Gab2 tyrosine phosphorylation by a pleckstrin homology domain-independent mechanism: role in epidermal growth factor-induced mitogenesis.
In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.
Kong
Mei
M
Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montréal, Québec, Canada.
Mounier
Catherine
C
Balbis
Alejandro
A
Baquiran
Gerry
G
Posner
Barry I
BI
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D046911:Macromolecular Substances; D008934:Mitogens / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D020893:Receptor, ErbB-3 / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001798:Blood Proteins; C490893:Gab2 protein, rat; D046911:Macromolecular Substances; D008934:Mitogens; D010750:Phosphoproteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C039077:platelet protein P47; D014443:Tyrosine; D004815:Epidermal Growth Factor; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D020893:Receptor, ErbB-3