ABSTRACT
ML-133 is a novel small molecule with potent antiproliferative activity, as shown in cancer cell lines and in a human colon tumor xenograft model. ML-133 reduces the concentration of intracellular labile zinc in HT-29 colon cancer cells, leading to induction of the Krüppel-like factor 4 transcription factor. Krüppel-like factor 4 displaces the positive regulator SP1 from the cyclin D1 promoter, thereby negatively regulating the expression of cyclin D1 and promoting the G(1)-S phase arrest of cell proliferation. The antiproliferative and antitumor activity of ML-133 described in the present study suggests modulation of intracellular zinc homeostasis as a potential strategy for the treatment of several cancer types, and ML-133 represents a promising new class of antitumor agents that deserves further development.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Division/drug effects , Homeostasis/drug effects , Imidazoles/pharmacology , Phenanthrolines/pharmacology , Zinc/metabolism , Base Sequence , Blotting, Western , Cell Cycle , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin D1/genetics , DNA Primers , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , HT29 Cells , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/biosynthesis , Polymerase Chain Reaction , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Up-Regulation/drug effectsABSTRACT
The Gab1 docking protein forms a platform for the assembly of a multiprotein signaling complex downstream from receptor tyrosine kinases. In general, recruitment of Gab1 occurs indirectly, via the adapter protein Grb2. In addition, Gab1 interacts with the Met/hepatocyte growth factor receptor in a Grb2-independent manner. This interaction requires a Met binding domain (MBD) in Gab1 and is essential for Met-mediated epithelial morphogenesis. The Gab1 MBD has been proposed to act as a phosphotyrosine binding domain that binds Tyr-1349 in the Met receptor. We show that a 16-amino acid motif within the Gab1 MBD is sufficient for interaction with the Met receptor, suggesting that it is unlikely that the Gab1 MBD forms a structured domain. Alternatively, the structural integrity of the Met receptor, and residues upstream of Tyr-1349 located in the C-terminal lobe of the kinase domain, are required for Grb2-independent interaction with the Gab1 MBD. Moreover, the substitution of Tyr-1349 with an acidic residue allows for the recruitment of the Gab1 MBD and for phosphorylation of Gab1. We propose that Gab1 and the Met receptor interact in a novel manner, such that the activated kinase domain of Met and the negative charge of phosphotyrosine 1349 engage the Gab1 MBD as an extended peptide ligand.
Subject(s)
Adaptor Proteins, Signal Transducing , Phosphoproteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-met/chemistry , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Blotting, Western , Cell Line , GRB2 Adaptor Protein , Glutathione Transferase/metabolism , Humans , Ligands , Microscopy, Fluorescence , Models, Biological , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Phosphoproteins/chemistry , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Transfection , Tyrosine/chemistryABSTRACT
The Gab family of docking proteins (Gab1 and Gab2) are phosphorylated in response to various cytokines and growth factors. Gab1 acts to diversify the signal downstream from the Met receptor tyrosine kinase through the recruitment of multiple signaling proteins, and is essential for epithelial morphogenesis. To determine whether Gab1 and Gab2 are functionally redundant, we have examined the role of Gab2 in epithelial cells. Both Gab1 and Gab2 are expressed in epithelial cells and localize to cell-cell junctions. However, whereas overexpression of Gab1 promotes a morphogenic response, the overexpression of Gab2 fails to induce this response. We show that Gab2 recruitment to the Met receptor is dependent on the Grb2 adapter protein. In contrast, Gab1 recruitment to Met is both Grb2 dependent and Grb2 independent. The latter requires a novel amino acid sequence present in the Met-binding domain of Gab1 but not Gab2. Mutation of these residues in Gab1 impairs both association with the Met receptor and the ability of Gab1 to promote a morphogenic response, whereas their insertion into Gab2 increases Gab2 association with Met, but does not confer on Gab2 the ability to promote epithelial morphogenesis. We propose that the Grb2-independent recruitment of Gab proteins to Met is necessary but not sufficient to promote epithelial morphogenesis.
