Control of Rapsyn Stability by the CUL-3-containing E3 Ligase Complex

doi:10.1074/jbc.M808230200

Control of Rapsyn Stability by the CUL-3-containing E3 Ligase Complex^*

^{^‡}Research Center for Cellulomics, Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, 151-742 Seoul, Korea, the ^{^§}Division of Nano Sciences (BK21), Ewha Womans University, 120-750 Seoul, Korea, ^{^¶}Protein Network Research Center, Department of Biochemistry, Yonsei University, 134 Shinchon, 120-749 Seoul, Korea

4 To whom correspondence should be addressed. Fax: 82-2-877-2661; E-mail: elegans{at}snu.ac.kr.

Abstract

Rapsyn is a postsynaptic protein required for clustering of nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. Here we report the mechanism for posttranslational control of rapsyn protein stability. We confirmed that C18H9.7-encoded RPY-1 is a rapsyn homolog in Caenorhabditis elegans by showing that human rapsyn rescued rpy-1 mutant phenotypes in nematodes, as determined by levamisole assays and micropost array behavioral assays. We found that RPY-1 was degraded in the absence of functional UNC-29, a non-α subunit of the receptor, in an allele-specific manner, but not in the absence of other receptor subunits. The cytoplasmic loop of UNC-29 was found to be critical for RPY-1 stability. Through RNA interference screening, we found that UBC-1, UBC-12, NEDD-8, and RBX-1 were required for degradation of RPY-1. We identified cullin (CUL)-3 as a component of E3 ligase and KEL-8 as the substrate adaptor of RPY-1. Mammalian rapsyn was ubiquitinated by the CUL3/KLHL8-containing E3 ligase in vitro, and the knockdown of KLHL-8, a mammalian KEL-8 homolog, inhibited rapsyn ubiquitination in vivo, implying evolutionary conservation of the rapsyn stability control machinery. kel-8 suppression and rpy-1 overexpression in C. elegans produced a phenotype similar to that of a loss-of-function mutation of rpy-1, suggesting that control of rapsyn abundance is important for proper function of the receptor. Our results suggest a link between the control of rapsyn abundance and congenital myasthenic syndromes.

Footnotes

↵⁵ The abbreviations used are: Ach, acetylcholine; nAChR, nicotinic acetylcholine receptor; MUSK, muscle-specific kinase; CL, cytoplasmic loop; CMS, congenital myasthenic syndrome; RNAi, RNA interference; CUL-3, cullin 3; WT, wild type; GFP, green fluorescent protein; PI, proteasome inhibitor; HA, hemagglutinin; Ub, ubiquitin.
↵^* This work was supported in part by Research Center for Cellulomics, Seoul National University funding from the Korea Science and Engineering Foundation (KOSEF), South Korea (to J. Lee), Molecular and Cellular BioDiscovery Research Program Grant M10748000241-07N4800-24100 from the Ministry of Science and Technology, Korea (to J. Lee), KRF grant KRF-2005-C00097 (to J. Lee), and KOSEF Grant M10536090002-05N3609-00210 funded by the Korean government (MEST) (to S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S8, Tables S1-S3, and Movies 1-7.
↵¹ Both authors contributed equally to this work.
↵² Supported by a postdoctoral grant from KOSEF (2004), the BK21 program (2006), and the Seoul R&BD Program (2006).
↵³ Supported by a postdoctoral grant from the Korean Research Foundation Grant 2005-037-C00027, BK21 program (2007), and the Seoul R&BD Program (2007).
- Received October 28, 2008.
- Revision received January 13, 2009.
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