| First Author | Yamada M | Year | 2001 |
| Journal | J Biochem | Volume | 130 |
| Issue | 1 | Pages | 157-65 |
| PubMed ID | 11432792 | Mgi Jnum | J:202593 |
| Mgi Id | MGI:5520050 | Doi | 10.1093/oxfordjournals.jbchem.a002955 |
| Citation | Yamada M, et al. (2001) Analysis of tyrosine phosphorylation-dependent protein-protein interactions in TrkB-mediated intracellular signaling using modified yeast two-hybrid system. J Biochem 130(1):157-65 |
| abstractText | 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. |
