Abstract
Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Androstadienes / pharmacology
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Animals
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Apoptosis
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Binding Sites
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Blotting, Western
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Caspase 3
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Caspases / metabolism
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Caspases / physiology
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Cell Division
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Cell Line
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Cell Movement
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Cell Survival
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DNA / metabolism
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DNA Damage
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DNA Fragmentation
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DNA Repair
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Dogs
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Doxorubicin / pharmacology
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Enzyme Inhibitors / pharmacology
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Epithelial Cells / metabolism
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GRB2 Adaptor Protein
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Genetic Vectors
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Hepatocyte Growth Factor / metabolism*
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Humans
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Models, Biological
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Mutation
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphoproteins / metabolism*
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Phosphoproteins / physiology*
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Phosphorylation
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Precipitin Tests
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Protein Binding
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Protein Serine-Threonine Kinases / metabolism
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Protein Structure, Tertiary
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Proteins / metabolism
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Proto-Oncogene Proteins c-met / metabolism*
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Recombinant Proteins / metabolism
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Signal Transduction
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Tetrazolium Salts / pharmacology
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Thiazoles / pharmacology
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Time Factors
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Transfection
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Wortmannin
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p21-Activated Kinases
Substances
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Adaptor Proteins, Signal Transducing
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Androstadienes
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Enzyme Inhibitors
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GAB1 protein, human
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GAB2 protein, human
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GRB2 Adaptor Protein
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GRB2 protein, human
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Phosphoproteins
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Proteins
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Proto-Oncogene Proteins
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Recombinant Proteins
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Tetrazolium Salts
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Thiazoles
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Hepatocyte Growth Factor
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Doxorubicin
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DNA
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Proto-Oncogene Proteins c-met
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AKT1 protein, human
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PAK1 protein, human
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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p21-Activated Kinases
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CASP3 protein, human
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Caspase 3
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Caspases
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thiazolyl blue
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Wortmannin