Dok6 By The Ptb Domain-dependent Tyrosine Phosphorylation In Combination With The The Trkc's Npqy Motif

Human Dok6 (downstream of tyrosine kinase/docking protein) gene consisting of eight exons was localized to chromosome 18q22.2. The open-reading frame of human Dok6 cDNA has 933 base pairs which encode 331 amino acids. The molecular weight of Dok6 protein is about 38.3 kDa. Dok6 protein was characterized to possess typical structure like other signal proteins: an N-terminal pleckstrin homology (PH) domain ( 8-114 amino acids), a central PTB domain (130-232 amino acids), and a variable C-terminal region ( 232-330 amino acids). Dok6 belongs to Dok4/5 subfamily in p62Dok family, a family of cellular adaptor proteins. Dok6 is highly expressed in the central nervous system, in particular, the fetal brain.Trk receptors, the high affinity receptors for neurotrophins, are cell membrane recetpor tyrosin kinases. There are three members in this family: TrkA, TrkB and TrkC. Neurotrophins stimulation will result in the dimeriztion and autophosphorylation of Trk receptors. The activated receptors then recruit multiple signal proteins and phosphorylate them, thus transduct the extemal signal into the cells. Our lab found Dok5 protein in Dok4/5 subfamily enhanced the activity level and prolong the activation time of MAPK pathway by the interacting with Trk receptor. It proved that Dok5 promotes TrkB/C-mediated signal transduction induced by BDNF and NT-3. The interaction between Dok5 and TrkB/C depend on the phosphorylation of receptor.Because of highly homology and expression in nervous system between Dok6 and DokS, it is possible that Dok6 also interacts with the Trk receptor. In order to confirm the deduction, firstly, we confirmed the interaction between Dok6 and Trk receptor using yeast two hybrid system, GST pulldown assay and Co-IP experiments. The result of research showed that Dok6 selectively interacted with TrkC, not with TrkA or TrkB, in Trk family. To identify the binding site and style of the interaction, PH, PTB domain and C terminal was divided. Each domain was examined to interact with TrkC receptor using yeast two hybrid system. The result showed that PTB domain, not PH domain or C terminal, coupled to TrkC receptor. Mutation analysis of five tyrosine residues and lysine residues which related with the activity of tyrosine kinase proved Dok6 PTB domain bound the NPQY motif of Trk receptor using two hybrid system, GST pulldown assay and Co-IP experiments.