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90K Glycoprotein Promotes Degradation of Mutant β -Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus 1 2

View Article: PubMed Central - PubMed

ABSTRACT

β-Catenin is a major transducer of the Wnt signaling pathway, which is aberrantly expressed in colorectal and other cancers. Previously, we showed that β-catenin is downregulated by the 90K glycoprotein via ISGylation-dependent degradation. However, the further mechanisms of β-catenin degradation by 90K-mediated ISGylation pathway were not investigated. This study aimed to identify the β-catenin domain responsible for the action of 90K and to compare the mechanism of 90K on β-catenin degradation with phosphorylation-dependent ubiquitinational degradation of β-catenin. The deletion mutants of β-catenin lacking N- or C-terminal domain or mutating the N-terminal lysine or nonlysine residue were employed to delineate the characteristics of β-catenin degradation by 90K-mediated ISGylation pathway. 90K induced Herc5 and ISG15 expression and reduced β-catenin levels in HeLa and CSC221 cells. The N-terminus of β-catenin is required for 90K-induced β-catenin degradation, but the N-terminus of β-catenin is not essential for interaction with Herc5. However, substituting lysine residues in the N-terminus of β-catenin with arginine or deleting serine or threonine residue containing domains from the N-terminus does not affect 90K-induced β-catenin degradation, indicating that the N-terminal 86 amino acids of β-catenin are crucial for 90K-mediated ISGylation/degradation of β-catenin in which the responsible lysine or nonlysine residues were not identified. Our present results highlight the action of 90K on promoting degradation of mutant β-catenin lacking the phosphorylation sites in the N-terminus. It provides further insights into the discrete pathway downregulating the stabilized β-catenin via acquiring mutations at the serine/threonine residues in the N-terminus.

No MeSH data available.


Substituting N-terminal lysine residues with arginine or deleting N-terminal serine- or threonine-containing domains from β-catenin. (A) Schematic illustration of the β-catenin deletion mutants and the amino acid sequence of the N-terminus of β-catenin. Lysine (K, red), serine (S, orange), and threonine (T, orange) residues within the N-terminus of β-catenin are shown in colors. Serine and threonine residues known to be targets for GSK-3β phosphorylation are shown in blue. (B-E) Substitution of N-terminal lysine residues with arginine or deleting N-terminal domains containing serine or threonine residues does not affect 90K-induced β-catenin degradation. The β-catenin mutants were transfected into HEK293T cells, which were then treated with either ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), endogenous β-catenin, and actin. Decreases in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the fold changes of relative GFP-β-catenin level compared with actin were depicted as a bar graph below the immunoblot data. Each bar represents mean ± SD for triplicate samples, and number under each gel corresponds to each bar graph. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (**P < .01; ***P < .001). (B) Introduction of arginine mutations does not affect 90K-induced β-catenin degradation. (C) Deleting serine- and threonine-containing domains between Lys-19 and Lys-49 does not affect 90K-induced β-catenin degradation. (D) Deleting serine- and threonine-containing domains beyond Lys-49 does not affect 90K-induced β-catenin degradation. (E) 90K-induced β-catenin degradation occurs if β-catenin harbors either half of its N-terminal domain.
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f0015: Substituting N-terminal lysine residues with arginine or deleting N-terminal serine- or threonine-containing domains from β-catenin. (A) Schematic illustration of the β-catenin deletion mutants and the amino acid sequence of the N-terminus of β-catenin. Lysine (K, red), serine (S, orange), and threonine (T, orange) residues within the N-terminus of β-catenin are shown in colors. Serine and threonine residues known to be targets for GSK-3β phosphorylation are shown in blue. (B-E) Substitution of N-terminal lysine residues with arginine or deleting N-terminal domains containing serine or threonine residues does not affect 90K-induced β-catenin degradation. The β-catenin mutants were transfected into HEK293T cells, which were then treated with either ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), endogenous β-catenin, and actin. Decreases in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the fold changes of relative GFP-β-catenin level compared with actin were depicted as a bar graph below the immunoblot data. Each bar represents mean ± SD for triplicate samples, and number under each gel corresponds to each bar graph. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (**P < .01; ***P < .001). (B) Introduction of arginine mutations does not affect 90K-induced β-catenin degradation. (C) Deleting serine- and threonine-containing domains between Lys-19 and Lys-49 does not affect 90K-induced β-catenin degradation. (D) Deleting serine- and threonine-containing domains beyond Lys-49 does not affect 90K-induced β-catenin degradation. (E) 90K-induced β-catenin degradation occurs if β-catenin harbors either half of its N-terminal domain.

