Testin and filamin-C downregulation by acetylated Siah2 increases invasiveness of Helicobacter pylori-infected gastric cancer cells

Kokate, Shrikant Babanrao; Dixit, Pragyesh; Poirah, Indrajit; Roy, Arjama Dhar; Chakraborty, Debashish; Rout, Niranjan; Singh, Shivaram Prasad; Ashktorab, Hassan; Smoot, Duane T.; Bhattacharyya, Asima

doi:10.1016/j.biocel.2018.07.012

Cited by 18 publications

(17 citation statements)

References 47 publications

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“…Western blotting was performed following standard protocols [7,9,20]. Briefly, total cell lysates were prepared from GECs (with or without H. pylori infection), run on SDS-PAGE and electro-transferred onto PVDF membrane.…”

Section: Western Blotting and Coimmunoprecipitation Assaysmentioning

confidence: 99%

“…Stably-transfected MKN45 cells were used to assess anchorage-independent growth following our previouslyestablished protocols [8,9]. Clonogenic survival assay pcDNA3.1+, WT, S6A and T279A siah2-expressing MKN45 stable cells were seeded in 24-well cell culture dishes with a density of 16.6 × 10 4 followed by infection for 12 h at 200 MOI.…”

Section: Soft Agar Assaymentioning

confidence: 99%

“…pcDNA3.1+, WT, S6A and T279A siah2-expressing AGS stable cells were seeded on cell culture inserts with 0.3 cm 2 polyethylene terephthalate membrane (8-μm pore size and 6.4-mm diameter; Falcon Corning, MA) coated with growth factor-reduced standard Matrigel matrix (Becton Dickinson, NJ) and further processed according to our previously reported protocol [9,22]. Images were captured from at least three different fields using an inverted microscope as already mentioned.…”

Section: In Vitro Invasion Assaymentioning

confidence: 99%

“…Our group identified that Siah2 was optimally induced by H. pylori 12 h after infection and at 200 MOI [7,9]. In order to identify kinases that interact with Siah2 and to enrich interaction signals, overexpression-immunoprecipitation assay was performed.…”

Section: H Pylori Infection Enhances Siah2 Phosphorylationmentioning

confidence: 99%

“…Increase in Siah2 aids in H. pylori-mediated GC progression [8]. Siah2 enhances invasiveness and progression of GC through the regulation of degradation of its various binding partners [9]. Siah2 function and subcellular localization are modulated by post-translational modifications.…”

Section: Introductionmentioning

confidence: 99%

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Helicobacter pylori-induced gastric cancer is orchestrated by MRCKβ-mediated Siah2 phosphorylation

et al. 2021

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Background 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. Methods 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. Results 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. Conclusions 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.

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Section: Western Blotting and Coimmunoprecipitation Assaysmentioning

confidence: 99%

Section: Soft Agar Assaymentioning

confidence: 99%

Section: In Vitro Invasion Assaymentioning

confidence: 99%

Section: H Pylori Infection Enhances Siah2 Phosphorylationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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