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-terminal pleckstrin homology (PH) domain, a central phosphotyrosine-binding (PTB) domain, and a C-terminal region .
Interestingly, although all the members share almost same structural feature, they exert different, even opposite, roles in different tissues and organs. Dok1 to Dok3, which are predominantly expressed in haematopoietic tissues, act as negative regulators [7–12]. The results from single or double knockout of Dok1/Dok2 mice provided solid evidence to indicate their negative roles in leukemogenesis [11, 13, 14]. Yasuda et al. also showed that Dok1 and Dok2 are negative regulators of the T cell receptor signal pathway . Dok3 exerts its negative role in the immunoreceptors of B cells and the macrophage signalling pathway by formation of Dok3-SHIP1 complex [1, 16].
The other four family members, Dok4 to Dok7, are expressed in non-haematopoietic tissues, functioning in different places and in different ways. Dok4 is widely expressed, especially in intestine, kidney and lung that are all epithelial in origin. Bedirian et al. showed that Dok4 may function as an inhibitor of the tyrosine kinase signal pathway , while Grimm et al. found that Dok4 and Dok5 enhanced c-Ret-dependent activation of mitogen-activated protein kinase (MAPK) and promoted neurite outgrowth in PC12 cells . Also, Uchida et al. showed that Dok4 may promote glial cell-derived neurotrophic factor (GDNF)-mediated neurite outgrowth in TGW cells through activation of the Rap1-Erk1/2 pathway .
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-α1 cells . Dok7 was identified recently and plays an important role in neuromuscular synaptogenesis by interaction with muscle specific kinase (MuSK). Mice lacking Dok7 fail to form acetylcholine receptor clusters and neuromuscular synapses . Patients who have mutations in Dok7 develop congenital myasthenic syndrome (CMS) associated with small neuromuscular synapses [20, 21].
Our previous work has shown that Dok5 is a substrate of tropomyosin-related kinase (Trk)B and TrkC receptors and is involved in neurotrophin-induced MAPK activation . Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5 (NT-4/5), and so on, are a family of growth factors implicated in several different functions in the nervous system [23, 24]. There are two types of receptors for neurotrophins: the Trk family are high affinity receptors, while p75 is a low affinity receptor. The Trk family receptors have three members, TrkA, TrkB and TrkC, that are structurally conserved and exhibit specificity to different neurotrophins. TrkA is the preferred receptor for NGF, TrkB prefers BDNF, while NT-3 is the preferred ligand of TrkC . Neurotrophins stimulation can induce the dimerisation and phosphorylation of Trk receptors, which then recruit and phosphorylate downstream adaptor proteins to mediate signal transduction [24–26]. It has been reported that neurotrophins and Trk receptors are involved in almost all stages of nervous system development, 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.