Mentions: Because Herc5 interacts with both FL- and ΔN86-β-catenin, N-terminal β-catenin likely harbors residues directly involved in the conjugation of ISG15. Therefore, to identify the residue(s) in the N-terminus of β-catenin that is(are) responsible for ISG15 conjugation, we compared the N-terminal aa sequence of β-catenin with the sequences published in previous studies reporting the consensus motif required for conjugation of ISG15. ISG15 conjugation occurs on the ε-amine group of lysine residues [17] or on cysteine residues [18]. As shown in Figure 3A, we identified two lysine residues (Lys-19 and Lys-49); however, the 86 aa N-terminus of β-catenin contains no cysteine residues. Although not reported for ISG15 conjugation, noncanonical residues such as serine or threonine are modified by ubiquitin even though lysine residues are the favored target for ubiquitin conjugation [19]. We identified nine serine residues (Ser-23, Ser-29, Ser-33, Ser-37, Ser-45, Ser-47, Ser-60, Ser-71, and Ser-73) and six threonine residues (Thr-3, Thr-40, Thr-41, Thr-42, Thr-59, and Thr-75) within the 86 aa N-terminus of β-catenin (Figure 3A).


90K Glycoprotein Promotes Degradation of Mutant β -Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus 1 2
Substituting N-terminal lysine residues with arginine or deleting N-terminal serine- or threonine-containing domains from β-catenin. (A) Schematic illustration of the β-catenin deletion mutants and the amino acid sequence of the N-terminus of β-catenin. Lysine (K, red), serine (S, orange), and threonine (T, orange) residues within the N-terminus of β-catenin are shown in colors. Serine and threonine residues known to be targets for GSK-3β phosphorylation are shown in blue. (B-E) Substitution of N-terminal lysine residues with arginine or deleting N-terminal domains containing serine or threonine residues does not affect 90K-induced β-catenin degradation. The β-catenin mutants were transfected into HEK293T cells, which were then treated with either ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), endogenous β-catenin, and actin. Decreases in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the fold changes of relative GFP-β-catenin level compared with actin were depicted as a bar graph below the immunoblot data. Each bar represents mean ± SD for triplicate samples, and number under each gel corresponds to each bar graph. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (**P < .01; ***P < .001). (B) Introduction of arginine mutations does not affect 90K-induced β-catenin degradation. (C) Deleting serine- and threonine-containing domains between Lys-19 and Lys-49 does not affect 90K-induced β-catenin degradation. (D) Deleting serine- and threonine-containing domains beyond Lys-49 does not affect 90K-induced β-catenin degradation. (E) 90K-induced β-catenin degradation occurs if β-catenin harbors either half of its N-terminal domain.
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f0015: Substituting N-terminal lysine residues with arginine or deleting N-terminal serine- or threonine-containing domains from β-catenin. (A) Schematic illustration of the β-catenin deletion mutants and the amino acid sequence of the N-terminus of β-catenin. Lysine (K, red), serine (S, orange), and threonine (T, orange) residues within the N-terminus of β-catenin are shown in colors. Serine and threonine residues known to be targets for GSK-3β phosphorylation are shown in blue. (B-E) Substitution of N-terminal lysine residues with arginine or deleting N-terminal domains containing serine or threonine residues does not affect 90K-induced β-catenin degradation. The β-catenin mutants were transfected into HEK293T cells, which were then treated with either ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), endogenous β-catenin, and actin. Decreases in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the fold changes of relative GFP-β-catenin level compared with actin were depicted as a bar graph below the immunoblot data. Each bar represents mean ± SD for triplicate samples, and number under each gel corresponds to each bar graph. