Abstract
Activated receptor tyrosine kinases induce a large number of tyrosine phosphorylation-dependent protein-protein interactions through which they mediate their various ligand-exerted functions including regulation of proliferation, differentiation and survival. TrkB receptor tyrosine kinase activated by binding of brain-derived neurotrophic factor (BDNF) also stimulates various protein interactions in a tyrosine phosphorylation-dependent manner in neuronal cells. To examine tyrosine phosphorylation-dependent interactions stimulated by active TrkB, we developed a modified yeast two-hybrid system, which we call the yeast two-and-a-half-hybrid system. In this system, yeast was engineered to express a tyrosine kinase domain of TrkB as an effector, in addition to two fusion proteins with GAL4 DNA-binding and GAL4 activation domains as bait and prey proteins, respectively. Using this system with Shp2 as the bait, we demonstrated that Shp2 interacts directly with BIT/SHPS-1 (also called SIRP) and Grb2 depending on tyrosine phosphorylation mediated by TrkB. Furthermore, we screened an adult human brain cDNA library with the yeast two-and-a-half-hybrid system in order to identify other Shp2-binding proteins in TrkB-stimulated tyrosine phosphorylation signaling. We found that fibroblast growth factor receptor substrate 2beta (FRS2beta), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2beta/SNT2. Therefore, we show that the two-and-a-half-hybrid system is a powerful tool for studying tyrosine phosphorylation-dependent protein-protein interactions in intracellular signaling pathways stimulated by TrkB receptor tyrosine kinase.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adaptor Proteins, Signal Transducing*
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Adult
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Antigens, Differentiation*
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Brain / metabolism
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Line
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Electrophoresis, Polyacrylamide Gel
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GRB2 Adaptor Protein
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Humans
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Intracellular Signaling Peptides and Proteins*
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Lipoproteins / genetics
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Lipoproteins / metabolism*
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / metabolism*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Neural Cell Adhesion Molecule L1*
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Neural Cell Adhesion Molecules / genetics
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Neural Cell Adhesion Molecules / metabolism*
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Phosphoproteins / genetics
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Phosphoproteins / metabolism*
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Phosphorylation
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Phosphotyrosine / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases / genetics
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Protein Tyrosine Phosphatases / metabolism*
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Proteins / genetics
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Proteins / metabolism*
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Receptor, trkB / chemistry
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Receptor, trkB / genetics
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Receptor, trkB / metabolism*
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Receptors, Immunologic*
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Signal Transduction
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Two-Hybrid System Techniques
Substances
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Adaptor Proteins, Signal Transducing
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Antigens, Differentiation
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Carrier Proteins
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FRS2 protein, human
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FRS3 protein, human
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GRB2 Adaptor Protein
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GRB2 protein, human
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Intracellular Signaling Peptides and Proteins
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Lipoproteins
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Membrane Glycoproteins
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Membrane Proteins
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Neural Cell Adhesion Molecule L1
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Neural Cell Adhesion Molecules
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Phosphoproteins
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Proteins
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Receptors, Immunologic
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Phosphotyrosine
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Receptor, trkB
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PTPN11 protein, human
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PTPN6 protein, human
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Protein Tyrosine Phosphatase, Non-Receptor Type 11
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases