Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase

Abstract

During development of the central and peripheral nervous systems, neurite extension mediated via glial-cell-line-derived neurotrophic factor (GDNF) and its receptor RET is critical for neuronal differentiation. In the present study, we investigated the role of the RET substrate Dok-4 in neurite outgrowth induced by the GDNF/RET signaling pathway. In TGW neuroblastoma cells, which endogenously express both RET and Dok-4, depletion of Dok-4 through treatment with small interfering RNA resulted in a marked decrease in GDNF-stimulated neurite outgrowth. By contrast, exogenous expression of wild-type Dok-4 induced sustained p44/42 mitogen-activated protein kinase (ERK1/2) activation and enhanced neurite outgrowth. Expression of Dok-4 mutants in which the tyrosine residues at codons 187, 220 and 270, conserved between Dok-4, -5, and -6, were each replaced with a phenylalanine inhibited sustained ERK1/2 activation and neurite outgrowth. We also found that Dok-4 induced a significant activation of the small G protein Rap1 and that expression of a dominant active Rap1 mutant restored neurite outgrowth in Dok-4-depleted cells. By contrast, expression of a dominant negative Rap1 mutant impaired GDNF-stimulated neurite outgrowth from TGW cells. Finally, we found that neurite formation in cultured rat hippocampal neurons was enhanced by the expression of Dok-4. Together, our results suggest that Dok-4, through activation of the Rap1-ERK1/2 pathway, regulates GDNF-mediated neurite outgrowth during neuronal development.

The GTPase-activating protein Rap1GAP: A new player to modulate Ret signaling

“…Taking advantages of this cellular model, Jiao et al show that Rap1GAP inhibits GDNF/Ret-induced neurite outgrowth by restricting both the activation of Rap1 (Rap1-GTP) and the sustained activation of ERK1/2 [4]. These findings are in agreement with previous data npg showing that GDNF-promoted neurite outgrowth during neuronal development involves prolonged activation of Rap1-ERK1/2 pathway via the adaptor protein, Dok-4, which is phosphorylated after Ret activation [7]. Interestingly, Dok-4 was identified as a direct interactor partner of Ret by yeast two-hybrid screen [8].…”

Roles of induced expression of MAPK phosphatase-2 in tumor development in RET-MEN2A transgenic mice

“…To investigate whether MKP-2 expression is induced by the activation of endogenous RET by GDNF, TGW neuroblastoma cells, which have been shown to express both endogenous RET and GFRa1 (Watanabe et al, 2002;Uchida et al, 2006), were treated with GDNF. MKP-2 was weakly expressed in TGW cells in their basal state, and biphasic induction of MKP-2 was observed 15 min and 6 h after GDNF stimulation Figure S1).…”

“…The siRNA-mediated knock down of MKP-2 protein expression was performed using a previously described method Uchida et al, 2006). The 21-nt synthetic duplexes were purchased from B-Bridge International Inc.…”

mRNA expression of DOK1-6 in human breast cancer

“…In particular, DOK-2 has recently been proposed as a potential marker of poor prognosis in patients with gastric cancer after curative resection [43] . The other sub-group of the DOK family, consisting of DOK-4, DOK-5 and DOK-6, are positive regulators of the MAPK pathway and promote neurite outgrowth [3,4,29,32,44] . DOK-4 and DOK-6 are phosphorylated by the tyrosine kinase encoded by the proto-oncogene RET (rearranged by transfection) [3,31,32,44] .…”

“…The other sub-group of the DOK family, consisting of DOK-4, DOK-5 and DOK-6, are positive regulators of the MAPK pathway and promote neurite outgrowth [3,4,29,32,44] . DOK-4 and DOK-6 are phosphorylated by the tyrosine kinase encoded by the proto-oncogene RET (rearranged by transfection) [3,31,32,44] . Over expression of RET is primarily observed in oestrogen-receptorpositive (ER+) breast cancers where, in models, it is seen to cause increased cell migration and proliferation.…”