1. Grb2 Is a Key Mediator of Helicobacter pylori CagA Protein Activities
Hitomi Mimuro, Toshihiko Suzuki, Jiro Tanaka, Momoyo Asahi, Rainer Haas, Chihiro Sasakawa 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1
The CagA Derivatives with the Tyr Residues in the PY Region Substituted with Phe Had the Same Ability as Wild-Type CagA to Induce Cell Scattering
(A) Schematic representation of CagA (NCTC11637 strain). The numbers are the amino acid positions. The black bar indicates the EPIYA motif.
(B) The lysate from the ΔcagA mutant of H. pylori NCTC11637 strains carrying the pHel3 shuttle vector containing the promoter region only (vec), promoter region with Y5, F2Y3, F3Y2, F4Y1, F5, or ΔPY-cagA were coincubated with AGS cell lysates. The samples were subjected to Western blot analysis using anti-CagA, anti-pY-CagA, or anti-pY antibodies. Arrowheads indicate the position of full-length (FL) or ΔPY CagA.
(C) AGS cells were infected with indicated H. pylori NCTC11637 strain for 5 hr. Cell lysates were subjected to Western blot analysis using anti-CagA or anti pY-CagA antibodies.
(D) AGS cells were infected with indicated H. pylori NCTC11637 strains for 5 hr. Infected AGS cells were fractionated into the cytosolic (C) and membrane (M) fractions before being analyzed by Western blot using anti-CagA, anti-UreA, or anti-Transferrin receptor antibodies. The whole-cell lysates of H. pylori-infected AGS (W) were also examined.
(E–G) AGS cells were infected for 5 hr with ΔcagA H. pylori NCTC11637 strain carrying the pHel3 shuttle vector containing promoter region only (E), promoter region with Y5 (F), or F5 (G) cagA. Bar, 100 μm.
(H) The AGS cells were infected for 5 hr with indicated H. pylori NCTC11637 strains. One hundred AGS cells were counted per experiment. The data shown are the means of triplicate experiments. The top bars show the standard deviation of the mean.
(I) AGS cells were transiently transfected with pEGFP-C1 plasmid (EGFP), pEGFP-C1 containing Y5, F5, A5, S5, ΔPY, ΔPY1, and ΔPY2-cagA. The GFP-overexpressing cells were observed by fluorescence. The data shown are the means of triplicate experiments. The top bars show the standard error of the mean.
(J–O) AGS cells were transiently transfected with pEGFP-C1 plasmid (J), pEGFP-C1 vector containing Y5 (K), F5 (L), A5 (M), S5 (N), and ΔPY (O)-cagA. The GFP-overexpressing cells were observed by fluorescence. Bar, 10 μm.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 In Vitro Binding of CagA with Grb2
(A) The AGS lysates with (+) or without (−) infection of H. pylori NCTC11637 wt prepared in the presence (+) or absence (−) of Na3VO4 were subjected to Western blot analysis with anti-CagA and anti-pY-CagA antibodies.
(B) GST-fusion proteins of Grb2, Nck, p85-PI3K-SH2, Gab1, and SHP-2 were immobilized on glutathione-Sepharose beads and incubated with the lysates showed in Figure 2A. Proteins bound to the beads were subjected to Western blot analysis with anti-CagA antibody. GST beads (−) were used for the controls.
(C) Schematic representation of Grb2. The numbers are the amino acid positions.
(D) GST-fusion proteins of the Grb2 A, B, C, BC, and full length (ABC) were immobilized on glutathione-Sepharose beads and incubated with H. pylori-infected AGS lysates (Lysate). The bound proteins were analyzed by Western blot with an anti-CagA antibody. The result using GST alone immobilized on glutathione-Sepharose beads (GST) is also shown as the negative controls.
(E) COS7 cells were transiently transfected with various pEGFP-C1 plasmids. The lysates were incubated with GST-Grb2 beads. The pulled down proteins (PD) and lysates (Lysate) were analyzed by Western blot with an anti-CagA antibody. The results using GST beads are also shown as the negative controls.
(F) H. pylori-infected AGS cell lysates (Lysate) were incubated with GST-Grb2 beads containing 10 μM recombinant Grb2 fragments of BC domain (BC) or A domain (A) as competitors. The bound proteins were analyzed by Western blot with anti-CagA antibody. The results using GST beads and without any competitor (−) were also shown as the control.
(G) GST-Grb2 beads were incubated with lysates from indicated H. pylori NCTC11637 strains. Proteins bound to the beads were subjected to Western blot analysis with anti-CagA antibody. Arrow heads indicate the position of full-length (FL) or ΔPY CagA.
(H) The indicated concentrations of Grb2 were incubated GST-CagA-C beads (0.75 pmol), and the Kd value was calculated by Scatchard analysis.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 Physiological Interaction of CagA and Grb2
(A) COS7 cells were cotransfected with pcDL-SRα-myc-grb2 and pEGFP-C1 plasmid (EGFP), pEGFP-C1 vector containing Y5, ΔPY, or F5-cagA. Cell lysates were subjected to immunoprecipitation (IP) with anti-CagA antibody. Immunoprecipitates and whole-cell lysates (Lysate) were subjected to Western blot analysis using anti-CagA or anti-Myc to visualize Myc-tagged Grb2. Preimmuno rabbit IgG was used for a control IgG.
