Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses.
Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.
Schaeper
U
U
Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany. uschaep@mdc-berlin.de
Gehring
N H
NH
Fuchs
K P
KP
Sachs
M
M
Kempkes
B
B
Birchmeier
W
W
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D051380:GRB2 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000502:physiology; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C082869:CRKL protein; D051380:GRB2 Adaptor Protein; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D015800:Protozoan Proteins; D055765:Shc Signaling Adaptor Proteins; D019859:Proto-Oncogene Proteins c-met; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases