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Helicobacter pylori-induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation
Journal of Biomedical Science ( IF 11.0 ) Pub Date : 2021-02-03 , DOI: 10.1186/s12929-021-00710-0 Pragyesh Dixit 1 , Shrikant B Kokate 1, 2 , Indrajit Poirah 1 , Debashish Chakraborty 1 , Duane T Smoot 3 , Hassan Ashktorab 4 , Niranjan Rout 5 , Shivaram P Singh 6 , Asima Bhattacharyya 1
Journal of Biomedical Science ( IF 11.0 ) Pub Date : 2021-02-03 , DOI: 10.1186/s12929-021-00710-0 Pragyesh Dixit 1 , Shrikant B Kokate 1, 2 , Indrajit Poirah 1 , Debashish Chakraborty 1 , Duane T Smoot 3 , Hassan Ashktorab 4 , Niranjan Rout 5 , Shivaram P Singh 6 , Asima Bhattacharyya 1
Affiliation
Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression. H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay. Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine6 (Ser6) and threonine279 (Thr279). Phosphorylation of Siah2 was mediated by MRCKβ, a Ser/Thr protein kinase. MRCKβ was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCKβ could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCKβ and use of proteasomal inhibitor MG132 could rescue MRCKβ from Siah2-mediated degradation. Ser6 and Thr279 phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells. Increased level of Ser6 and Thr279-phosphorylated-Siah2 and downregulated MRCKβ were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCKβ-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.
中文翻译:
幽门螺杆菌诱导的胃癌由 MRCKβ 介导的 Siah2 磷酸化调控
幽门螺杆菌介导的胃癌发生是由受感染的胃上皮细胞 (GEC) 中的大量信号事件引发的。E3 泛素连接酶 7 in absentia homolog 2 (Siah2) 在 GEC 中被诱导以响应幽门螺杆菌感染。Siah2 的翻译后修饰协调其功能和稳定性。本研究的目的是评估幽门螺杆菌感染影响下的 Siah2 磷酸化状态及其对胃癌进展的影响。通过蛋白质印迹或免疫荧光显微镜评估幽门螺杆菌感染的各种 GEC、来自幽门螺杆菌感染的 GC 患者和 H. felis 感染的 C57BL/6 小鼠的胃组织的 Siah2 磷酸化。进行免疫共沉淀测定和质谱分析以确定与 Siah2 相互作用的激酶。Siah2 的磷酸化位点是通过使用定点诱变生成的各种质粒结构来鉴定的。蛋白酶体抑制剂 MG132 用于研究蛋白酶体降解事件。Siah2 磷酸化对感染细胞致瘤性的重要性通过使用磷酸化无效突变体和野生型 Siah2 稳定转染细胞,然后进行克隆形成测定、细胞增殖测定、贴壁非依赖性生长和 transwell 侵袭测定来检测。Siah2 在幽门螺杆菌感染的 GEC 以及转移性 GC 组织中的丝氨酸 6 (Ser6) 和苏氨酸 279 (Thr279) 残基处被磷酸化。Siah2 的磷酸化由 MRCKβ 介导,MRCKβ 是一种 Ser/Thr 蛋白激酶。MRCKβ 在未感染的 GEC 和非癌性胃组织中持续表达,但其在感染的 GEC 和转移组织中的水平降低,而转移组织中的 Siah2 表达增强。感染的小鼠胃组织显示出相似的结果。MRCKβ 可以使 Siah2 磷酸化,但它本身会从这种相互作用中泛素化,导致 MRCKβ 的蛋白酶体降解,使用蛋白酶体抑制剂 MG132 可以从 Siah2 介导的降解中拯救 MRCKβ。Ser6 和 Thr279 磷酸化的 Siah2 比其非磷酸化的对应物更稳定和致瘤,如稳定转染细胞的增殖、侵袭、迁移能力和贴壁非依赖性生长所揭示的。Ser6 和 Thr279-磷酸化-Siah2 水平升高和 MRCKβ 下调是幽门螺杆菌感染的胃上皮细胞和转移性人类 GC 的显着组织学特征。MRCKβ 依赖性 Siah2 磷酸化稳定了 Siah2,从而促进了 GEC 的不依赖锚定的存活和增殖潜力。Siah2 的无磷酸化突变体(S6A 和 T279A)表现出减弱的致瘤性。
更新日期:2021-02-03
中文翻译:
幽门螺杆菌诱导的胃癌由 MRCKβ 介导的 Siah2 磷酸化调控
幽门螺杆菌介导的胃癌发生是由受感染的胃上皮细胞 (GEC) 中的大量信号事件引发的。E3 泛素连接酶 7 in absentia homolog 2 (Siah2) 在 GEC 中被诱导以响应幽门螺杆菌感染。Siah2 的翻译后修饰协调其功能和稳定性。本研究的目的是评估幽门螺杆菌感染影响下的 Siah2 磷酸化状态及其对胃癌进展的影响。通过蛋白质印迹或免疫荧光显微镜评估幽门螺杆菌感染的各种 GEC、来自幽门螺杆菌感染的 GC 患者和 H. felis 感染的 C57BL/6 小鼠的胃组织的 Siah2 磷酸化。进行免疫共沉淀测定和质谱分析以确定与 Siah2 相互作用的激酶。Siah2 的磷酸化位点是通过使用定点诱变生成的各种质粒结构来鉴定的。蛋白酶体抑制剂 MG132 用于研究蛋白酶体降解事件。Siah2 磷酸化对感染细胞致瘤性的重要性通过使用磷酸化无效突变体和野生型 Siah2 稳定转染细胞,然后进行克隆形成测定、细胞增殖测定、贴壁非依赖性生长和 transwell 侵袭测定来检测。Siah2 在幽门螺杆菌感染的 GEC 以及转移性 GC 组织中的丝氨酸 6 (Ser6) 和苏氨酸 279 (Thr279) 残基处被磷酸化。Siah2 的磷酸化由 MRCKβ 介导,MRCKβ 是一种 Ser/Thr 蛋白激酶。MRCKβ 在未感染的 GEC 和非癌性胃组织中持续表达,但其在感染的 GEC 和转移组织中的水平降低,而转移组织中的 Siah2 表达增强。感染的小鼠胃组织显示出相似的结果。MRCKβ 可以使 Siah2 磷酸化,但它本身会从这种相互作用中泛素化,导致 MRCKβ 的蛋白酶体降解,使用蛋白酶体抑制剂 MG132 可以从 Siah2 介导的降解中拯救 MRCKβ。Ser6 和 Thr279 磷酸化的 Siah2 比其非磷酸化的对应物更稳定和致瘤,如稳定转染细胞的增殖、侵袭、迁移能力和贴壁非依赖性生长所揭示的。Ser6 和 Thr279-磷酸化-Siah2 水平升高和 MRCKβ 下调是幽门螺杆菌感染的胃上皮细胞和转移性人类 GC 的显着组织学特征。MRCKβ 依赖性 Siah2 磷酸化稳定了 Siah2,从而促进了 GEC 的不依赖锚定的存活和增殖潜力。Siah2 的无磷酸化突变体(S6A 和 T279A)表现出减弱的致瘤性。