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First published online 4 July 2006
doi: 10.1242/jcs.03043

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

¹ Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
² Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan
³ Department of Pathology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
⁴ Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
⁵ Division of Surgical Pathology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
⁶ Department of Medical Technology, Nagoya University of Health Sciences, Higashi-ku, Nagoya 461-8673, Japan
⁷ Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

^* Author for correspondence (e-mail: mtakaha{at}med.nagoya-u.ac.jp)

Accepted 9 May 2006

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.

Key words: Dok-4, GDNF, RET tyrosine kinase, Rap1, MAPK, Neuronal differentiation