Tyrosine-phosphorylated β4 interacts with Shp2. (A) Schematic representation of β4 structure. The cytoplasmic domain contains two pairs of type III fibronectin-like repeats (FNIII) separated by a connecting segment (CS). The consensus sequences for Shp2 and tyrosine positions are indicated. TM, transmembrane domain. (B) β4–Shp2 association by far-Western analysis. COS-7 cells were cotransfected with wild-type (WT) or kinase-dead (K⁻) Met and with wild-type β4 or β4 mutants bearing single (β4-ΔShp2^S; Y1257F), double (β4-ΔShp2^D; Y1257F and Y1494F), and triple (β4-ΔShp2^T; Y1257F, Y1440F, and Y1494F) phenylalanine substitutions of the potential docking tyrosines for Shp2. Lysates from each experimental point were subdivided into two aliquots and immunoprecipitated (IP) in parallel with antibodies to β4; Western blots (WB) were probed with antiphosphotyrosine (P-Tyr) antibodies (left) or with a GST-Shp2 fusion protein followed by anti-GST antibodies (right). The blots were then reprobed with anti-β4 antibodies. A fraction of the lysates (1/50) was loaded to detect Met from total extracts. In COS-7 cells, exogenous Met is expressed as both the precursor (top band) and mature form (bottom band). The weak band observable in the sixth lane corresponds to endogenous Met. (C) A β4 mutant with a single phenylalanine permutation of Tyr1440 (β4-Y1440F) displays reduced but detectable tyrosine phosphorylation and can bind Shp2 in far-Western analysis. (D) Coimmunoprecipitation experiment showing that wild-type Met but not a Met mutant with phenylalanine substitutions of the two tyrosines of the multidocking site (Met^D) associates with Shp2. (E) Coimmunoprecipitation of β4 and Shp2 from lysates of COS-7 cells overexpressing Shp2 and the indicated forms of Met and β4. (F and G) Coimmunoprecipitation of β4 and Shp2 in cells with the endogenous expression of both proteins. (F) Reciprocal coimmunoprecipitation experiment in GTL16 cells. Cell lysates were immunoprecipitated with antibodies to myc (negative control), Shp2 (positive control), or β4. Blots were probed with antibodies against Shp2 (top), β4 (middle), or phosphotyrosine (P-Tyr; bottom). Shp2 is present in β4 immunoprecipitates (top), and β4 is present in Shp2 immunoprecipitates (middle). Immunopurified β4 and Shp2-associated β4 appear to be constitutively tyrosine phosphorylated. (G) FG2 cells were either left untreated or were treated with 100 ng/ml HGF for 10 min. Lysates were immunoprecipitated with antibodies to myc or β4, and blots were probed as indicated. Shp2 can be recovered from β4 immunoprecipitates after HGF stimulation (top). Treatment with HGF induces the tyrosine phosphorylation of β4 (bottom).

β4-dependent activation of Src leads to the selective association of Gab1 with Grb2 and to the increased activation of ERK/MAPKs. (A) HGF stimulation results in the increased association of Gab1 with Grb2 in β4 cells. MDA-MB-435 mock and β4 cells were transiently transfected with Gab1 and stimulated with 100 ng/ml HGF for 30 min. Lysates were immunoprecipitated with antibodies to Gab1, and blots were probed as indicated. Densitometric analysis of the blots obtained in four experiments indicates that the HGF-dependent association of Grb2 with Gab1 in mock cells is increased 1.26-fold (± 0.48) compared with nonstimulated cells, whereas the HGF-dependent association of Gab1 with Grb2 in β4 cells is increased 4.7-fold (± 0.98; P < 0.01). (B) Abrogation of Shp2 binding or Src inhibition with PP2 impairs the coimmunoprecipitation of Gab1 with Grb2 in β4 cells. Dotted lines indicate the grouping of images from different parts of the same gel. (C) Far-Western analysis showing the association between immunoprecipitated Gab1 and GST-SH2-Grb2. In accordance with the coimmunoprecipitation data, HGF-dependent Gab1–Grb2 interaction is increased in β4 cells compared with mock and β4^ΔShp2 transfectants. PP2-mediated inhibition of Src results in decreased Gab1–Grb2 interaction in β4 but not in mock cells. (D) HGF activates ERK/MAPKs longer and more efficiently in β4 cells than in mock or β4^ΔShp2 cells. Cells were treated with 100 ng/ml HGF for the indicated times, and total lysates were probed on blots as indicated. Band intensities were quantitated by densitometric analysis with respect to total protein content.

β4 sustains HGF-induced anchorage-independent growth through the recruitment of Shp2. Representative images and quantitation of soft agar assays in MDA-MB-231 cells displaying the artificial manipulation of β4 levels as shown in Fig. 2 E. Data are the means ± SEM (error bars) of two independent experiments performed in triplicate. **, P < 0.01 with respect to all other transfectants. WT, wild type. Bar, 400 μm.

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 (β4^ΔShp2) 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 β4^ΔShp2 (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 β4^ΔShp2 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 β4^ΔShp2 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 β4^Δextra 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 (Shp2^DN; G) or Src (Src^DN; 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.

β4 sustains HGF-induced anchorage-independent growth through the activation of Shp2, Src, and ERK. (A) Western blot analysis showing expression of the indicated cDNAs in MDA-MB-435 cells subjected to soft agar assays. Filters probed with anti-Shp2 and -Src antibodies were deliberately underexposed to better visualize the overexpression of exogenous proteins. (B) Representative images and quantitation of soft agar assays in MDA- MB-435 cells with ectopic expression of the indicated proteins in the absence (NS) or presence of 30 ng/ml HGF. Pictures show colony aspect at high magnification, whereas the graph reports colony numbers as counted at low magnification over the entire microscopic field. Light bars, no stimulation; dark bars, HGF. (C) Representative images and quantitation of soft agar assays in MDA-MB-435 cells expressing β4 that were either left untreated (NS) or were treated with HGF in the absence or presence of the Src inhibitor PP2 or the ERK inhibitor PD98059. Data are the means ± SEM (error bars) of two independent experiments performed in triplicate. **, P < 0.01 with respect to all other transfectants. Bars, 400 μm.