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

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-␣ 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.

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15

“…GFP-KLHL15 recovered in FLAG-BЈ␤ immunoprecipitates migrated close to its predicted molecular weight, as well as a smear near the stacker/running gel interface. This suggests that KLHL15 undergoes autoubiquitylation, as was reported for other KLHL proteins (15,(33)(34)(35)(36).…”

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

“…Our data indicate that agrin induced-AChR clustering is impaired in absence of cullin activity. Interestingly, Rpy-1, the ortholog of Rapsyn in C. elegans , can be degraded by a cullin-RING ligase [73], suggesting unexplored functions for cullin-RING ligases (CRL) and their substrates in the formation of NMJ. Our data indicate that a defective ubiquitylation activity through CRL inhibition might either interfere with the abundance of proteins that are important for neuromuscular junction development, or with cytoskeletal remodeling that has been shown to be important for the agrin-induced AchR clustering process [74–79].…”

Regulation of muscle acetylcholine receptor turnover by β subunit tyrosine phosphorylation

“…Fifth, rapsyn's own turnover is more rapid (3 days) than the AChR and is highly regulated, placing it in a unique position to regulate receptor turnover (Bruneau and Akaaboune, 2007;Bruneau and Akaaboune, 2010). Regulation of rapsyn levels includes stabilization by binding to HSP90b and CUL3/KLHL8-dependent ubiquitination and degradation by the proteasome (Nam et al, 2009). Sixth, rapsyn interacts with several postsynaptic scaffolding proteins including the MuSK receptor complex, b-dystroglycan, ACF7, and a-actinin (DeChiara et al, 1996;Apel et al, 1995Apel et al, , 1997Cartaud et al, 1998;Bartoli et al, 2001;Antolik et al, 2007;Dobbins et al, 2008); thus, it could anchor the receptor to the MuSK scaffold, dystrophin-associated glycoprotein complex (DGC) or actin cytoskeleton.…”