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Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJ, Hansen TH - J. Cell Biol. (2009)

Bottom Line: Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD).In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates.However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

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The kK3 and kK5 RINGs in the context of mK3 interact with Ube2j2 to promote the formation of K48-linked Ub chain on S residues of the HCs. (A) kK3 or kK5 RING-mK3 chimeric molecules were expressed in WT3–Ld 1S cells. After permeabilization and depletion of cytosolic proteins, aliquots of these cells were incubated with no enzyme, E1 only, or E1 plus one of the two E2s in the presence of ATP and HA-Ub. Ld HCs were immunoprecipitated and blotted as indicated. (B) Aliquots of the cells used in A were incubated with ATP, E1, and WT or mutant HA-Ub in an ubiquitination assay of permeabilized cells as described in A. Immunoprecipitation (IP) of Ld and blotting by anti-HA (top) or anti-Ub (middle) antibodies shows newly formed Ub-Ld HCs and total Ub-Ld HCs, respectively. The conjugates formed by HA-Ub are in red. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot.
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fig6: The kK3 and kK5 RINGs in the context of mK3 interact with Ube2j2 to promote the formation of K48-linked Ub chain on S residues of the HCs. (A) kK3 or kK5 RING-mK3 chimeric molecules were expressed in WT3–Ld 1S cells. After permeabilization and depletion of cytosolic proteins, aliquots of these cells were incubated with no enzyme, E1 only, or E1 plus one of the two E2s in the presence of ATP and HA-Ub. Ld HCs were immunoprecipitated and blotted as indicated. (B) Aliquots of the cells used in A were incubated with ATP, E1, and WT or mutant HA-Ub in an ubiquitination assay of permeabilized cells as described in A. Immunoprecipitation (IP) of Ld and blotting by anti-HA (top) or anti-Ub (middle) antibodies shows newly formed Ub-Ld HCs and total Ub-Ld HCs, respectively. The conjugates formed by HA-Ub are in red. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot.

Mentions: Unlike mK3, kK3 and kK5 ligases ubiquitinate HCs in a post-ER compartment, resulting in their rapid endocytosis and lysosomal degradation (Lehner et al., 2005). The cognate E2s of kK3 have been identified as Ubc5b/c (Ube2d2/3) and Ubc13 (Ube2n), which catalyze a K63-linked poly-Ub chain on the tail of the HCs (Duncan et al., 2006). Moreover, both kK3 and kK5 can facilitate Ub conjugation of C and K residues (Cadwell and Coscoy, 2005, 2008) and not S/T residues like mK3. Whether the same or different E2s facilitate a C ubiquitination versus a K ubiquitination by kK3 and kK5 has not yet been reported. Nevertheless, the fact that mK3 and kK3/kK5 have similar overall structures with highly homologous RING-CH domains make them valuable probes for understanding the determinants and consequences of specific E2–E3 interactions with each other and with substrates. To determine the importance of the RING-CH domain in E2 selection and the potential to ubiquitinate hydroxylated residues, the RING domains of kK3 and kK5 were each swapped into mK3. Each of these RING swap constructs was introduced into cells expressing WT Ld or Ld mutants with only C, K, S, or T residues available in the tail. As expected, both kK3 and kK5 RINGs in the context of mK3-mediated robust ubiquitination of K, S, or T but not C in tails of the HC in these cells (unpublished data). Their lack of ability to efficiently conjugate C residues is not caused by the position of the C residue because other positions were tested, including sites proximal to the C terminus of the tail, as favored by intact kK3 ligase (Herr et al., 2009). Thus, in the context of mK3, the RING domains of kK3 or kK5 no longer supported ubiquitination of C residues like their intact ligases but facilitated S or T ubiquitination like intact mK3. Furthermore, in our permeabilized cell system, Ube2j2 and not Ube2n interacted with kK3 or kK5 RING swaps to greatly enhance the ubiquitination on an S residue of the tail of HC (Fig. 6 A, left and right, respectively). Accordingly, the Ub chains formed on the tail of Ld 1S molecules in kK3 or kK5 swap–expressing cells were Ub K48 and not K63 dependent (Fig. 6 B), which is the same as what was observed with WT mK3 (Fig. S4). Collectively, these data demonstrate that sequences outside the RING domain of mK3 ligase are critical determinants for which E2 is recruited, which non-K residues can be ubiquitinated, and which linkage is used for poly-Ub chain assembly.


Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

Wang X, Herr RA, Rabelink M, Hoeben RC, Wiertz EJ, Hansen TH - J. Cell Biol. (2009)

The kK3 and kK5 RINGs in the context of mK3 interact with Ube2j2 to promote the formation of K48-linked Ub chain on S residues of the HCs. (A) kK3 or kK5 RING-mK3 chimeric molecules were expressed in WT3–Ld 1S cells. After permeabilization and depletion of cytosolic proteins, aliquots of these cells were incubated with no enzyme, E1 only, or E1 plus one of the two E2s in the presence of ATP and HA-Ub. Ld HCs were immunoprecipitated and blotted as indicated. (B) Aliquots of the cells used in A were incubated with ATP, E1, and WT or mutant HA-Ub in an ubiquitination assay of permeabilized cells as described in A. Immunoprecipitation (IP) of Ld and blotting by anti-HA (top) or anti-Ub (middle) antibodies shows newly formed Ub-Ld HCs and total Ub-Ld HCs, respectively. The conjugates formed by HA-Ub are in red. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot.
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Related In: Results  -  Collection

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fig6: The kK3 and kK5 RINGs in the context of mK3 interact with Ube2j2 to promote the formation of K48-linked Ub chain on S residues of the HCs. (A) kK3 or kK5 RING-mK3 chimeric molecules were expressed in WT3–Ld 1S cells. After permeabilization and depletion of cytosolic proteins, aliquots of these cells were incubated with no enzyme, E1 only, or E1 plus one of the two E2s in the presence of ATP and HA-Ub. Ld HCs were immunoprecipitated and blotted as indicated. (B) Aliquots of the cells used in A were incubated with ATP, E1, and WT or mutant HA-Ub in an ubiquitination assay of permeabilized cells as described in A. Immunoprecipitation (IP) of Ld and blotting by anti-HA (top) or anti-Ub (middle) antibodies shows newly formed Ub-Ld HCs and total Ub-Ld HCs, respectively. The conjugates formed by HA-Ub are in red. (A and B) Molecular mass is indicated in kilodaltons. IB, immunoblot.
Mentions: Unlike mK3, kK3 and kK5 ligases ubiquitinate HCs in a post-ER compartment, resulting in their rapid endocytosis and lysosomal degradation (Lehner et al., 2005). The cognate E2s of kK3 have been identified as Ubc5b/c (Ube2d2/3) and Ubc13 (Ube2n), which catalyze a K63-linked poly-Ub chain on the tail of the HCs (Duncan et al., 2006). Moreover, both kK3 and kK5 can facilitate Ub conjugation of C and K residues (Cadwell and Coscoy, 2005, 2008) and not S/T residues like mK3. Whether the same or different E2s facilitate a C ubiquitination versus a K ubiquitination by kK3 and kK5 has not yet been reported. Nevertheless, the fact that mK3 and kK3/kK5 have similar overall structures with highly homologous RING-CH domains make them valuable probes for understanding the determinants and consequences of specific E2–E3 interactions with each other and with substrates. To determine the importance of the RING-CH domain in E2 selection and the potential to ubiquitinate hydroxylated residues, the RING domains of kK3 and kK5 were each swapped into mK3. Each of these RING swap constructs was introduced into cells expressing WT Ld or Ld mutants with only C, K, S, or T residues available in the tail. As expected, both kK3 and kK5 RINGs in the context of mK3-mediated robust ubiquitination of K, S, or T but not C in tails of the HC in these cells (unpublished data). Their lack of ability to efficiently conjugate C residues is not caused by the position of the C residue because other positions were tested, including sites proximal to the C terminus of the tail, as favored by intact kK3 ligase (Herr et al., 2009). Thus, in the context of mK3, the RING domains of kK3 or kK5 no longer supported ubiquitination of C residues like their intact ligases but facilitated S or T ubiquitination like intact mK3. Furthermore, in our permeabilized cell system, Ube2j2 and not Ube2n interacted with kK3 or kK5 RING swaps to greatly enhance the ubiquitination on an S residue of the tail of HC (Fig. 6 A, left and right, respectively). Accordingly, the Ub chains formed on the tail of Ld 1S molecules in kK3 or kK5 swap–expressing cells were Ub K48 and not K63 dependent (Fig. 6 B), which is the same as what was observed with WT mK3 (Fig. S4). Collectively, these data demonstrate that sequences outside the RING domain of mK3 ligase are critical determinants for which E2 is recruited, which non-K residues can be ubiquitinated, and which linkage is used for poly-Ub chain assembly.

Bottom Line: Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD).In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates.However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

Show MeSH