Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins

210

164

The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His 6 -tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His 6 -tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.The small ubiquitin-like modifier (SUMO) 1 is linked to target proteins by post-translational conjugation and may thereby influence protein function and/or localization (1-3). The name derives from the relationship of SUMO to the better characterized post-translational modifier ubiquitin (4). They are 18% identical at the amino acid level and show a clear similarity in their respective three-dimensional structures (5). Whereas yeast and nematodes have a single SUMO gene, in humans and mice, the SUMO family consists of three members, SUMO-1, -2, and -3, which are encoded by separate genes. The mature forms of SUMO-2 and SUMO-3 are similar (ϳ95% identical), but less closely related to SUMO-1 (ϳ50% identical) (6 -8).Despite their close similarity, there is evidence that SUMO-1 and SUMO-2/3 are preferentially conjugated to distinct sets of target proteins. Whereas RanGAP1 is modified by SUMO-1, many as yet unidentified proteins are modified by SUMO-2/3 after exposure of cells to various stress stimuli (7).Several groups independently identified SUMO-1, which is also known as PIC1, GMP1, SMT3C, UBL1, and sentrin (9 -13). In contrast to most polyubiquitinylated proteins, sumoylated proteins are not thereby targeted for degradation. SUMO-1 can even compete with ubiquitin for the same acceptor lysine in the target protein. In the case of IB␣, SUMO modification serves to protect the protein from ubiquitin-mediated degradation (14), whereas in yeast, DNA repair can proceed only after proliferating cell nuclear antigen is desumoylated to allow ubiquitination. Here, ubiquitination does not target the protein for degradation, but functions as a switch to activate the DNA repair function of proliferating cell nuclear antigen (15). Therefore, despite their structural relationship, it appears that SUMO and ubiquitin conjugation can play distinct roles in modulating target proteins. Sumoylation can also regulate the subcellular localization of target proteins. For example, SUMO conjugation is required for both the nuclear pore and kinetochore localization of RanGAP1 (1...

mentioning

confidence: 70%

“…The single SUMO gene in both Saccharomyces cerevisiae (51) and Caenorhabditis elegans (52,53) is essential for viability, indicating that sumoylation is an essential process. Consistent with this, several genes encoding SUMO-conjugating enzymes and SUMO proteases are also essential (22,42,52,53).…”

mentioning

confidence: 99%

A Proteomic Study of SUMO-2 Target Proteins

Vertegaal

Ogg

Jaffray

et al. 2004

210

164

“…UbcH5B/C may thus be required for the ubiquitination of a specific subset of proteins in vivo. The human genome encodes for more E2s than that of S. cerevisiae and contains several homologues of S. cerevisiae Ubc4/5 (11,56). This provides the scope for both divergence and for greater redundancy of E2 function in humans.…”

Section: Discussionmentioning

confidence: 99%

“…A ubiquitin ligase (E3) then facilitates transfer of ubiquitin from an E2 to the substrate. There is one human ubiquitin E1 and multiple ubiquitin E2s (10,11). Mdm2 acts as an E3 ligase for p53 (12) and can promote its ubiquitination and degradation in vivo (13,14).…”

mentioning

confidence: 99%

Regulation of p53 by the Ubiquitin-conjugating Enzymes UbcH5B/C in Vivo

Saville¹,

Sparks²,

Xirodimas³

et al. 2004

139

117

p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNAmediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity.

“…We investigated all available UBC genes by using the previously described feeding method (46). We used an RNAi library purchased from MRC (Cambridge, UK).…”

Section: Methodsmentioning

confidence: 99%

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

Nam

Min

Hwang

et al. 2009

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.