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (**P < .01; ***P < .001). (B) Introduction of arginine mutations does not affect 90K-induced β-catenin degradation. (C) Deleting serine- and threonine-containing domains between Lys-19 and Lys-49 does not affect 90K-induced β-catenin degradation. (D) Deleting serine- and threonine-containing domains beyond Lys-49 does not affect 90K-induced β-catenin degradation. (E) 90K-induced β-catenin degradation occurs if β-catenin harbors either half of its N-terminal domain.
Mentions: Because Herc5 interacts with both FL- and ΔN86-β-catenin, N-terminal β-catenin likely harbors residues directly involved in the conjugation of ISG15. Therefore, to identify the residue(s) in the N-terminus of β-catenin that is(are) responsible for ISG15 conjugation, we compared the N-terminal aa sequence of β-catenin with the sequences published in previous studies reporting the consensus motif required for conjugation of ISG15. ISG15 conjugation occurs on the ε-amine group of lysine residues [17] or on cysteine residues [18]. As shown in Figure 3A, we identified two lysine residues (Lys-19 and Lys-49); however, the 86 aa N-terminus of β-catenin contains no cysteine residues. Although not reported for ISG15 conjugation, noncanonical residues such as serine or threonine are modified by ubiquitin even though lysine residues are the favored target for ubiquitin conjugation [19]. We identified nine serine residues (Ser-23, Ser-29, Ser-33, Ser-37, Ser-45, Ser-47, Ser-60, Ser-71, and Ser-73) and six threonine residues (Thr-3, Thr-40, Thr-41, Thr-42, Thr-59, and Thr-75) within the 86 aa N-terminus of β-catenin (Figure 3A).

View Article: PubMed Central - PubMed

ABSTRACT

&beta;-Catenin is a major transducer of the Wnt signaling pathway, which is aberrantly expressed in colorectal and other cancers. Previously, we showed that &beta;-catenin is downregulated by the 90K glycoprotein via ISGylation-dependent degradation. However, the further mechanisms of &beta;-catenin degradation by 90K-mediated ISGylation pathway were not investigated. This study aimed to identify the &beta;-catenin domain responsible for the action of 90K and to compare the mechanism of 90K on &beta;-catenin degradation with phosphorylation-dependent ubiquitinational degradation of &beta;-catenin. The deletion mutants of &beta;-catenin lacking N- or C-terminal domain or mutating the N-terminal lysine or nonlysine residue were employed to delineate the characteristics of &beta;-catenin degradation by 90K-mediated ISGylation pathway. 90K induced Herc5 and ISG15 expression and reduced &beta;-catenin levels in HeLa and CSC221 cells. The N-terminus of &beta;-catenin is required for 90K-induced &beta;-catenin degradation, but the N-terminus of &beta;-catenin is not essential for interaction with Herc5. However, substituting lysine residues in the N-terminus of &beta;-catenin with arginine or deleting serine or threonine residue containing domains from the N-terminus does not affect 90K-induced &beta;-catenin degradation, indicating that the N-terminal 86 amino acids of &beta;-catenin are crucial for 90K-mediated ISGylation/degradation of &beta;-catenin in which the responsible lysine or nonlysine residues were not identified. Our present results highlight the action of 90K on promoting degradation of mutant &beta;-catenin lacking the phosphorylation sites in the N-terminus. It provides further insights into the discrete pathway downregulating the stabilized &beta;-catenin via acquiring mutations at the serine/threonine residues in the N-terminus.

No MeSH data available.