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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

CPT-induced nuclear foci formation. ICC of 3T3 cells after CPT treatment (5 μM for 4 h) and in situ cell fractionation reveals Ubc13 nuclear foci (4E11 as primary antibody and Alexa488 as secondary antibody). These foci are compared, by merging images, with those of Mre11 (A), Rad51 (B), and BrdU (C) and in 3T3 cells transfected with Mms2-myc (D), Uev1A-myc (E), and Uev1AΔ30-myc (F) viewed by using specific primary antibodies and the Alexa546 secondary antibody, except in C, in which Alexa546 is directly conjugated with anti-BrdU. Bar, 5 μm.
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fig4: CPT-induced nuclear foci formation. ICC of 3T3 cells after CPT treatment (5 μM for 4 h) and in situ cell fractionation reveals Ubc13 nuclear foci (4E11 as primary antibody and Alexa488 as secondary antibody). These foci are compared, by merging images, with those of Mre11 (A), Rad51 (B), and BrdU (C) and in 3T3 cells transfected with Mms2-myc (D), Uev1A-myc (E), and Uev1AΔ30-myc (F) viewed by using specific primary antibodies and the Alexa546 secondary antibody, except in C, in which Alexa546 is directly conjugated with anti-BrdU. Bar, 5 μm.

Mentions: Because Ubc13 and Uev are thought to be freely diffusible between the cytoplasm and the nucleus, we attempted to refine their localization by using an in situ cell fractionation procedure before fixation, which is a method frequently applied to identify nuclear localization of DNA repair proteins (Andegeko et al., 2001). After CPT treatment, nuclear foci positive for Mre11 (Fig. 4 A), Rad51 (Fig. 4 B), and Ubc13 (Fig. 4, A and B) immunoreactivity were observed after detergent extraction under a stringent condition capable of releasing the diffuse Ubc13 nuclear staining in the S phase cells (not depicted). Surprisingly, CPT-induced Ubc13 nuclear foci exhibit morphological distributions distinct from both Rad51 and Mre11 foci. It was found that within 1 h after CPT treatment, Rad51 and Mre11 foci become very apparent in a significant number of cells as fine nuclear foci; however, Ubc13 foci were not apparent until nearly 4 h after CPT treatment. A time course analysis indicates that as time progressed nearly all the cells retained Ubc13 immunoreactivity (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502113/DC1). Furthermore, Ubc13 foci appeared as punctuate structures distinctly larger than the fine granular foci of either Rad51 or Mre11 and were colocalized with those of BrdU incorporation (Fig. 4 C), implying that Ubc13 is involved in DNA synthesis under DNA damage conditions. The punctuate pattern of Ubc13 foci and BrdU incorporation agrees with a previous finding (Sakamoto et al., 2001) that Rad51 nuclear foci are distinct from the distribution pattern of BrdU incorporation after CPT treatment.


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)

CPT-induced nuclear foci formation. ICC of 3T3 cells after CPT treatment (5 μM for 4 h) and in situ cell fractionation reveals Ubc13 nuclear foci (4E11 as primary antibody and Alexa488 as secondary antibody). These foci are compared, by merging images, with those of Mre11 (A), Rad51 (B), and BrdU (C) and in 3T3 cells transfected with Mms2-myc (D), Uev1A-myc (E), and Uev1AΔ30-myc (F) viewed by using specific primary antibodies and the Alexa546 secondary antibody, except in C, in which Alexa546 is directly conjugated with anti-BrdU. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171356&req=5

fig4: CPT-induced nuclear foci formation. ICC of 3T3 cells after CPT treatment (5 μM for 4 h) and in situ cell fractionation reveals Ubc13 nuclear foci (4E11 as primary antibody and Alexa488 as secondary antibody). These foci are compared, by merging images, with those of Mre11 (A), Rad51 (B), and BrdU (C) and in 3T3 cells transfected with Mms2-myc (D), Uev1A-myc (E), and Uev1AΔ30-myc (F) viewed by using specific primary antibodies and the Alexa546 secondary antibody, except in C, in which Alexa546 is directly conjugated with anti-BrdU. Bar, 5 μm.
Mentions: Because Ubc13 and Uev are thought to be freely diffusible between the cytoplasm and the nucleus, we attempted to refine their localization by using an in situ cell fractionation procedure before fixation, which is a method frequently applied to identify nuclear localization of DNA repair proteins (Andegeko et al., 2001). After CPT treatment, nuclear foci positive for Mre11 (Fig. 4 A), Rad51 (Fig. 4 B), and Ubc13 (Fig. 4, A and B) immunoreactivity were observed after detergent extraction under a stringent condition capable of releasing the diffuse Ubc13 nuclear staining in the S phase cells (not depicted). Surprisingly, CPT-induced Ubc13 nuclear foci exhibit morphological distributions distinct from both Rad51 and Mre11 foci. It was found that within 1 h after CPT treatment, Rad51 and Mre11 foci become very apparent in a significant number of cells as fine nuclear foci; however, Ubc13 foci were not apparent until nearly 4 h after CPT treatment. A time course analysis indicates that as time progressed nearly all the cells retained Ubc13 immunoreactivity (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502113/DC1). Furthermore, Ubc13 foci appeared as punctuate structures distinctly larger than the fine granular foci of either Rad51 or Mre11 and were colocalized with those of BrdU incorporation (Fig. 4 C), implying that Ubc13 is involved in DNA synthesis under DNA damage conditions. The punctuate pattern of Ubc13 foci and BrdU incorporation agrees with a previous finding (Sakamoto et al., 2001) that Rad51 nuclear foci are distinct from the distribution pattern of BrdU incorporation after CPT treatment.

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