Volume 132, Issue 4, Pages 1309-1319 (April 2007) Activation of Abl by Helicobacter pylori: A Novel Kinase for CagA and Crucial Mediator of Host Cell Scattering  Ina Tammer, Sabine Brandt, Roland Hartig, Wolfgang König, Steffen Backert  Gastroenterology  Volume 132, Issue 4, Pages 1309-1319 (April 2007) DOI: 10.1053/j.gastro.2007.01.050 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Inhibition of Abl blocks Hp-induced cell scattering and CagA phosphorylation. AGS cells were infected with Hp in the (A) absence or (B) presence of the Abl inhibitors SKI-DV2-43 (1 μmol/L) or STI571 (2.5 μmol/L). These inhibitors block Hp-induced cell scattering and elongation. (C) The number of elongated cells was quantitated in 10 different 0.25-mm2 fields in triplicate samples. (D) The effect of the inhibitors on CagA phosphorylation was determined by immunoblotting (arrows). α-glyceraldehyde-3-phosphate dehydrogenase staining served as loading control. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Knockdown of c-Abl and Arg inhibits phosphorylation of CagA, and scattering and elongation of AGS cells. (A) AGS cells were transfected with a c-Abl-shRNA vector and/or Arg-small interfering RNA, followed by infection with Hp for 6 hours. Immunoblotting with α-c-Abl and α-Arg antibodies confirmed down-regulation of the proteins. The effect of c-Abl/Arg gene silencing on the phosphorylation of CagA was examined using α-PY-99 and α-CagA antibodies (arrows). α-glyceraldehyde-3-phosphate dehydrogenase staining served as loading control (bottom). (B) The number of elongated cells in each experiment was quantitated in 10 different 0.25-mm2 fields in triplicate. (C) Phase-contrast microscopy of c-Abl/Arg knockdown or shRNA control cells infected with Hp. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 In vitro phosphorylation of CagA by Abl or Src family kinases. (A) Lysates of fibroblasts derived from SYF cells and SYF + c-Src were used to perform in vitro CagA phosphorylation assays. To determine the role of Abl and Src kinases in phosphorylating CagA, the reactions were performed in the presence or absence of the inhibitors SKI-DV2-43 (1 μmol/L) or PP2 (10 μmol/L). Immunoblotting using α-PY-99 and α-CagA antibodies (arrows) indicate that both c-Src and Abl can phosphorylate CagA. As controls, the blots were reprobed with α-c-Abl and α-c-Src antibodies (bottom). (B) Incubation of Hp lysates with recombinant human c-Src and Abl resulted in specific phosphorylation of wt CagA but not the CagAY899/918/972F mutant (arrows). Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Sustained activation of Abl and transient activation of c-Src in Hp-infected AGS cells. (A) Kinase activity of Abl and Src during infection for the indicated time intervals. Activation of Abl or Src was determined by immunostaining with (B) α-Abl-PY-412 and (C) α-c-Src-PY-416 antibodies, respectively. Each value was calculated from the immunoblotting data using a luminescence image analyzer. The blots were reprobed with (C) α-c-Src-PY-527, (D) α-PY-99, and (E) α-glyceraldehyde-3-phosphate dehydrogenase as loading control. (F) The number of elongated cells was quantitated in 10 different 0.25-mm2 fields in triplicate. (A) ▩, c-Abl; ■, c-Src. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 CagA phosphorylation is dominated by Src and Abl tyrosine kinases at different time points of infection, and CagA phosphorylation/dephosphorylation reactions are rapid and highly dynamic. (A) AGS cells were infected with Hp for 120 or 360 minutes and then treated for 20 minutes with 10 μmol/L PP2 or 1 μmol/L SKI-DV2-43, respectively. Effect of the inhibitors on the phosphorylation of CagA was determined by immunoblotting (arrows). (B) The number of elongated AGS cells was quantitated in 10 different 0.25-mm2 fields in triplicate samples. Phase-contrast microscopy of infected AGS cells (C) before and (D) after treatment with SKI-DV2-43. