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90K Glycoprotein Promotes Degradation of Mutant ホイ -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.


Related in: MedlinePlus

ホイ-Catenin degradation by 90K is Herc5 dependent, and ホ年86-ホイ-catenin interacts with Herc5. (A) Herc5 silencing diminishes 90K-induced ホイ-catenin degradation. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against endogenous ホイ-catenin, Herc5, and actin. The endogenous ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative ホイ-catenin level (ホイ-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) Both FL-ホイ-catenin and ホ年86-ホイ-catenin interact with Herc5. HEK293T cells transfected with FL or ホ年86 deletion mutant of ホイ-catenin together with Ube1L and UbcH8 were treated with either ctrl/CM or 90K/CM, followed by immunoprecipitation with an anti-GFP antibody. Resolved bound proteins were then immunoblotted with antibodies against Herc5 and ホイ-catenin. Total cell lysates were immunoblotted with the corresponding antibodies to show the input amount of each protein. The immunoprecipitated Herc5 and ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes (Herc5/ホイ-ctn) between ctrl/CM and 90K/CM groups were indicated below each gel lane.
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f0010: ホイ-Catenin degradation by 90K is Herc5 dependent, and ホ年86-ホイ-catenin interacts with Herc5. (A) Herc5 silencing diminishes 90K-induced ホイ-catenin degradation. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against endogenous ホイ-catenin, Herc5, and actin. The endogenous ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative ホイ-catenin level (ホイ-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) Both FL-ホイ-catenin and ホ年86-ホイ-catenin interact with Herc5. HEK293T cells transfected with FL or ホ年86 deletion mutant of ホイ-catenin together with Ube1L and UbcH8 were treated with either ctrl/CM or 90K/CM, followed by immunoprecipitation with an anti-GFP antibody. Resolved bound proteins were then immunoblotted with antibodies against Herc5 and ホイ-catenin. Total cell lysates were immunoblotted with the corresponding antibodies to show the input amount of each protein. The immunoprecipitated Herc5 and ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes (Herc5/ホイ-ctn) between ctrl/CM and 90K/CM groups were indicated below each gel lane.

Mentions: Because deletion of the N-terminal 86 aa of ホイ-catenin completely abolished the effect of 90K, the N-terminus may either contain residue(s) required for ISG15-conjugation or play a crucial role in the association with ISG15 E3 ligase. As shown in Figure 2A, Herc5 is essential for 90K-induced ホイ-catenin degradation. To determine whether the N-terminus of ホイ-catenin is required for the interaction with Herc5 (the E3 ligase required for ISG15-conjugation to ホイ-catenin), we performed an immunoprecipitation assay to measure the interaction between Herc5 and full-length (FL) or ホ年86 mutants of ホイ-catenin. To rule out the involvement of unexpected possibility in mediating Herc5 interaction, ISG15 E1 and E2 enzyme, Ube1L and UbcH8, respectively, were overexpressed. We observed a clear interaction between Herc5 and ホ年86-ホイ-catenin (Figure 2). These results suggest that the N-terminal 86 aa of ホイ-catenin are not essential for its interaction with the E3 ligase, Herc5.


90K Glycoprotein Promotes Degradation of Mutant ホイ -Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus 1 2
ホイ-Catenin degradation by 90K is Herc5 dependent, and ホ年86-ホイ-catenin interacts with Herc5. (A) Herc5 silencing diminishes 90K-induced ホイ-catenin degradation. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against endogenous ホイ-catenin, Herc5, and actin. The endogenous ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative ホイ-catenin level (ホイ-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) Both FL-ホイ-catenin and ホ年86-ホイ-catenin interact with Herc5. HEK293T cells transfected with FL or ホ年86 deletion mutant of ホイ-catenin together with Ube1L and UbcH8 were treated with either ctrl/CM or 90K/CM, followed by immunoprecipitation with an anti-GFP antibody. Resolved bound proteins were then immunoblotted with antibodies against Herc5 and ホイ-catenin. Total cell lysates were immunoblotted with the corresponding antibodies to show the input amount of each protein. The immunoprecipitated Herc5 and ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes (Herc5/ホイ-ctn) between ctrl/CM and 90K/CM groups were indicated below each gel lane.
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f0010: ホイ-Catenin degradation by 90K is Herc5 dependent, and ホ年86-ホイ-catenin interacts with Herc5. (A) Herc5 silencing diminishes 90K-induced ホイ-catenin degradation. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against endogenous ホイ-catenin, Herc5, and actin. The endogenous ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative ホイ-catenin level (ホイ-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane. (B) Both FL-ホイ-catenin and ホ年86-ホイ-catenin interact with Herc5. HEK293T cells transfected with FL or ホ年86 deletion mutant of ホイ-catenin together with Ube1L and UbcH8 were treated with either ctrl/CM or 90K/CM, followed by immunoprecipitation with an anti-GFP antibody. Resolved bound proteins were then immunoblotted with antibodies against Herc5 and ホイ-catenin. Total cell lysates were immunoblotted with the corresponding antibodies to show the input amount of each protein. The immunoprecipitated Herc5 and ホイ-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes (Herc5/ホイ-ctn) between ctrl/CM and 90K/CM groups were indicated below each gel lane.
Mentions: Because deletion of the N-terminal 86 aa of ホイ-catenin completely abolished the effect of 90K, the N-terminus may either contain residue(s) required for ISG15-conjugation or play a crucial role in the association with ISG15 E3 ligase. As shown in Figure 2A, Herc5 is essential for 90K-induced ホイ-catenin degradation. To determine whether the N-terminus of ホイ-catenin is required for the interaction with Herc5 (the E3 ligase required for ISG15-conjugation to ホイ-catenin), we performed an immunoprecipitation assay to measure the interaction between Herc5 and full-length (FL) or ホ年86 mutants of ホイ-catenin. To rule out the involvement of unexpected possibility in mediating Herc5 interaction, ISG15 E1 and E2 enzyme, Ube1L and UbcH8, respectively, were overexpressed. We observed a clear interaction between Herc5 and ホ年86-ホイ-catenin (Figure 2). These results suggest that the N-terminal 86 aa of ホイ-catenin are not essential for its interaction with the E3 ligase, Herc5.

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.


Related in: MedlinePlus