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- Title
Analysis of Tyrosine Phosphorylation-Dependent Protein-Protein Interactions in TrkB-Mediated Intracellular Signaling Using Modified Yeast Two-Hybrid System1.
- Authors
Yamada, Masashi; Suzuki, Kenji; Mizutani, Masaharu; Asada, Akiko; Matozaki, Takashi; Ikeuchi, Toshihiko; Koizumi, Shinichi; Hatanaka, Hiroshi
- 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 phosphoryla-tion-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 2β (FRS2β), also called SNT2, interacts with Shp2 dependently on TrkB-mediated tyrosine phosphorylation of FRS2β/ 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 intracellu-lar signaling pathways stimulated by TrkB receptor tyrosine kinase.
- Subjects
PROTEIN-tyrosine kinases; PROTEIN-protein interactions; BRAIN-derived neurotrophic factor; PHOSPHORYLATION; GROWTH factors
- Publication
Journal of Biochemistry, 2001, Vol 130, Issue 1, p157
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a002955