(B–I) AGS cells were infected with H. pylori NCTC11637 wt (B–E) or ΔcagA mutant (F–I) and immunostained using anti-pY-CagA antibody (B and F), anti-Grb2 antibody (C and G), and TO-PRO3 (D and H). Phase contrast images are also shown (E and I). Bar, 10 μm.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4
Overexpression of a Grb2 Mutant Unable to Bind with Sos Inhibited CagA-Induced Cell Scattering
(A) GST, GST-Grb2 wt, or ACm beads were incubated with AGS lysates (upper panel) or H. pylori wt lysates (lower panel). The bound proteins were analyzed by Western blot with anti-Sos or anti-CagA antibodies.
(B–K) AGS cells transiently overexpressing wt (B, D, F, H, and J) or ACm (C, E, G, I, and K) of Myc-tagged Grb2 were infected with H. pylori (D–K) and immunostained using anti-Myc antibody (B, C, F, and G), anti-pY-CagA antibody (D and E) and TO-PRO3. Bars, 10 μm.
(L) The AGS cells overexpressing pcDL-SRα-myc plasmid (−), grb2 wt, and ACm were coincubated with (+) or without (−) H. pylori for 5 hr. Over 40 Myc-tagged Grb2 overexpressing AGS cells were counted per experiment. The data shown are the means of triplicate experiments. The top bars show the standard deviation of the mean.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
CagA-Induced Activation of Ras and MEK Is Important for Cell Scattering
(A) AGS cells were infected with indicated H. pylori NCTC11637 strains or without bacteria (−) for 5 hr. The lysates were incubated with GST-Raf-1 RBD beads, then the bound proteins were subjected to Western blot using anti-Ras antibody (upper panel). Whole-cell lysates from the same experiments were probed with anti-Ras antibody to assess the Ras input level (lower panel).
(B) AGS cells were infected for the indicated times with H. pylori. Total cell lysates were subjected to Western blot with anti-phospho-MEK antibody, and the levels of activated MEK were quantified by densitometry. The same samples were also probed with anti-MEK antibody to assess the total MEK levels and with anti-UreA to assess input levels of the bacteria (data not shown).
(C–F) AGS cells were infected with H. pylori (D and F) for 5 hr in the absence (C and D) or presence of 50 μM PD98059 (E and F). Bar, 100 μm.
(G) AGS cells were coincubated with (+) or without (−) H. pylori wt for 5 hr in the absence (−) or presence (+) of 50 μM PD One hundred AGS cells were counted per experiment. The data shown are the means of triplicate experiments. The top bars show the standard deviation of the mean.
(H) AGS cells were infected with H. pylori (+) for 5 hr in the absence (−) or presence (+) of 50 μM PD Total cell lysates were subjected to Western blot using anti-CagA (upper panel) or anti-pY-CagA (lower panel) antibodies to assess both the total and intracellular levels of CagA.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
CagA PY-Region Is Important for Inducing MEK Activation, Cell Scattering, and Proliferation in MDCK Cells Stably Expressing CagA
(A) Lysates from MDCK cells containing the plasmid of pEGFP-C1 vector alone (Mock), or pEGFP-C1 vector containing Y5, F5, or ΔPY-cagA were subjected to Western blot using anti-CagA, anti-phospho-MEK, and anti-MEK antibodies.
(B–J) MDCK cells stably expressing EGFP (Mock) (B, C, and D), EGFP-Y5 (E and F), F5 (G and H), or ΔPY-cagA (I and J) were incubated in the absence (−) (B, E, G, and I) or presence of 50 μM PD98059 (C, F, H, and J) or 10 ng/ ml HGF (D) for 16 hr. Bar, 50 μm.
(K) MDCK cells of 1 × 104 with stably expression of EGFP alone (Mock), EGFP-tagged Y5, F5, or ΔPY-cagA were subjected to the cell proliferation assay using a Cell Counting Kit-8 (WAKO). All values were normalized to that of Mock. The data shown are the means of triplicate experiments. The top bars show the standard deviation of the mean.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
Intracellular CagA May Act as a Multiple Scaffold Protein Inducing the MAPK Cascade via Binding to Grb2
CagA anchored in the plasma membrane by unknown mechanism(s) (X) may recruit Grb2 independent from the Tyr phosphorylation. Next, CagA activates the MAPK cascade by recruiting the Grb2-Sos complex to the cell membrane in close proximity to the Ras GTPase. Activated Ras-GTP promotes the activation of the mitogen-activated protein kinase kinase kinase (MAPKKK) Raf-1, which phosphorylates MEK (MAPKK), which in turn activates ERK (MAPK).
Molecular Cell  , DOI: ( /S (02) )