Dok6 In Primary Cultured Mouse Cortical Neurons Neurite Outgrowth Process In Functional Study

Trk (tropomyosin-related kinase) is an important tyrosine kinase family which locates on cell membrane. It exerts pivotal roles in neuron survival, proliferation, differentiation, migration, apoptosis, learning and memory. Trk belong to RTK (receptor tyrosine kinase) family and have 3 members:TrkA, TrkB and TrkC. They are high affinity receptors for Neurotrophins family which includ NGF, BDNF, NT-3 and NT-4. Trk receptors could occur dimerization and activation upon binding to Neurotrophins. Then they could recruit and phosphorylate some adaptor proteins to mediate signal transduction.Dok (downstream of tyrosine kinase/Docking protein) is an adaptor protein family that plays important roles in cell signal transduction. There are 7 family members: Dokl-Dok7. All 7 family members share the similar structural topology:N-terminal pleckstrin homology (PH) domain, central phosphor-tyrosine binding (PTB) domain and C-terminal region. That is the typical structure topology of adaptor proteins and it is why Dok could be recruited and phosphorylated as substrate of RTKs to mediate cell signal transduction.Our previous works have demonstrated that Dok5 could work as substrate of TrkB and TrkC to activate MAPK pathway. As both of Dok5 and Dok6 are highly expressed in nervous system and they share the highest homology in 7 members, we hypothesized that Dok6 is involved in Trk receptors signal transduction in nervous system. So, we used yeast two-hybrid system to confirm that Dok6 could bind to TrkC ICD, but not to TrkA or TrkB. We further constructed serial region clones of Dok6 to demonstrate that Dok6 bind to TrkC through its PTB domain. We achieved consistent results in GST pull-down and CoIP experiments. Then we used amino acid mutation techniques to confirm that Dok6 PTB binds to the NPQY motif in TrkC ICD and this interaction depends on TrkC receptor kinase activity.In order to confirm these results in nervous system, we used immuno-staining method to demonstrate that TrkC and Dok6 colocalize in primary mouse cortex neurons. IP experiments further confirmed their interaction in vivo which indicated that Dok6 exerts pivotal roles as a substrate of TrkC receptor in nervous system. By knockdown of Dok6 in mouse cortex neurons, we demonstrated that Dok6 plays an important role in neurite outgrowth in primary cortex neurons. Then upon stimulation of NT-3, we confirmed that Dok6 is involved in NT-3 mediated neurite outgrowth as a substrate of TrkC receptor in mouse primary cortex neurons. At last, we identified 26 proteins that could interact with Dok6 by IP following with mass spectrum method. After bioinformatic analysis, we focus on 9 proteins which are involved in neural development and cell signal transduction.In summary, we have demonstrated that Dok6 binds to NPQY motif in TrkC ICD through the PTB domain in a kinase-activity-dependent manner and plays an important role in NT-3 mediated neurite outgrowth in mouse primary cortex neurons. These results gave new clews about Dok6 functions in vivo and shed lights on the treatments of nervous system deficiency diseases.