Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

abstract: ...Background The downstream of tyrosine kinase/docking protein (Dok) adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk) receptor family, which has three members (TrkA, TrkB and TrkC), are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST) precipitation assays and coimmunoprecipitation (Co-IP) experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB) domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3) stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

... signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST) precipitation assays and coimmunoprecipitation (Co-IP) experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB) domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3) stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons. Background P62Dok/Dok1, which was identified by Carpino et al . and Yamanashi and Baltimore in 1997, was the first identified member of the downstream of tyrosine kinase/docking protein (Dok) family. Since then, six other members have been identified: p56Dok/Dok2/DokR, Dok3/DokL, Dok4, Dok5, Dok6/Dok5 like and Dok7 [ 1 - 4 ]. Dok family proteins are adaptor proteins that act as substrates of multiple receptor tyrosine kinases and non-receptor tyrosine kinases [ 5 , 6 ]. All seven family members share a similar structural topology: an N

...-Erk1/2 pathway [ 18 ]. Dok5 is mainly expressed in nervous system. It acts as a substrate for the insulin receptor (IR) and GDNF receptor c-Ret, and can promote GDNF-induced neurite outgrowth in PC12 cells [ 2 , 19 ]. For Dok6, in situ hybridisation results indicated that it is also specifically expressed in the nervous system, especially in the cortex and dorsal root ganglia (DRG). Crowder et al . showed that Dok6 could promote Ret-mediated neurite outgrowth in N2A-1 cells [ 3 ]. Dok7 was

..., such as survival, proliferation, differentiation, migration, axon and dendrite projection, and neuron patterning [ 23 , 27 - 30 ]. To date, Dok6's functions in the nervous system have been poorly understood. In this study, we identified Dok6 as a novel substrate of the TrkC receptor. We confirmed that Dok6 selectively binds to the NPQY motif of TrkC through its PTB domain in a kinase activity-dependent manner. Immunostaining results showed that TrkC and Dok6 colocalise in E18.5 mouse cortical neurons. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon NT-3 stimulation, which indicates that Dok6 is involved in NT-3-mediated neuronal development. Results Dok6 binds to the TrkC intracellular domain (ICD) but not TrkA or TrkB through the PTB domain in a yeast two-hybrid system Our previous work indicated that Dok5, a member of the p62Dok family, directly interacted with TrkB and TrkC receptors to act as a downstream substrate to induce MAPK activation [ 22 ]. We used DNAMAN software (Lynnon Corporation, Pointe-Claire, Quebec, Canada H9R 2A3) to analyse the homology of Dok family proteins and found that Dok6 and Dok5 are the most conserved among all seven family members (Figure 1a ), which means Dok6 may be more like Dok5 than any other members in the family. In consideration of the same specific neural expression pattern of Trk receptors and Dok6 in embryonic mice [ 3 ], we hypothesised that Dok6 may bind to Trk receptors directly to act as a downstream substrate, as Dok5

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Gene: DOK6 - docking protein 6 - (NCBI)

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