Role of intracellular localization and phosphorylation of CagA in host cell activation.

胃幽門桿菌(Helicobacter pylori)可造成胃癌、胃炎以及胃潰瘍等疾病，而CagA被認為是主要的致病分子，CagA是一個120-145 kDa的蛋白質，可以透過胃幽門桿菌的線毛及其他cag PAI上分子的協助，利用第四型分泌系統(type IV secretion system)注入胃上皮細胞中，被細胞中的磷酸酶Src family kinase和Abl磷酸酶磷酸化，而被磷酸化的CagA可與SHP-2結合，形成活化態SHP-2，引起SHP-2將focal adhesion kinase(FAK)去磷酸化，也透過E-cadherin和β-catenin等路徑，改變細胞與細胞間的交互作用，造成細胞型態的改變，成為延長狀偽足的表現型。而SHP-2不正常持續的活化情形被證實與癌症的生成有關，因此，認為CagA是一個可引起癌症的致癌蛋白。我們實驗室發現CagA可以轉運至B淋巴細胞內，同樣的在細胞質中被磷酸化形成phospho-CagA，phospho-CagA也會與SHP-2交互作用，而在CagA的影響下，活化p38、Erk、抗凋亡分子Bcl-2和Bcl-XL。 但是，目前對於CagA進入B淋巴細胞後所存在的胞內位置及作用仍不清楚。而H. pylori+黏膜相關淋巴組織林巴瘤患者組織切片染色發現CagA表現在細胞的細胞核區域，因此，本研究想探討CagA進入細胞內的位置以及CagA是否會進入B細胞核內。我們將B細胞株轉染CagA質體DNA或與胃幽門桿菌共同培養，皆觀察到CagA有在細胞質和細胞核中，證明了CagA分布在細胞質與細胞核內，同樣於胃上皮細胞AGS細胞株中觀察到CagA分佈在細胞核內的結果，同時也發現到細胞核中的CagA表現量比細胞質多，代表CagA在細胞核內具有一定的角色。再製作不同片段的CagA質體DNA轉染進AGS細胞中，觀察CagA進核的方式，我們發現到 CagA的磷酸化修飾的有無對於CagA進入細胞核中並沒有影響，胺基端序列對於CagA進入細胞核有重要性的角色，我們再利用CagA抗體作免疫沉澱法，發現細胞核內phospho-CagA與SHP-2可共沉澱在一起，顯示它們具有交互作用，但是，幫助CagA進入細胞核確切的片段和CagA在細胞核中主要的角色仍需要深入探討。希望將來我們的研究結果對於CagA如何造成B淋巴細胞的活化將具有重要的啟發與貢獻。Helicobacter pylori could cause gastritis, duodenal ulcer, and gastric cancer. CagA is regarded as a master virulent factor. CagA of H. pylori, a 120-145 kDa protein, is injected into gastric epithelial cells cytoplasm by type Ⅳ secretion system, which is accomplished by pilus and the cag PAI genes. Furthermore, the cytoplasmic CagA is phosphorylated by eukaryotic Src family kinase and Abl kinase. The phospho-CagA interacts with SHP-2 which becomes constitutive activated. The activated SHP-2 not only dephosphorylates focal adhesion kinase but also disrupts the E-cadherin and β-catenin pathway, leading to destroy the epithelial cell-cell junction. It is characterized by the spreading and elongated filopodia called hummingbird phenotype. The aberrant constitutive activation of SHP-2 is associated with tumor formation, therefore, CagA is considered as an oncoprotein. In the previous study, our lab firstly demonstrates that CagA could be directly translocated into human B lymphocytes from Helicobacter pylori. CagA is also phosphorylated in the cytoplasm, and then the phospho-CagA could bind to the phosphatase SHP-2. CagA activates p38, Erk, and anti-apoptosis molecules, Bcl-2 and Bcl-XL. However, it is unclear about the intracellular location and the function of translocated CagA in B lymphocytes. In our previous study, it showed that CagA co-localized in nucleus in the immunohistochemistry staining of H. pylori+ mucosa-associated lymphoid tissue (MALT) lymphoma patients. Therefore, we want to study the intracellular location of CagA and to prove whether CagA would translocate into nucleus in B lymphocytes. We used B cells transfected with CagA plasmid or co-cultured with H. pylori, isolating the cytosolic and nuclear fraction. Our results indicated CagA could be detected in both cytoplasm and nucleus in B lymphocytes. Similarly, we also observed CagA could be translocated in nucleus of gastric epithelial cell lines, AGS cells. The expression of CagA is more abundant in nucleus than in the cytoplasm. Our results suggest that CagA may have an important role in the nucleus. By transfecting different truncated CagA plasmids into AGS cells, we reveal phosphorylation is not required for CagA nuclear translocation. CagA nuclear translocation is dependent on the fragment of CagA amine-terminus. We also isolated the nuclear fraction, and we demonstrated that nuclear translocated CagA co-immunoprecipitated with SHP-2. But the specific amine-terminal sequences for nuclear translocation and the role of CagA in nucleus is still needed to be investigated. Our results in this study will provide a crucial insight and contribution on CagA-induced B lymphocytes activation.