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Activation of Abl on infection of AGS cells leads to the phosphorylation of CrkII at Y-221 in vitro. Total Abl was purified by IP from infected cell lysates followed by in vitro kinase assays using 1 μg CrkII-GST in the presence or absence of 1 μmol/L SKI-DV2-43. Immunoblotting with α-CrkII-PY-221 confirmed that Abl phosphorylated CrkII. α-c-Abl, and α-GST blots served as loading control. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 CagA PY forms a complex with activated Abl and CrkII in vivo, which is dependent on a functional T4SS in Hp. (A) AGS cells were infected with P12wt or isogenic cagL mutant Hp for 6 hours followed by pulldown of CrkII. The IPs were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with the indicated antibodies showing the presence of CrkIIPY, CagPY, and c-AblPY in one complex, which was not detected in the mutant-infected or noninfected control. (B) AGS cells were infected with wt or isogenic cagA and cagL mutants for 6 hours followed by pulldown of c-Abl. The IPs were probed with the indicated antibodies showing that the isogenic cagA mutant also substantially stimulates c-Abl and the phosphorylation CrkII. Quantification of c-Abl activity in wt- and mutant-infected AGS cells using the luminescence image analyzer were performed in triplicate IP experiments. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Abl kinase activity and phosphorylation of CrkII at Y-221 is important for Hp-induced phenotypic responses. (A) AGS cells were transfected for 36 hours with either empty vector, wt c-Abl, kinase-inactive c-Abl-K290M, wt CrkII, or dominant-negative CrkII-R38V, CrkII-W169L, and CrkII-Y221F mutants. Transfected cells then were infected with Hp for 4 hours. The number of elongated cells was quantitated in triplicate experiments. (B) Expression of the constructs was verified by immunoblotting using α-c-Abl or α-CrkII antibodies. (C) Phase-contrast microscopy of infected AGS cells expressing either wt c-Abl or c-Abl-K290M. (D) Immunofluorescence of cells transfected with either wt or CrkII-W169L (red) and infected with green fluorescence protein (GFP)-expressing Hp (green). These results show that activation of c-Abl kinase activity and CrkII plays a crucial role in Hp-induced AGS cell elongation. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 9 Transient expression of activated Abl and CagA induce AGS cell elongation in a phosphorylation-dependent manner. (A) AGS cells were transfected for 36 hours with either empty vector, constitutive-active c-Abl (c-Abl-PP), wt CagA, or phosphorylation-deficient CagA. Expression of the constructs was verified by immunoblotting using the indicated antibodies. (B) The number of elongated cells was quantitated in triplicate experiments. The results show that both activated c-AblPY and CagAPY are required to induce AGS cell elongation. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions

Figure 10 Model for successive CagA phosphorylation and CagA-induced signaling leading to the cytoskeletal rearrangements of infected cells. Hp translocates CagA by a T4SS-dependent process. At early time points of infection (1–2 h) Src is active when phosphorylated at Y-416, resulting in rapid phosphorylation of translocated CagA in the EPIYA repeats. CagAPY then inactivates c-Src by a negative-feedback loop mechanism involving direct binding of CagAPY to Src, phosphorylation of the negatively regulatory site Y-527, and dephosphorylation of Y-416.7 Phosphorylation of Y-527 is achieved by Csk.13 Src inactivation prevents succeeding tyrosine phosphorylation of CagA and other Src targets such as cortactin.7 In contrast to Src, Abl kinases are activated continuously by Hp at late time points of infection. Activated Abl (phosphorylated at Y-412) continues phosphorylating CagA and phosphorylates CrkII at Y-221. We propose that the phosphorylated forms of CagA, Abl, and CrkII create a signaling complex to trigger downstream signaling, leading to actin-cytoskeletal rearrangements and to host cell scattering. Gastroenterology 2007 132, 1309-1319DOI: (10.1053/j.gastro.2007.01.050) Copyright © 2007 AGA Institute Terms and Conditions