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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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Ube2j2 is required for mK3-induced HC ubiquitination and degradation. (A) WT3 cells expressing WT Ld and mK3 were transduced by lentivirus-based shRNA targeting one of the following Ub-conjugating enzymes: Ube2d1, Ube2g1, Ube2g2, Ube2j1, or Ube2j2 (S#1). After incubation with 50 µM MG132 for 2 h, Ld HCs were precipitated from these cells and followed by SDS-PAGE and blotting with anti-Ub and anti-Ld antibodies. The β-actin and mK3 blots in the middle panel were included to show a similar amount of input lysate and mK3 in each cell line. In the right panel, selected cell lines from the left panel were incubated for 24 h with 125 U/ml IFN-γ, pulse labeled with [35S]Cys/Met, and chased for the indicated times with unlabeled Cys/Met. Ld precipitates were resolved by SDS-PAGE and visualized by autoradiography. Relative band intensities from the gels are plotted as a percentage of the intensity at time 0 for each cell line. Data are representative of three independent experiments. (B) WT3 cells expressing WT Ld and mK3 were transduced by retrovirus-based shRNA targeting Ube2j2 (S#2) or Ube2n or by an empty vector. The ubiquitination and degradation of Ld HCs were determined by immunoblotting and pulse–chase experiments as in A. Data are representative of three independent experiments. (C) Selected cells used in A and B were permeabilized, cytosol depleted, and incubated with E1 or E1 + Ube2j2 in the presence of ATP, HA-Ub, and Ub-Al. Precipitated Ld molecules were resolved by SDS-PAGE and blotted for newly ubiquitinated Ld by anti-HA. An anti-Ld blot was included to show the level of Ld molecules in each cell line. (D) Same as in C except that WT3 cells expressing 1S Ld and mK3 were used. (A–D) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
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fig3: Ube2j2 is required for mK3-induced HC ubiquitination and degradation. (A) WT3 cells expressing WT Ld and mK3 were transduced by lentivirus-based shRNA targeting one of the following Ub-conjugating enzymes: Ube2d1, Ube2g1, Ube2g2, Ube2j1, or Ube2j2 (S#1). After incubation with 50 µM MG132 for 2 h, Ld HCs were precipitated from these cells and followed by SDS-PAGE and blotting with anti-Ub and anti-Ld antibodies. The β-actin and mK3 blots in the middle panel were included to show a similar amount of input lysate and mK3 in each cell line. In the right panel, selected cell lines from the left panel were incubated for 24 h with 125 U/ml IFN-γ, pulse labeled with [35S]Cys/Met, and chased for the indicated times with unlabeled Cys/Met. Ld precipitates were resolved by SDS-PAGE and visualized by autoradiography. Relative band intensities from the gels are plotted as a percentage of the intensity at time 0 for each cell line. Data are representative of three independent experiments. (B) WT3 cells expressing WT Ld and mK3 were transduced by retrovirus-based shRNA targeting Ube2j2 (S#2) or Ube2n or by an empty vector. The ubiquitination and degradation of Ld HCs were determined by immunoblotting and pulse–chase experiments as in A. Data are representative of three independent experiments. (C) Selected cells used in A and B were permeabilized, cytosol depleted, and incubated with E1 or E1 + Ube2j2 in the presence of ATP, HA-Ub, and Ub-Al. Precipitated Ld molecules were resolved by SDS-PAGE and blotted for newly ubiquitinated Ld by anti-HA. An anti-Ld blot was included to show the level of Ld molecules in each cell line. (D) Same as in C except that WT3 cells expressing 1S Ld and mK3 were used. (A–D) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.

Mentions: To determine whether Ube2j2 and/or Ube2d1 is required for mK3-mediated HC ubiquitination and degradation, shRNAs targeting specific E2s were introduced into WT3-Ld-mK3 cells individually. Target gene expression was evaluated by quantitative RT-PCR (Fig. S3). shRNAs that yielded >70% inhibition were selected for comparisons (Table S1). Compared with controls, the extent of ubiquitination of WT Ld in cells expressing Ube2j2 shRNA (S#1) was lower, especially in the HC bands containing more than two Ub moieties (Fig. 3 A, left). The specificity of this effect was confirmed by another Ube2j2 shRNA (S#2) that targets a different sequence on Ube2j2 (Fig. 3 B, left). In contrast to Ube2j2, inhibiting Ube2g1, Ube2g2, Ube2j1, Ube2k, and Ube2n had no effect on the levels of Ld ubiquitination (Fig. 3, A [left] and B [left]; and not depicted), although comparable levels of mK3 were expressed in each cell line (Fig. 3, A [middle] and B [middle]). Concordant with the reduced level of ubiquitination, the half-life of the Ld molecules was dramatically increased in cells with depleted levels of Ube2j2 (Fig. 3, A [right] and B [right]). Thus, selective inhibition of Ube2j2 by shRNA resulted in impaired Ub conjugation and degradation of the HC substrates by mK3. In contrast, suppression of Ube2d1 by shRNA had no obvious effect on ubiquitination and stability of HCs (Fig. 3 A). Also, there was no additive effect observed when Ube2d1 shRNA was coexpressed with Ube2j2 shRNA (unpublished data). Thus, Ube2j2 is the uniquely required E2 supporting mK3 function in vivo.


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)

Ube2j2 is required for mK3-induced HC ubiquitination and degradation. (A) WT3 cells expressing WT Ld and mK3 were transduced by lentivirus-based shRNA targeting one of the following Ub-conjugating enzymes: Ube2d1, Ube2g1, Ube2g2, Ube2j1, or Ube2j2 (S#1). After incubation with 50 µM MG132 for 2 h, Ld HCs were precipitated from these cells and followed by SDS-PAGE and blotting with anti-Ub and anti-Ld antibodies. The β-actin and mK3 blots in the middle panel were included to show a similar amount of input lysate and mK3 in each cell line. In the right panel, selected cell lines from the left panel were incubated for 24 h with 125 U/ml IFN-γ, pulse labeled with [35S]Cys/Met, and chased for the indicated times with unlabeled Cys/Met. Ld precipitates were resolved by SDS-PAGE and visualized by autoradiography. Relative band intensities from the gels are plotted as a percentage of the intensity at time 0 for each cell line. Data are representative of three independent experiments. (B) WT3 cells expressing WT Ld and mK3 were transduced by retrovirus-based shRNA targeting Ube2j2 (S#2) or Ube2n or by an empty vector. The ubiquitination and degradation of Ld HCs were determined by immunoblotting and pulse–chase experiments as in A. Data are representative of three independent experiments. (C) Selected cells used in A and B were permeabilized, cytosol depleted, and incubated with E1 or E1 + Ube2j2 in the presence of ATP, HA-Ub, and Ub-Al. Precipitated Ld molecules were resolved by SDS-PAGE and blotted for newly ubiquitinated Ld by anti-HA. An anti-Ld blot was included to show the level of Ld molecules in each cell line. (D) Same as in C except that WT3 cells expressing 1S Ld and mK3 were used. (A–D) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
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fig3: Ube2j2 is required for mK3-induced HC ubiquitination and degradation. (A) WT3 cells expressing WT Ld and mK3 were transduced by lentivirus-based shRNA targeting one of the following Ub-conjugating enzymes: Ube2d1, Ube2g1, Ube2g2, Ube2j1, or Ube2j2 (S#1). After incubation with 50 µM MG132 for 2 h, Ld HCs were precipitated from these cells and followed by SDS-PAGE and blotting with anti-Ub and anti-Ld antibodies. The β-actin and mK3 blots in the middle panel were included to show a similar amount of input lysate and mK3 in each cell line. In the right panel, selected cell lines from the left panel were incubated for 24 h with 125 U/ml IFN-γ, pulse labeled with [35S]Cys/Met, and chased for the indicated times with unlabeled Cys/Met. Ld precipitates were resolved by SDS-PAGE and visualized by autoradiography. Relative band intensities from the gels are plotted as a percentage of the intensity at time 0 for each cell line. Data are representative of three independent experiments. (B) WT3 cells expressing WT Ld and mK3 were transduced by retrovirus-based shRNA targeting Ube2j2 (S#2) or Ube2n or by an empty vector. The ubiquitination and degradation of Ld HCs were determined by immunoblotting and pulse–chase experiments as in A. Data are representative of three independent experiments. (C) Selected cells used in A and B were permeabilized, cytosol depleted, and incubated with E1 or E1 + Ube2j2 in the presence of ATP, HA-Ub, and Ub-Al. Precipitated Ld molecules were resolved by SDS-PAGE and blotted for newly ubiquitinated Ld by anti-HA. An anti-Ld blot was included to show the level of Ld molecules in each cell line. (D) Same as in C except that WT3 cells expressing 1S Ld and mK3 were used. (A–D) Molecular mass is indicated in kilodaltons. IB, immunoblot; IP, immunoprecipitation.
Mentions: To determine whether Ube2j2 and/or Ube2d1 is required for mK3-mediated HC ubiquitination and degradation, shRNAs targeting specific E2s were introduced into WT3-Ld-mK3 cells individually. Target gene expression was evaluated by quantitative RT-PCR (Fig. S3). shRNAs that yielded >70% inhibition were selected for comparisons (Table S1). Compared with controls, the extent of ubiquitination of WT Ld in cells expressing Ube2j2 shRNA (S#1) was lower, especially in the HC bands containing more than two Ub moieties (Fig. 3 A, left). The specificity of this effect was confirmed by another Ube2j2 shRNA (S#2) that targets a different sequence on Ube2j2 (Fig. 3 B, left). In contrast to Ube2j2, inhibiting Ube2g1, Ube2g2, Ube2j1, Ube2k, and Ube2n had no effect on the levels of Ld ubiquitination (Fig. 3, A [left] and B [left]; and not depicted), although comparable levels of mK3 were expressed in each cell line (Fig. 3, A [middle] and B [middle]). Concordant with the reduced level of ubiquitination, the half-life of the Ld molecules was dramatically increased in cells with depleted levels of Ube2j2 (Fig. 3, A [right] and B [right]). Thus, selective inhibition of Ube2j2 by shRNA resulted in impaired Ub conjugation and degradation of the HC substrates by mK3. In contrast, suppression of Ube2d1 by shRNA had no obvious effect on ubiquitination and stability of HCs (Fig. 3 A). Also, there was no additive effect observed when Ube2d1 shRNA was coexpressed with Ube2j2 shRNA (unpublished data). Thus, Ube2j2 is the uniquely required E2 supporting mK3 function in vivo.

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