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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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Related in: MedlinePlus

Requirement of Ubc13 and Uev1A for LPS-induced p65 translocation. Phase contrast, p65-ICC, and DAPI staining were performed 4 d after transfection of mouse microglia with RNAi constructs as indicated, followed by a 1.5-h exposure to 1 μg/ml LPS and fixation. Merged images indicate colocalization of p65 immunostaining with nuclei. Identical color adjustment was made to all merged images to enhance differential colocalization. Bar, 10 μm.
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fig7: Requirement of Ubc13 and Uev1A for LPS-induced p65 translocation. Phase contrast, p65-ICC, and DAPI staining were performed 4 d after transfection of mouse microglia with RNAi constructs as indicated, followed by a 1.5-h exposure to 1 μg/ml LPS and fixation. Merged images indicate colocalization of p65 immunostaining with nuclei. Identical color adjustment was made to all merged images to enhance differential colocalization. Bar, 10 μm.

Mentions: The bacterial endotoxin LPS stimulates NF-κB activation in microglia (Bonaiuto et al., 1997). To determine the physiological significance of the Ubc13–Uev complex in this pathway, we monitored the nuclear translocation of the p65 subunit of NF-κB in response to LPS in primary murine microglia cells. In untreated cells, p65 resided mainly in the cytoplasm, whereas upon LPS treatment, p65 rapidly translocated into the nucleus (unpublished data). As shown in Fig. 7, RNAi directed to reduce Mms2 did not affect p65 translocation to the nucleus after LPS treatment. However, after LPS treatment, the number of microglia nuclei containing significant p65 immunoreactivity was reduced from nearly 100% to ∼30% in cells transfected with RNAi constructs directed to reduce either Ubc13 or Uev1. This result indicates that the activities of Ubc13 and Uev1A, but not Mms2, are indeed required in the NF-κB signaling 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)

Requirement of Ubc13 and Uev1A for LPS-induced p65 translocation. Phase contrast, p65-ICC, and DAPI staining were performed 4 d after transfection of mouse microglia with RNAi constructs as indicated, followed by a 1.5-h exposure to 1 μg/ml LPS and fixation. Merged images indicate colocalization of p65 immunostaining with nuclei. Identical color adjustment was made to all merged images to enhance differential colocalization. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171356&req=5

fig7: Requirement of Ubc13 and Uev1A for LPS-induced p65 translocation. Phase contrast, p65-ICC, and DAPI staining were performed 4 d after transfection of mouse microglia with RNAi constructs as indicated, followed by a 1.5-h exposure to 1 μg/ml LPS and fixation. Merged images indicate colocalization of p65 immunostaining with nuclei. Identical color adjustment was made to all merged images to enhance differential colocalization. Bar, 10 μm.
Mentions: The bacterial endotoxin LPS stimulates NF-κB activation in microglia (Bonaiuto et al., 1997). To determine the physiological significance of the Ubc13–Uev complex in this pathway, we monitored the nuclear translocation of the p65 subunit of NF-κB in response to LPS in primary murine microglia cells. In untreated cells, p65 resided mainly in the cytoplasm, whereas upon LPS treatment, p65 rapidly translocated into the nucleus (unpublished data). As shown in Fig. 7, RNAi directed to reduce Mms2 did not affect p65 translocation to the nucleus after LPS treatment. However, after LPS treatment, the number of microglia nuclei containing significant p65 immunoreactivity was reduced from nearly 100% to ∼30% in cells transfected with RNAi constructs directed to reduce either Ubc13 or Uev1. This result indicates that the activities of Ubc13 and Uev1A, but not Mms2, are indeed required in the NF-κB signaling 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