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Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

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Ablation of Uev1A suppresses TRAF-induced NF-κB activity and NEMO polyubiquitination in 293T cells. (A) Western blot analysis confirms RNAi specificity and efficacy of target suppression. (B) Modulation of IKKβ-, TRAF2-, and TRAF6-induced NF-κB activity by ablation of Ubc13, Uev1, or Mms2. The relative NF-κB activity was calculated against transfected cells without siRNA treatment. (C) Effects of Mms2 or Uev1 ablation by siRNA on NEMO polyubiquitination. s, scrambled nonspecific siRNA; M, siMms2; U, siUev1. The arrow points to TRAF6.
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fig6: Ablation of Uev1A suppresses TRAF-induced NF-κB activity and NEMO polyubiquitination in 293T cells. (A) Western blot analysis confirms RNAi specificity and efficacy of target suppression. (B) Modulation of IKKβ-, TRAF2-, and TRAF6-induced NF-κB activity by ablation of Ubc13, Uev1, or Mms2. The relative NF-κB activity was calculated against transfected cells without siRNA treatment. (C) Effects of Mms2 or Uev1 ablation by siRNA on NEMO polyubiquitination. s, scrambled nonspecific siRNA; M, siMms2; U, siUev1. The arrow points to TRAF6.

Mentions: The TNF associated factors 2 and 6 (TRAF2 and TRAF6) are involved in the signaling cascades initiated by TNF receptors, Toll-like receptors, and several interleukin receptors (Sun and Chen, 2004). Both TRAF2 (Shi and Kehrl, 2003) and TRAF6 (Deng et al., 2000) have been implicated as E3s for Ubc13-mediated NF-κB activation; however, it remains unclear which Uev is required for Ubc13 function in these signaling pathways. To address this issue, we used synthetic small interference RNA (siRNA) to specifically inhibit the expression of either Mms2 or Uev1. Because 2H11 monoclonal antibody recognizes both Mms2 and Uev1, we verified the specificity of our targeting siRNAs for either Mms2 or Uev1 by examining their effects in knocking down the expressions of FLAG-Mms2 and/or myc-Uev1A. As shown in Fig. 6 A, siRNA against Mms2 (lanes 2 and 8) or Uev1 (lanes 6 and 9) specifically inhibits its target gene expression but does not display a cross inhibitory effect. Consistent with our previous findings (Zhou et al., 2004), siRNA-targeting Ubc13 significantly reduced TRAF2- and TRAF6-stimulated NF-κB activation (Fig. 6 B). Moreover, siRNA-targeting Uev1 but not Mms2 inhibited TRAF2- and TRAF6-stimulated NF-κB activation. This effect is apparently specific for TRAF2- and TRAF6-dependent ubiquitination to activate NF-κB because neither Ubc13 nor Uev1 silencing affected IKKβ-mediated NF-κB activation (Fig. 6 B). These results highlight a specific role for Uev1 but not Mms2 in cooperating with Ubc13 in activating the NF-κB pathway.


Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Ablation of Uev1A suppresses TRAF-induced NF-κB activity and NEMO polyubiquitination in 293T cells. (A) Western blot analysis confirms RNAi specificity and efficacy of target suppression. (B) Modulation of IKKβ-, TRAF2-, and TRAF6-induced NF-κB activity by ablation of Ubc13, Uev1, or Mms2. The relative NF-κB activity was calculated against transfected cells without siRNA treatment. (C) Effects of Mms2 or Uev1 ablation by siRNA on NEMO polyubiquitination. s, scrambled nonspecific siRNA; M, siMms2; U, siUev1. The arrow points to TRAF6.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171356&req=5

fig6: Ablation of Uev1A suppresses TRAF-induced NF-κB activity and NEMO polyubiquitination in 293T cells. (A) Western blot analysis confirms RNAi specificity and efficacy of target suppression. (B) Modulation of IKKβ-, TRAF2-, and TRAF6-induced NF-κB activity by ablation of Ubc13, Uev1, or Mms2. The relative NF-κB activity was calculated against transfected cells without siRNA treatment. (C) Effects of Mms2 or Uev1 ablation by siRNA on NEMO polyubiquitination. s, scrambled nonspecific siRNA; M, siMms2; U, siUev1. The arrow points to TRAF6.
Mentions: The TNF associated factors 2 and 6 (TRAF2 and TRAF6) are involved in the signaling cascades initiated by TNF receptors, Toll-like receptors, and several interleukin receptors (Sun and Chen, 2004). Both TRAF2 (Shi and Kehrl, 2003) and TRAF6 (Deng et al., 2000) have been implicated as E3s for Ubc13-mediated NF-κB activation; however, it remains unclear which Uev is required for Ubc13 function in these signaling pathways. To address this issue, we used synthetic small interference RNA (siRNA) to specifically inhibit the expression of either Mms2 or Uev1. Because 2H11 monoclonal antibody recognizes both Mms2 and Uev1, we verified the specificity of our targeting siRNAs for either Mms2 or Uev1 by examining their effects in knocking down the expressions of FLAG-Mms2 and/or myc-Uev1A. As shown in Fig. 6 A, siRNA against Mms2 (lanes 2 and 8) or Uev1 (lanes 6 and 9) specifically inhibits its target gene expression but does not display a cross inhibitory effect. Consistent with our previous findings (Zhou et al., 2004), siRNA-targeting Ubc13 significantly reduced TRAF2- and TRAF6-stimulated NF-κB activation (Fig. 6 B). Moreover, siRNA-targeting Uev1 but not Mms2 inhibited TRAF2- and TRAF6-stimulated NF-κB activation. This effect is apparently specific for TRAF2- and TRAF6-dependent ubiquitination to activate NF-κB because neither Ubc13 nor Uev1 silencing affected IKKβ-mediated NF-κB activation (Fig. 6 B). These results highlight a specific role for Uev1 but not Mms2 in cooperating with Ubc13 in activating the NF-κB pathway.

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

Show MeSH
Related in: MedlinePlus