LINGO-1 Interacts with WNK1 to Regulate Nogo-induced Inhibition of Neurite Extension

Zhaohuan Zhang,Xiaohui Xu,Yong Zhang,Jianfeng Zhou,Zhongwang Yu,Cheng He

doi:10.1074/jbc.m808751200

Zhaohuan Zhang, Xiaohui Xu + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m808751200

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Abstract

LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1-(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation. Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.

Highlights

LINGO-1 is a transmembrane protein that contains a leucine-rich repeat, an immunoglobulin domain, and a short intracellular tail [11]
WNK1 Is Identified as a LINGO-1-binding Protein in a Yeast Two-hybrid Screening—In an attempt to identify intracellular proteins involved in the regulation of LINGO-1 signaling, a yeast two-hybrid screening was performed with LINGO-1ICD, the intracellular domain of LINGO-1, as bait (Fig. 1A)
Many axon growth inhibitors have been identified in myelin, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and Nogo account for most of the central nervous system myelin inhibitory activities [7, 41]

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construct—The full-length LINGO-1 was generated by PCR from a human brain cDNA library and inserted into the YFP or GFP fusion vector pEYFP-N1 or pEGFP-N1 to generate LINGO-1-YFP or LINGO-1-GFP. FRET Measurements with Three-channel Microscopy—PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5% fetal bovine serum and 10% heat-inactivated horse serum (HyClone) in a humidified 5% CO2 atmosphere at 37 °C. The siRNA sequences are: WNK1si-1, GCAACAGGATGATATCGAA; WNK1si-2, GCCAGAGCCTAATGGAATT These WNK1si sequences were constructed into pSUPER vector to generate shRNAs. The WNK1si constructs were transfected into PC12 cells, and cells were subjected to reverse transcription-PCR or Western blot 48 h later. Neurite Outgrowth Assay—PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5% fetal bovine serum and 10% heat-inactivated horse serum (HyClone) in a 37 °C and 5% CO2 incubator. Forty-eight hours after transfection cells were observed under a fluorescent microscope (Olympus, excitation 454 nm), and the length of the longest neurite of GFP-positive neurons was quantified by MetaMorph image analysis software. The data were analyzed by Student’s t test, and each value was the mean Ϯ S.D

RESULTS

When normalized with GAPDH

DISCUSSION