Figure 6: C-terminus of CagA binds to GSK-3β through the Axin binding site.

(a) Schematic diagram of GFP-Flag-fusion constructs for CagA C-terminus. Arrows and arrowheads denote EPIYA and multimerization domain on AF202923, respectively. (b) The 293 cells were either transfected with control or with Flag-tagged GFP-fusion constructs as indicated in combination with HA-tagged GSK-3β expression vector for a 48 h period. The GSK-3β binding affinity of GFP-CagA fusion constructs was detected by immunoprecipitation with anti-Flag agarose following immunoblot analysis with either anti-Flag (GFP-CagA) or anti-HA (GSK-3β) antibody. (c) The Flag-tagged GFP-EPIYA fusion constructs or Axin2 were co-transfected with various types of HA-GSK-3β mutant in 293 cells as indicated for 48 h. The lysates were subjected to immunoprecipitation with anti-Flag agarose and the GSK-3β binding ability to CagA or Axin2 was detected with immunoblot analysis. (d) The wt or mutant CagA expression vectors were co-transfected with a HA-tagged GSK-3β (wt) or N-terminal deleted GSK-3β (Δ9) expression vector in 293 cells. The binding affinity of GSK-3β to CagA was assessed by immunoprecipitation with anti-flag agarose and immunoblot analysis against anti-HA antibody. (e) Immunoblot analysis of bacterial CagA protein in 60190 or ΔCagA strain (left panel). His-tagged recombinant GSK-3β from sf9 insect cell was incubated with bacterial lysates of 60190 or ΔCagA strain for 1 h, followed by 100 ng of Axin peptide competition on GSK-3 binding. The eluted CagA and GSK-3β on Ni-Ti beads were detected by immunoblot analysis (right panels). (f) Schematic diagram of CagA-mediated GSK-3 depletion and subsequent activation of EMT and other oncogenic pathways. Following transduction of CagA into the cells via a type IV secretion system, the cytoplasmic GSK-3 is rapidly insolubilized and downregulated, resulting in the activation of the GSK-3-dependent oncogenic pathway.