School of Biological Sciences, National Institute of Science Education and Research (NISER) Bhubaneswar, HBNI, P.O. Bhimpur-Padanpur, Via Jatni, Khurda, 752050, Odisha, India.
Institute of Biotechnology, University of Helsinki, P.O. Box 56, 0014, Helsinki, Finland.
J Biomed Sci. 2021 Feb 3;28(1):12. doi: 10.1186/s12929-021-00710-0.
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 serine (Ser) and threonine (Thr). 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. Ser and Thr 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 Ser and Thr-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.
幽门螺杆菌介导的胃癌发生是由感染胃上皮细胞（GECs）中的大量信号事件引发的。E3 泛素连接酶七缺失同源物 2（Siah2）在 GECs 中响应 H. pylori 感染而被诱导。Siah2 的翻译后修饰协调其功能和稳定性。本研究旨在评估 H. pylori 感染下 Siah2 的磷酸化状态及其在胃癌进展中的作用。
通过 Western blot 或免疫荧光显微镜检测 H. pylori 感染的各种 GECs、H. pylori 感染 GC 患者的胃组织和 H. felis 感染的 C57BL/6 小鼠中的 Siah2 磷酸化。通过免疫沉淀测定结合质谱分析鉴定与 Siah2 相互作用的激酶。通过使用定点突变产生的各种质粒构建体鉴定 Siah2 的磷酸化位点。使用蛋白酶体抑制剂 MG132 研究蛋白酶体降解事件。通过使用磷酸化缺失突变体和野生型 Siah2 稳定转染细胞进行集落形成、细胞增殖、非锚定生长和 Transwell 侵袭测定，检测感染细胞肿瘤发生中 Siah2 磷酸化的重要性。
Siah2 在 H. pylori 感染的 GECs 以及转移性 GC 组织中的丝氨酸（Ser）和苏氨酸（Thr）残基上发生磷酸化。Siah2 的磷酸化由 Ser/Thr 蛋白激酶 MRCKβ介导。MRCKβ 在未感染的 GECs 和非癌性胃组织中持续表达，但在感染的 GECs 和转移性组织中其水平降低，这些组织中 Siah2 的表达增强。感染的鼠胃组织也显示出类似的结果。MRCKβ 可以磷酸化 Siah2，但自身会因此相互作用而被泛素化，导致 MRCKβ 被蛋白酶体降解，使用蛋白酶体抑制剂 MG132 可以从 Siah2 介导的降解中拯救 MRCKβ。与非磷酸化的 Siah2 相比，磷酸化的 Ser 和 Thr-Siah2 更稳定且具有致瘤性，这一点通过稳定转染细胞的增殖、侵袭、迁移能力和非锚定生长能力揭示。
Ser 和 Thr 磷酸化 Siah2 水平升高和 MRCKβ 下调是幽门螺杆菌感染胃上皮组织和转移性人类 GC 的突出组织学特征。MRCKβ 依赖性 Siah2 磷酸化稳定了 Siah2，促进了 GECs 的非锚定生存和增殖潜能。Siah2 的磷酸化缺失突变体（S6A 和 T279A）显示出减弱的致瘤性。
