Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system

J Biochem. 2001 Jul;130(1):157-65. doi: 10.1093/oxfordjournals.jbchem.a002955.


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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Adult
  • Antigens, Differentiation*
  • Brain / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line
  • Electrophoresis, Polyacrylamide Gel
  • GRB2 Adaptor Protein
  • Humans
  • Intracellular Signaling Peptides and Proteins*
  • Lipoproteins / genetics
  • Lipoproteins / metabolism*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Neural Cell Adhesion Molecule L1*
  • Neural Cell Adhesion Molecules / genetics
  • Neural Cell Adhesion Molecules / metabolism*
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphotyrosine / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • Protein Tyrosine Phosphatase, Non-Receptor Type 6
  • Protein Tyrosine Phosphatases / genetics
  • Protein Tyrosine Phosphatases / metabolism*
  • Proteins / genetics
  • Proteins / metabolism*
  • Receptor, trkB / chemistry
  • Receptor, trkB / genetics
  • Receptor, trkB / metabolism*
  • Receptors, Immunologic*
  • Signal Transduction
  • Two-Hybrid System Techniques


  • Adaptor Proteins, Signal Transducing
  • Antigens, Differentiation
  • Carrier Proteins
  • FRS2 protein, human
  • FRS3 protein, human
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Intracellular Signaling Peptides and Proteins
  • Lipoproteins
  • Membrane Glycoproteins
  • Membrane Proteins
  • Neural Cell Adhesion Molecule L1
  • Neural Cell Adhesion Molecules
  • Phosphoproteins
  • Proteins
  • Receptors, Immunologic
  • Phosphotyrosine
  • Receptor, trkB
  • PTPN11 protein, human
  • PTPN6 protein, human
  • Protein Tyrosine Phosphatase, Non-Receptor Type 11
  • Protein Tyrosine Phosphatase, Non-Receptor Type 6
  • Protein Tyrosine Phosphatases