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. 2000 Nov;20(22):8513-25.
doi: 10.1128/MCB.20.22.8513-8525.2000.

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

C R Maroun  1 , M A Naujokas, M Holgado-Madruga, A J Wong, M Park
Affiliations

The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase

C R Maroun et al. Mol Cell Biol. 2000 Nov.

Abstract

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

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Figures

FIG. 1
FIG. 1
SHP-2 association with Gab1 is tyrosine phosphorylation dependent. (A) MDCK cells stably transfected with vector or constructs that express HA-Gab1 were stimulated for 15 min with 100 U of HGF/ml. SHP-2, Grb2, SOS, and Shc were immunoprecipitated with specific antibodies. Proteins resolved by SDS-PAGE were transferred to a nitrocellulose membrane and immunoblotted with anti-HA. (B) Lysates from HA-Gab1-expressing cells, stimulated or not with 100 U of HGF/ml, were subjected to immunoprecipitation as indicated. The resulting supernatants were subjected to a second round of immunoprecipitation with the indicated antibodies, followed by a third round of immunoprecipitation with anti-HA. (C) Lysates from HA-Gab1-expressing MDCK cells, stimulated or not with 100 U of HGF/ml, were subjected to a pull-down assay using GST-SHP-2 SH2 domain fusion proteins. Proteins were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-HA. ip, immunoprecipitate; WT, wild type.
FIG. 2
FIG. 2
Gab1 can function as a substrate for SHP-2 phosphatase activity. (A) 293T cells were transiently transfected with vectors encoding CSF-Met together with either wild-type (WT) SHP-2 or a catalytically inactive SHP-2 C/S mutant. Lysates were subjected to immunoprecipitation with anti-SHP-2 and Western blotting with anti-PY, anti-Met, or anti-Gab1 as indicated. (B) 293T cells were transfected with vectors encoding CSF-Met, together with either wild-type SHP-2 or the catalytically inactive SHP-2 C/S mutant, and Gab1. Gab1, Met, and SHP-2 were immunoprecipitated and immunoblotted, as indicated, with specific antibodies. ip, immunoprecipitate.
FIG. 3
FIG. 3
Gab1 ΔSHP-2 mutant fails to rescue branching tubulogenesis downstream from a Met receptor mutant. (A) 293T cells were transfected with vectors encoding Gab1 mutants at Y637F and/or Y659F in the absence or presence of CSF-Met. Lysates were subjected to immunoprecipitation with anti-HA followed by Western blotting with either anti-SHP-2, anti-PY or anti-HA. (B) MDCK cells expressing the CSF-Met receptor mutant N1358H were stably transfected with vectors encoding wild-type (WT) the Gab1 or Gab1 ΔSHP-2 mutant. Proteins from cell lysates were immunoprecipitated with anti-HA and resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-HA. (C) Cells expressing wild-type Gab1 (clone 3) and cells expressing the Gab1 ΔSHP-2 mutant protein (clone 5) were grown in collagen for 5 days, during which time they formed cysts. RhCSF-1 or HGF, both at 5 U/ml, were added, and 14 days later branching tubules were visualized at a magnification of ×10. (D) Quantitation of the tubulogenic response following stimulation with HGF and CSF was performed as described in Materials and Methods. The responses are plotted as the percentage of cysts that have undergone branching tubulogenesis. The values were derived from three independent experiments. ip, immunoprecipitate.
FIG. 4
FIG. 4
Overexpression of a Gab1 ΔSHP-2 inhibits branching tubulogenesis downstream from the endogenous Met receptor. (A) Proteins from lysates of MDCK cells expressing wild-type (WT) Gab1 (clone 9) and MDCK cells expressing Gab1 ΔSHP-2 mutant proteins (clones 6B and 8B) were subjected to immunoprecipitation and Western blotting with anti-HA. (B) Quantitation of the tubulogenic response was performed as described in Materials and Methods. The responses are plotted as the percentage of cysts that have undergone branching tubulogenesis. The values are derived from three independent experiments. (C) Cells were grown in collagen for 5 days, during which they formed cysts. HGF (5 U/ml) was added, and 14 days later branching tubules were visualized at a magnification of ×10. ip, immunoprecipitate.
FIG. 5
FIG. 5
The cellular localization of Gab1 is not altered in the absence of a Gab1-SHP-2 interaction. (A) MDCK cells (104 cells) expressing either wild-type (WT) Gab1 or Gab1 ΔSHP-2 mutant proteins were grown for 72 h on glass coverslips in DMEM containing 10% FBS. Cells were fixed in 2% paraformaldehyde and were subjected to indirect immunofluorescence using anti-HA, followed by CY3-conjugated anti-mouse antibody. Photographs were taken at a magnification of ×60. (B) MDCK cells (104 cells) expressing either wild-type Gab1 or Gab1 ΔSHP-2 mutant proteins were grown overnight on glass coverslips in DMEM containing 10% FBS. Cells were stimulated with 50 U of HGF/ml prior to fixation and indirect immunofluorescence with anti-HA, followed by CY3-conjugated anti-mouse antibody. Results were visualized by confocal microscopy at a magnification of ×63.
FIG. 6
FIG. 6
The association of wild-type Gab1 and Gab1 ΔSHP-2 with Met and with p120 RasGAP and Grb2 fusion proteins. (A) 293T cells were transfected with vectors encoding wild type (WT), N1358H, or Y1356F CSF-Met receptors together with either wild-type Gab1 or the Gab1 ΔSHP-2 mutant. Lysates were subjected to immunoprecipitation with anti-HA and blotting with anti-Met, anti-PY, or anti-SHP-2 and immunoprecipitation with anti-Met, followed by blotting with anti-PY. Fifty micrograms of total cellular proteins (TCP) was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-HA or anti-Met. (B) Lysates from A were subjected to a pull-down experiment using GST-p120 RasGAP N-SH2 and GST-Grb2 SH2 domain fusion proteins. Associated proteins were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-HA. ip, immunoprecipitate.
FIG. 7
FIG. 7
Activation of Erk is altered in cells expressing Gab1 ΔSHP-2 mutant proteins. (A) Cell lines expressing either wild-type (WT) Gab1 (clone 9) or a Gab1 ΔSHP-2 mutant (clone 8B) were stimulated with 100 U of HGF/ml for the indicated times at 37°C. Fifty micrograms of total cellular proteins was resolved by SDS–10% PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-Erk. Blots were stripped and reprobed with anti-total Erk. (B) Lysates were subjected to immunoprecipitation with anti-HA followed by Western blotting with anti-PY, anti-SHP-2, anti-p85 or anti-HA as indicated. (C) Fifty micrograms of total cellular proteins was resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phospho-AKT. Blots were stripped and reprobed with anti-total AKT. ip, immunoprecipitate.
FIG. 8
FIG. 8
Catalytic activity of SHP-2 is required for branching tubulogenesis and sustained Erk activation induced by HGF. MDCK cells stably expressing SHP-2 C/S were generated (clones C/S-6 and -11). (A) Cells were grown in collagen for 5 days, during which they formed cysts. HGF (5 U/ml) was added, and 14 days later branching tubules were visualized at a magnification of ×10. (B) Quantitation of the tubulogenic response following stimulation with HGF was performed as described in Materials and Methods. The responses are plotted as the percentage of cysts that have undergone branching tubulogenesis. The values are derived from three independent experiments. (C) MDCK cells expressing vector control or SHP-2 C/S mutant proteins were stimulated with 100 U of HGF/ml for the indicated time. Fifty micrograms of total cellular proteins was resolved on an SDS–10% PAGE gel, transferred to a nitrocellulose membrane, and blotted for anti-phospho-Erk. Gels were stripped and reprobed with anti-total Erk.
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