Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells
Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells
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DOI：
10.1046/j.1365-2958.2001.02649.x
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发表时间：
2001-11-01
影响因子：
3.6
通讯作者：
Meyer, TF
中科院分区：
文献类型：
--
作者：
Backert, S;Moese, S;Meyer, TF
幽门螺杆菌在人体胃内定植，是多种胃病的病原体。细菌附着后，H.幽门螺杆菌CagA蛋白转位到胃上皮细胞中并酪氨酸磷酸化。这一过程与特征性的细胞骨架重排有关，导致散射因子样（“蜂鸟”）表型。在这项研究中，使用cagA突变体补充野生型cagA和瞬时表达CagA在AGS细胞中，我们已经证明，易位CagA是必要的肌动蛋白细胞骨架的重排发生。抗磷酸酪氨酸免疫印迹研究和治疗感染的细胞与磷酸酪氨酸激酶抑制剂表明，不仅易位，而且CagA的磷酸化是重要的，在这个过程中。CagA-绿色荧光蛋白（GFP）融合蛋白的瞬时表达和CagA蛋白种类的二维凝胶电泳证明了C-末端的酪氨酸磷酸化。CagA的定点突变显示，酪氨酸残基972是诱导细胞表型所必需的。我们还证明，CagA的移位和磷酸化对于诱导AGS细胞中的蜂鸟表型是必要的，但还不足以，这表明涉及尚未鉴定的细菌因子。
Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor-like ('hummingbird') phenotype. In this study, using a cagA mutant complemented with wild-type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Antiphosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA-green fluorescent protein (GFP) fusion proteins and two-dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C-terminus. Site-directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).