Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase.
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.
Uchida
Mayumi
M
Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Enomoto
Atsushi
A
Fukuda
Toshifumi
T
Kurokawa
Kei
K
Maeda
Kengo
K
Kodama
Yoshinori
Y
Asai
Naoya
N
Hasegawa
Taisaku
T
Shimono
Yohei
Y
Jijiwa
Mayumi
M
Ichihara
Masatoshi
M
Murakumo
Yoshiki
Y
Takahashi
Masahide
M
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004789:Enzyme Activation; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C486625:DOK4 protein, human; C432911:Dok4 protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D051096:Proto-Oncogene Proteins c-ret; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020827:rap1 GTP-Binding Proteins