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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.


N-Terminus of β-catenin is required for 90K-induced β-catenin degradation. (A) 90K/CM induced Herc5 and ISG15 expression and reduced β-catenin levels in HEK293T, HeLa, and CSC221 cells. Cells were treated with either control conditioned medium (ctrl/CM) or 90K/CM for 48 hours, followed by immunoblotting with antibodies against β-catenin, ISG15, Herc5, or actin (loading control). These endogenous signals were measured by densitometry in triplicate experiments, and the fold changes of relative β-catenin level [A(i)], Herc5 level [A(ii)], and ISG15 [A(iii)] level compared with actin were depicted as a bar graph among three cell lines (right side). Each bar represents mean ± SD for triplicate samples. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (***P < .001). (B, C) Deleting the N-terminal 86 amino acids of β-catenin abolished the ability of 90K to degrade β-catenin. GFP-tagged deletion mutants of β-catenin lacking the N-terminus (ΔN86: deletion of aa 1-86), C-terminus (ΔC: deletion of aa 659-781), or both the N- and C-termini (ARM) were used to determine the domain responsible for 90K-induced β-catenin degradation. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the mean fold changes of relative GFP-β-catenin level (GFP/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) The β-catenin deletion 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. Reductions in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The levels of FL- and ΔC-β-catenin decreased in 90K/CM-treated cells, whereas those of ΔN86- and ARM-β-catenin remained constant. For the effect of 90K on FL-β-catenin, another enhanced image is shown as an inset. (C) The β-catenin deletion mutants were transfected into stable 90K-myc expressing HEK293T cells together with either scrambled or 90K-specific siRNA, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), myc (90K-myc), and actin. The levels of FL- and ΔC-β-catenin increased in si-90K transfected cells. The asterisk indicates ARM β-catenin mutants, which are shown after immunoblotting of the membrane with an anti-GFP antibody.
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f0005: N-Terminus of β-catenin is required for 90K-induced β-catenin degradation. (A) 90K/CM induced Herc5 and ISG15 expression and reduced β-catenin levels in HEK293T, HeLa, and CSC221 cells. Cells were treated with either control conditioned medium (ctrl/CM) or 90K/CM for 48 hours, followed by immunoblotting with antibodies against β-catenin, ISG15, Herc5, or actin (loading control). These endogenous signals were measured by densitometry in triplicate experiments, and the fold changes of relative β-catenin level [A(i)], Herc5 level [A(ii)], and ISG15 [A(iii)] level compared with actin were depicted as a bar graph among three cell lines (right side). Each bar represents mean ± SD for triplicate samples. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (***P < .001). (B, C) Deleting the N-terminal 86 amino acids of β-catenin abolished the ability of 90K to degrade β-catenin. GFP-tagged deletion mutants of β-catenin lacking the N-terminus (ΔN86: deletion of aa 1-86), C-terminus (ΔC: deletion of aa 659-781), or both the N- and C-termini (ARM) were used to determine the domain responsible for 90K-induced β-catenin degradation. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the mean fold changes of relative GFP-β-catenin level (GFP/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) The β-catenin deletion 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. Reductions in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The levels of FL- and ΔC-β-catenin decreased in 90K/CM-treated cells, whereas those of ΔN86- and ARM-β-catenin remained constant. For the effect of 90K on FL-β-catenin, another enhanced image is shown as an inset. (C) The β-catenin deletion mutants were transfected into stable 90K-myc expressing HEK293T cells together with either scrambled or 90K-specific siRNA, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), myc (90K-myc), and actin. The levels of FL- and ΔC-β-catenin increased in si-90K transfected cells. The asterisk indicates ARM β-catenin mutants, which are shown after immunoblotting of the membrane with an anti-GFP antibody.

Mentions: We previously reported that glycoprotein 90K suppresses the Wnt/β-catenin signal in colorectal cancer tissues by promoting ISGylational (ISG15-conjugation) degradation of β-catenin [7]. 90K treatment increases expression of ISG15 mRNA in 293T, HCT116, and Caco2 cells and promotes association of the HECT E3 ligase, Herc5, with β-catenin. Here, we examined the effects of 90K on HeLa (cervical cancer) and CSC221 (colorectal adenocarcinoma–enriched cancer stem cell) cells. As shown in Figure 1A, 90K/CM treatment significantly induced Herc5 and ISG15 expression and significantly reduced β-catenin levels in both cell lines, indicating that 90K treatment can promote ISGylational degradation of β-catenin in cervical cancer cells and CRC cells–enriched cancer stem cell, in addition to CRC cells.


