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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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Ub chain building. Mms2 and Uev1A were assayed for Ub chain building activity using an in vitro ubiquitination reaction. The components of the reaction are noted in Materials and methods. The concentration of wild-type hUbc13 (Wt), hUbc13K92R (K92R), hMms2, Uev1A, and Uev1AΔ30 is 250 nM. The positions of free Ub (Ub), di-Ub (Ub2), Ubc13-Ub (Wt-Ub), and multi-Ub chains (Ubn) are indicated. Major contaminant bands from the 35S-Ub preparation, including the background band comigrating with Ub2, are indicated by an asterisk.
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fig8: Ub chain building. Mms2 and Uev1A were assayed for Ub chain building activity using an in vitro ubiquitination reaction. The components of the reaction are noted in Materials and methods. The concentration of wild-type hUbc13 (Wt), hUbc13K92R (K92R), hMms2, Uev1A, and Uev1AΔ30 is 250 nM. The positions of free Ub (Ub), di-Ub (Ub2), Ubc13-Ub (Wt-Ub), and multi-Ub chains (Ubn) are indicated. Major contaminant bands from the 35S-Ub preparation, including the background band comigrating with Ub2, are indicated by an asterisk.

Mentions: Given that both Uev1A and Mms2 are able to form a stable complex with Ubc13 in vivo and in vitro, but their biological functions are distinct, we hypothesize that the structural and sequence differences between Uev1A and Mms2 are responsible for their distinct intracellular signaling pathways. As the first step toward understanding how Uev1A and Mms2 are involved in distinct cellular processes, we performed an in vitro polyubiquitination activity assay using highly purified components. To prevent E2 self-ubiquitination, a Ubc13 derivative (Ubc13K92R) was used in the assay in combination with either Mms2 or Uev1A. We have previously shown that both Ubc13 and Ubc13K92R were able to form di-Ub conjugates with equal efficiency (McKenna et al., 2001); however, neither (Fig. 8, lanes 1 and 2) is able to carry out Ub chain assembly in the absence of Uev. The addition of Mms2 could only support di-Ub formation (Fig. 8, lane 3), in contrast to the poly-Ub formation in the presence of Uev1A (Fig. 8, lane 5). Uev1A differs from Mms2 in two aspects, namely, the nonconserved NH2-terminal 30 amino acids and <10% sequence variation in the remaining core domain (Fig. 1). To determine which difference is responsible for the observed poly-Ub versus di-Ub formation, a 30-residue truncation was made in Uev1A such that it resembled the core region of Mms2. Incubating Uev1AΔ30 with Ubc13K92R resulted in only di-Ub formation (Fig. 8, lane 4) in a manner similar to that observed with the addition of Mms2. These observations suggest that the additional NH2-terminal region of Uev1A is responsible for Ubc13-mediated poly-Ub chain assembly through Lys63-Gly76 linkage in vitro and that the differential polyubiquitination activity between Uev1A and Mms2 may play a role in their distinct cellular functions.


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)

Ub chain building. Mms2 and Uev1A were assayed for Ub chain building activity using an in vitro ubiquitination reaction. The components of the reaction are noted in Materials and methods. The concentration of wild-type hUbc13 (Wt), hUbc13K92R (K92R), hMms2, Uev1A, and Uev1AΔ30 is 250 nM. The positions of free Ub (Ub), di-Ub (Ub2), Ubc13-Ub (Wt-Ub), and multi-Ub chains (Ubn) are indicated. Major contaminant bands from the 35S-Ub preparation, including the background band comigrating with Ub2, are indicated by an asterisk.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171356&req=5

fig8: Ub chain building. Mms2 and Uev1A were assayed for Ub chain building activity using an in vitro ubiquitination reaction. The components of the reaction are noted in Materials and methods. The concentration of wild-type hUbc13 (Wt), hUbc13K92R (K92R), hMms2, Uev1A, and Uev1AΔ30 is 250 nM. The positions of free Ub (Ub), di-Ub (Ub2), Ubc13-Ub (Wt-Ub), and multi-Ub chains (Ubn) are indicated. Major contaminant bands from the 35S-Ub preparation, including the background band comigrating with Ub2, are indicated by an asterisk.
Mentions: Given that both Uev1A and Mms2 are able to form a stable complex with Ubc13 in vivo and in vitro, but their biological functions are distinct, we hypothesize that the structural and sequence differences between Uev1A and Mms2 are responsible for their distinct intracellular signaling pathways. As the first step toward understanding how Uev1A and Mms2 are involved in distinct cellular processes, we performed an in vitro polyubiquitination activity assay using highly purified components. To prevent E2 self-ubiquitination, a Ubc13 derivative (Ubc13K92R) was used in the assay in combination with either Mms2 or Uev1A. We have previously shown that both Ubc13 and Ubc13K92R were able to form di-Ub conjugates with equal efficiency (McKenna et al., 2001); however, neither (Fig. 8, lanes 1 and 2) is able to carry out Ub chain assembly in the absence of Uev. The addition of Mms2 could only support di-Ub formation (Fig. 8, lane 3), in contrast to the poly-Ub formation in the presence of Uev1A (Fig. 8, lane 5). Uev1A differs from Mms2 in two aspects, namely, the nonconserved NH2-terminal 30 amino acids and <10% sequence variation in the remaining core domain (Fig. 1). To determine which difference is responsible for the observed poly-Ub versus di-Ub formation, a 30-residue truncation was made in Uev1A such that it resembled the core region of Mms2. Incubating Uev1AΔ30 with Ubc13K92R resulted in only di-Ub formation (Fig. 8, lane 4) in a manner similar to that observed with the addition of Mms2. These observations suggest that the additional NH2-terminal region of Uev1A is responsible for Ubc13-mediated poly-Ub chain assembly through Lys63-Gly76 linkage in vitro and that the differential polyubiquitination activity between Uev1A and Mms2 may play a role in their distinct cellular functions.

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