Development of an efficient "substrate-trapping" mutant of Src homology phosphotyrosine phosphatase 2 and identification of the epidermal growth factor receptor, Gab1, and three other proteins as target substrates.
Src homology containing phosphotyrosine phosphatase 2 (SHP2) is a positive effector of growth factor, cytokine, and integrin signaling. However, neither its physiological substrate nor its mechanism of action in tyrosine kinase signaling has been demonstrated. We reasoned that the identification of physiological substrates of SHP2 would be a stepping stone in elucidating its mechanism of action, and, thus, we constructed a potent trapping mutant of SHP2. Surprisingly, the frequently used Asp to Ala substitution did not give rise to a trapping mutant. However, we were able to develop an efficient trapping mutant of SHP2 by introducing Asp to Ala and Cys to Ser double mutations. The double mutant (DM) protein identified the epidermal growth factor receptor (EGFR), the Grb2 binder 1, and three other, as yet unidentified, phosphotyrosyl proteins as candidate physiological substrates. Given that substrate trapping occurred in intact cells and that the interaction was very specific, it is highly likely that EGFR and Gab1 represent physiological SHP2 substrates. Therefore, the DM protein would serve as an important tool in future SHP2 studies, including identification of p190, p150, and p90.
Agazie
Yehenew M
YM
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, 11794-5222, USA.
Hayman
Michael J
MJ
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D019556:COS Cells; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D015801:Helminth Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C098587:GAB1 protein, human; C490897:Gab1 protein, mouse; D015801:Helminth Proteins; D010750:Phosphoproteins; C095048:microfilarial sheath protein, Helminth; D001224:Aspartic Acid; D012694:Serine; D066246:ErbB Receptors; D010744:Phosphoric Monoester Hydrolases; D003545:Cysteine; D000409:Alanine