90K Glycoprotein Promotes Degradation of Mutant β -Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus 1 2
N-Terminus of β-catenin is required for 90K-induced β-catenin degradation. (A) 90K/CM induced Herc5 and ISG15 expression and reduced β-catenin levels in HEK293T, HeLa, and CSC221 cells. Cells were treated with either control conditioned medium (ctrl/CM) or 90K/CM for 48 hours, followed by immunoblotting with antibodies against β-catenin, ISG15, Herc5, or actin (loading control). These endogenous signals were measured by densitometry in triplicate experiments, and the fold changes of relative β-catenin level [A(i)], Herc5 level [A(ii)], and ISG15 [A(iii)] level compared with actin were depicted as a bar graph among three cell lines (right side). Each bar represents mean ± SD for triplicate samples. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (***P < .001). (B, C) Deleting the N-terminal 86 amino acids of β-catenin abolished the ability of 90K to degrade β-catenin. GFP-tagged deletion mutants of β-catenin lacking the N-terminus (ΔN86: deletion of aa 1-86), C-terminus (ΔC: deletion of aa 659-781), or both the N- and C-termini (ARM) were used to determine the domain responsible for 90K-induced β-catenin degradation. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the mean fold changes of relative GFP-β-catenin level (GFP/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) The β-catenin deletion 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. Reductions in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The levels of FL- and ΔC-β-catenin decreased in 90K/CM-treated cells, whereas those of ΔN86- and ARM-β-catenin remained constant. For the effect of 90K on FL-β-catenin, another enhanced image is shown as an inset. (C) The β-catenin deletion mutants were transfected into stable 90K-myc expressing HEK293T cells together with either scrambled or 90K-specific siRNA, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), myc (90K-myc), and actin. The levels of FL- and ΔC-β-catenin increased in si-90K transfected cells. The asterisk indicates ARM β-catenin mutants, which are shown after immunoblotting of the membrane with an anti-GFP antibody.
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f0005: N-Terminus of β-catenin is required for 90K-induced β-catenin degradation. (A) 90K/CM induced Herc5 and ISG15 expression and reduced β-catenin levels in HEK293T, HeLa, and CSC221 cells. Cells were treated with either control conditioned medium (ctrl/CM) or 90K/CM for 48 hours, followed by immunoblotting with antibodies against β-catenin, ISG15, Herc5, or actin (loading control). These endogenous signals were measured by densitometry in triplicate experiments, and the fold changes of relative β-catenin level [A(i)], Herc5 level [A(ii)], and ISG15 [A(iii)] level compared with actin were depicted as a bar graph among three cell lines (right side). Each bar represents mean ± SD for triplicate samples. The asterisk (*) indicates a significant difference between ctrl/CM and 90K/CM groups (***P < .001). (B, C) Deleting the N-terminal 86 amino acids of β-catenin abolished the ability of 90K to degrade β-catenin. GFP-tagged deletion mutants of β-catenin lacking the N-terminus (ΔN86: deletion of aa 1-86), C-terminus (ΔC: deletion of aa 659-781), or both the N- and C-termini (ARM) were used to determine the domain responsible for 90K-induced β-catenin degradation. The exogenous β-catenin levels (GFP-β-catenin) were measured by densitometry in triplicate experiments, and the mean fold changes of relative GFP-β-catenin level (GFP/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) The β-catenin deletion 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. Reductions in endogenous β-catenin levels are shown as a positive control for the effects of 90K. The levels of FL- and ΔC-β-catenin decreased in 90K/CM-treated cells, whereas those of ΔN86- and ARM-β-catenin remained constant. For the effect of 90K on FL-β-catenin, another enhanced image is shown as an inset. (C) The β-catenin deletion mutants were transfected into stable 90K-myc expressing HEK293T cells together with either scrambled or 90K-specific siRNA, followed by immunoblotting with antibodies against GFP (exogenous β-catenin), myc (90K-myc), and actin. The levels of FL- and ΔC-β-catenin increased in si-90K transfected cells. The asterisk indicates ARM β-catenin mutants, which are shown after immunoblotting of the membrane with an anti-GFP antibody.
Mentions: We previously reported that glycoprotein 90K suppresses the Wnt/β-catenin signal in colorectal cancer tissues by promoting ISGylational (ISG15-conjugation) degradation of β-catenin [7]. 90K treatment increases expression of ISG15 mRNA in 293T, HCT116, and Caco2 cells and promotes association of the HECT E3 ligase, Herc5, with β-catenin. Here, we examined the effects of 90K on HeLa (cervical cancer) and CSC221 (colorectal adenocarcinoma–enriched cancer stem cell) cells. As shown in Figure 1A, 90K/CM treatment significantly induced Herc5 and ISG15 expression and significantly reduced β-catenin levels in both cell lines, indicating that 90K treatment can promote ISGylational degradation of β-catenin in cervical cancer cells and CRC cells–enriched cancer stem cell, in addition to CRC cells.

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.