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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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Amino acid sequence comparison of yeast Mms2 and its human homologues. The amino acid sequences are obtained from the following sources: Mms2 (Broomfield et al., 1998); hMms2 (Xiao et al., 1998); Uev1A and Uev1B (Rothofsky and Lin, 1997). The corrected Uev1A sequence as reported previously (Deng et al., 2000) was used for alignment.
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fig1: Amino acid sequence comparison of yeast Mms2 and its human homologues. The amino acid sequences are obtained from the following sources: Mms2 (Broomfield et al., 1998); hMms2 (Xiao et al., 1998); Uev1A and Uev1B (Rothofsky and Lin, 1997). The corrected Uev1A sequence as reported previously (Deng et al., 2000) was used for alignment.

Mentions: Poly-Ub chains attached to a substrate can also be linked through different lysines within Ub, and it has become clear that the signaling properties of ubiquitination depend on the topology of poly-Ub chains. For example, it has been well established that poly-Ub chains linked through Lys48 are the principal signal to target a substrate for proteolysis by 26S proteasomes, whereas Lys63-linked chains play a regulatory role in diverse signaling pathways in a nonproteolytic fashion (Pickart, 2001b). The molecular basis for determining such specificity in chain assembly and recognition, however, remains poorly understood. So far only one E2, Ubc13, has been shown to mediate the assembly of Lys63-linked poly-Ub chains, and this activity requires a Ubc variant (Uev) as a cofactor; neither Ubc13 nor Uev alone is able to promote Lys63 poly-Ub chains (Hofmann and Pickart, 1999; McKenna et al., 2001). Uev is defined as a protein that resembles Ubc in structure and amino acid sequence, but does not contain a cysteine residue in the presumptive active site, rendering the protein catalytically inactive (Broomfield et al., 1998; Sancho et al., 1998). The prototype Uev, Mms2, was first isolated and characterized from the budding yeast Saccharomyces cerevisiae and is required for error-free postreplication repair (PRR; Broomfield et al., 1998). The crystal structure (Moraes et al., 2001; VanDemark et al., 2001) and NMR analysis (McKenna et al., 2001) of the Ubc13–Mms2 heterodimer show that Mms2 binds the acceptor Ub in an orientation that allows only Lys63, and not Lys48, to approach the active site on Ubc13. Subsequent studies indicate that many other proteins contain Uev domains. Collectively called the Uev family of proteins, they are as ancient as the Ubc family of proteins (Villalobo et al., 2002) and they are highly conserved in the eukaryotic kingdom, from protists to human (Brown et al., 2002). Higher eukaryotes, including plants and mammals, contain an increasing number of Uev proteins (Wong et al., 2003), suggesting that the Uev family of proteins may have evolved to increase diversity and selectivity in Ub conjugation. Nevertheless, it remains unknown whether these evolved Uev proteins are functionally redundant or each plays a specific role in discrete cellular processes. We addressed this question in the current study and found that Mms2 and Uev1, two mammalian homologues of the yeast Mms2, although sharing >90% amino acid sequence identity to each other in their core domain (Fig. 1) and both capable of cooperating with Ubc13 to promote ubiquitination in vitro, are involved in distinct biological activities in vivo. Specifically, we demonstrated that Mms2 is required in Ubc13-dependent DNA damage response but not NF-κB activation, whereas Uev1A is involved in Ubc13-dependent NF-κB activation but not DNA damage response. Thus, our study provides novel insight that Uev family proteins may have evolved to differentially regulate E2 functions in diverse cellular processes.


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)

Amino acid sequence comparison of yeast Mms2 and its human homologues. The amino acid sequences are obtained from the following sources: Mms2 (Broomfield et al., 1998); hMms2 (Xiao et al., 1998); Uev1A and Uev1B (Rothofsky and Lin, 1997). The corrected Uev1A sequence as reported previously (Deng et al., 2000) was used for alignment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171356&req=5

fig1: Amino acid sequence comparison of yeast Mms2 and its human homologues. The amino acid sequences are obtained from the following sources: Mms2 (Broomfield et al., 1998); hMms2 (Xiao et al., 1998); Uev1A and Uev1B (Rothofsky and Lin, 1997). The corrected Uev1A sequence as reported previously (Deng et al., 2000) was used for alignment.
Mentions: Poly-Ub chains attached to a substrate can also be linked through different lysines within Ub, and it has become clear that the signaling properties of ubiquitination depend on the topology of poly-Ub chains. For example, it has been well established that poly-Ub chains linked through Lys48 are the principal signal to target a substrate for proteolysis by 26S proteasomes, whereas Lys63-linked chains play a regulatory role in diverse signaling pathways in a nonproteolytic fashion (Pickart, 2001b). The molecular basis for determining such specificity in chain assembly and recognition, however, remains poorly understood. So far only one E2, Ubc13, has been shown to mediate the assembly of Lys63-linked poly-Ub chains, and this activity requires a Ubc variant (Uev) as a cofactor; neither Ubc13 nor Uev alone is able to promote Lys63 poly-Ub chains (Hofmann and Pickart, 1999; McKenna et al., 2001). Uev is defined as a protein that resembles Ubc in structure and amino acid sequence, but does not contain a cysteine residue in the presumptive active site, rendering the protein catalytically inactive (Broomfield et al., 1998; Sancho et al., 1998). The prototype Uev, Mms2, was first isolated and characterized from the budding yeast Saccharomyces cerevisiae and is required for error-free postreplication repair (PRR; Broomfield et al., 1998). The crystal structure (Moraes et al., 2001; VanDemark et al., 2001) and NMR analysis (McKenna et al., 2001) of the Ubc13–Mms2 heterodimer show that Mms2 binds the acceptor Ub in an orientation that allows only Lys63, and not Lys48, to approach the active site on Ubc13. Subsequent studies indicate that many other proteins contain Uev domains. Collectively called the Uev family of proteins, they are as ancient as the Ubc family of proteins (Villalobo et al., 2002) and they are highly conserved in the eukaryotic kingdom, from protists to human (Brown et al., 2002). Higher eukaryotes, including plants and mammals, contain an increasing number of Uev proteins (Wong et al., 2003), suggesting that the Uev family of proteins may have evolved to increase diversity and selectivity in Ub conjugation. Nevertheless, it remains unknown whether these evolved Uev proteins are functionally redundant or each plays a specific role in discrete cellular processes. We addressed this question in the current study and found that Mms2 and Uev1, two mammalian homologues of the yeast Mms2, although sharing >90% amino acid sequence identity to each other in their core domain (Fig. 1) and both capable of cooperating with Ubc13 to promote ubiquitination in vitro, are involved in distinct biological activities in vivo. Specifically, we demonstrated that Mms2 is required in Ubc13-dependent DNA damage response but not NF-κB activation, whereas Uev1A is involved in Ubc13-dependent NF-κB activation but not DNA damage response. Thus, our study provides novel insight that Uev family proteins may have evolved to differentially regulate E2 functions in diverse cellular processes.

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