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. 2006 Dec 18;175(6):993–1003. doi: 10.1083/jcb.200605114

Figure 2.

Figure 2.

Association between β4 and Shp2 leads to the increased phosphorylation of Gab1 through a Src-dependent mechanism. (A) Expression of β4 or the β4 mutant unable to bind Shp2 (β4ΔShp2) in MDA-MB-435 cells. Both molecules are exposed at the cell surface, as assessed by surface biotinylation (top), and are expressed at comparable levels, as shown in total cell extracts (middle). Equal loading was verified by probing the blot with antiactin antibodies (bottom). Surface biotinylation detects the entire α6β4 integrin complex. (B) In vitro kinase assay measuring Src activation upon stimulation with 100 ng/ml HGF at different time points in mock (blue), β4 (green), and β4ΔShp2 (red) MDA-MB-435 cells. Data are the means ± SEM (error bars) of five independent experiments performed in duplicate. (C) Differential Gab1 phosphorylation upon HGF stimulation for different times in mock, β4, and β4ΔShp2 cells. MDA-MB-435 transfectants were stimulated with 50 ng/ml HGF, and lysates were immunoprecipitated and probed as indicated. Dotted lines indicate the grouping of images from different parts of the same gel. Gab1 phosphorylation was quantitated by densitometric analysis (OD) relative to Gab1 protein content in immunoprecipitates. (D) Differential Gab1 phosphorylation upon stimulation with different doses of HGF for 30 min in mock, β4, and β4ΔShp2 cells. (E) Western blot analysis showing β4 expression in lysates from MDA-MB-231 cells infected with a scrambled (ctr) siRNA or with a β4-specific siRNA; β4 siRNA cells were reinfected with wild-type β4Δextra or with a ΔShp2 mutant. In all transfectants, Met expression was unaffected. (F) Differential Gab1 phosphorylation in the different MDA-MB-231 transfectants. (G–I) Inhibition of Shp2 and Src impairs the HGF-dependent phosphorylation of Gab1 in MDA-MB-435–β4 but not in mock cells. Cells were transiently transfected with a control vector (empty) or with catalytically inactive isoforms of either Shp2 (Shp2DN; G) or Src (SrcDN; I).The exogenous expression of Shp2 and Src is shown in the bottom panels (G and I). In H, cells were pretreated with 5 μM PP2 for 30 min. The PP2-mediated inhibition of Src was assessed with an antibody against active Src (bottom; H). HGF was given at 50 ng/ml for 30 min.