Figure 3

Downstream of tyrosine kinase/docking protein (Dok)6 binds to the NPQY motif of tropomyosin-related kinase (Trk)C in a kinase-activity-dependent manner and was phosphorylated on neurotrophin 3 (NT-3) stimulation. (a) Alignment of amino acid sequences around the NPQY motif of Trk family receptors (homology level: red, 100%; green, ≥50%), and schematic diagram showing the important amino acids of TrkC. (b) Mutation analysis of the key amino acids in the TrkC receptor for Dok6 binding. A yeast two-hybrid assay was performed with pACT2-Dok6-phosphotyrosine-binding (PTB) and TrkC intracellular domain (ICD) mutants using the pAS2-1 vector, including CM1(K572A), CM2(Y516F), CM3(Y705A), CM4(Y709D), CM5(Y710E), CM6(Y820F). (c) Dok6 binds to the NPQY motif of the TrkC receptor in a kinase activity-dependent manner in glutathione S-transferase (GST) precipitation assays. Human embryonic kidney (HEK)293 cells were transfected with wild-type TrkC, CM1, CM2 and CM6. After stimulation with NT-3, cell lysates were collected for GST precipitation assays. (d) Dok6 is phosphorylated by the TrkC receptor, which is activated upon NT-3 stimulation. HEK293 cells were cotransfected with V5-TrkC and Flag-Dok6, and cells were treated with/without NT-3 before harvest. Cell lysates were then analysed by western blotting with anti-pTyr antibody.