Phosphorylation of ADAM 17 at T735 is cell cycle dependant and induced by H. pylori and TNF-α

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Examination of the localisation of the three phosphorylated forms of ADAM17 (p-T735, p-S719 and p-S819) in unstimulated AGS wild type cells and AGS cells over expressing ADAM17 was undertaken by immunofluorescent microscopy (Figure 2A and B) and  FACS analysis (Figure 2C and D). High levels of ADAM17 phosphorylated at T735 were present in cells that were undergoing mitosis in both wild type AGS gastric epithelial cells (Figure 2A)  and ADAM17 over expressing cells (Figure 2B) but this was not observed with ADAM p-S719 or ADAM p-S819 immunofluoresence microscopy (data not shown).

To quantify further the phosphorylation of ADAM17 at these three sites in relation to cell cycle, FACS analysis of cell cycle using propidium iodide (PI) was performed along with staining for each of the phosphorylated forms of ADAM17 in wild type AGS cells (Figure 2C) and  ADAM17 over expressing AGS cells (Figure 2D). In AGS gastric epithelial cells treated with PI and the Alexa 488 conjugated secondary antibody only, there was a signal from the Alexa 488 indicating that there was some non-specific binding of the secondary antibody but the intensity of this staining was much lower than that for any of the primary phospho-ADAM17 antibody treated cells (Figure 2C and D). The PI intensity of AGS cells plotted against the intensity of ADAM17 phosphorylated at S791 or S819 indicated that phosphorylation of ADAM17 occurs at these sites but the AGS cells fell within one cluster suggesting phosphorylation at S791 and S819 was not linked to specific phases of the cell cycle. In contrast, co-staining with PI and an antibody against phospho-T735 identified two populations of cells in both wild type AGS cells (Figure 2C) and AGS ADAM17 over expressing cells (Figure 2D). Phospho-T735 was detected in the majority of cells and these cells were in all phases of the cell cycle, however a population of cells with high PI staining in G2/M phase of the cell cycle had much higher expression of p-T735 (Figure 2C and 2D). Unstimulated AGS wild type cells and AGS cells over expressing ADAM17 had respectively 0.95 + 0.16 (mean + SEM) % and 3.4 + 0.77 % (n = 4 independent experiments) of total cells with high expression of p-T735 in G2/M phase of the cell cycle (p <0.02).  These data confirm the findings of the immunofluorescence microscopy where high levels of p-T735 were detected in AGS cells undergoing mitosis. This suggests that either phosphorylation of ADAM17 at T735 may have a role in regulating the cell cycle or that ADAM17 phosphorylated at T735 plays some role in cell division.

To investigate whether H. pylori induced phosphorylation of ADAM17 in AGS gastric epithelial cells, AGS  cells were cultured with H. pylori strain NCTC 11637 for 90 min and TNF-α (50ng/ml) for 15 min and cells were immunolabelled for ADAM17 and  phospho-T735 and phospho-S719 ADAM17 (Figure 2E and F) and phospho-S819 ADAM17. Phosphorylation of ADAM17 T735 (Figure 2E) and ADAM17 S791 (Figure 2F) was stimulated by both H. pylori NCTC 11637 strain and TNF-α in AGS gastric epithelial cells. Similar results were observed with H. pylori G27 strain. Background levels of ADAM phospho-T735 positivity in unstimulated AGS cells were lower than that of phospho-S791 ADAM17. Pre-incubation of permeabilised cells with phosphatase prior to immunolabelling confirmed specificity of phospho-T735 and S791 ADAM17 antibodies for phosphorylation epitopes. No specific upregulation of phospho-S819 ADAM17 was observed with H. pylori or TNF-α stimulation (data not shown).