas介导的凋亡具有抗性，表明FAP-1在Fas介导的死亡信号中起负调节剂的作用。然而，FAP-1抑制细胞凋亡的机制仍不清楚。为了确定FAP-1如何影响Fas介导的信号传导，我们着手鉴定FAP-1的底物。为此，我们制备了FAP-1的催化结构域（C-末端399个氨基酸）或其非活性形式（Cys 2408-> Ser）与谷胱甘肽-S-转移酶（GST）融合的合成蛋白。使用体外去磷酸化反应，我们发现FAP-1使I κ B α去磷酸化。此外，发现底物捕获突变体结合酪氨酸磷酸化的I κ B α。综上所述，我们的数据证实I κ B α是FAP-1的底物。
Fas (APO-1/CD95), a member of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) superfamily, is a cell-surface molecule that induces apoptosis upon activation. Fas-associated phosphatase-1 (FAP-1) is a 250-kDa protein tyrosine phosphatase (PTP) that is associated with the negative regulatory domain of Fas (C-terminal 15 amino acids). Human tumor cell lines become resistant to Fas-mediated apoptosis when transfected with FAP-1, indicating that FAP-1 functions as a negative regulator in Fas-mediated death signaling. However, the mechanisms by which FAP-1 inhibits apoptosis are still unclear. In order to determine how FAP-1 affects the signaling mediated by Fas, we set out to identify substrates of FAP-1. Toward this end, we prepared synthetic proteins with either the catalytic domain of FAP-1 (C-terminal 399 amino acids) or its inactive form (Cys2408 --> Ser) fused to glutathione-S-transferase (GST). Using an in vitro dephosphorylation reaction, we found that FAP-1 dephosphorylates I kappaB alpha. Furthermore, a substrate trapping mutant was found to bind tyrosine-phosphorylated I kappaB alpha. Taken together, our data confirm that I kappaB alpha is a substrate of FAP-1.