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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.


Related in: MedlinePlus

Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (A, B) 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. (A) Deleting N-terminal 19-75 aa domain of β-catenin does not affect 90K-induced β-catenin degradation. (B) Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (C) The degradations of β-catenin mutants are dependent on Herc5. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then transfected with β-catenin mutants. Then, cells were treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against β-catenin, Herc5, and actin. The exogenous and endogenous β-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative β-catenin level (exo. and endo. β-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane.
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f0020: Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (A, B) 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. (A) Deleting N-terminal 19-75 aa domain of β-catenin does not affect 90K-induced β-catenin degradation. (B) Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (C) The degradations of β-catenin mutants are dependent on Herc5. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then transfected with β-catenin mutants. Then, cells were treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against β-catenin, Herc5, and actin. The exogenous and endogenous β-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative β-catenin level (exo. and endo. β-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane.

Mentions: Next, we assumed that 90K-induced β-catenin degradation would occur if there was a lysine or serine residue at the N-terminus of β-catenin and asked whether the N-terminal 19-75 aa domain, which contains mostly lysine, serine, and threonine residues, is required for 90K-induced β-catenin degradation. To examine this, we generated deletion mutants lacking 19-75 aa (Δ19-75), Δ19-74 (harboring Thr-75), and Δ20-75 (harboring Lys-19). All the deletion mutants were degraded by 90K (Figure 4A). These mutants contained the Thr-3 residue; therefore, we introduced the T3A mutation. Surprisingly, all the β-catenin mutants were still degraded by 90K (Figure 4B). Thus, even though the N-terminus of β-catenin plays an essential role in 90K-induced β-catenin degradation, the specific residues responsible for this activity remain unknown. At least from Figure 4C, the results suggest that degradations of these β-catenin mutants are dependent on Herc5 and implicate that β-catenin N-terminus is not responsible for Herc5 interaction.


90K Glycoprotein Promotes Degradation of Mutant β -Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus 1 2
Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (A, B) 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. (A) Deleting N-terminal 19-75 aa domain of β-catenin does not affect 90K-induced β-catenin degradation. (B) Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (C) The degradations of β-catenin mutants are dependent on Herc5. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then transfected with β-catenin mutants. Then, cells were treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against β-catenin, Herc5, and actin. The exogenous and endogenous β-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative β-catenin level (exo. and endo. β-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane.
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f0020: Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (A, B) 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. (A) Deleting N-terminal 19-75 aa domain of β-catenin does not affect 90K-induced β-catenin degradation. (B) Removing all lysine, serine, and threonine residue-containing domains from the N-terminus of β-catenin does not affect 90K-induced β-catenin degradation. (C) The degradations of β-catenin mutants are dependent on Herc5. HEK293T cells transfected with either scramble or Herc5 siRNA were incubated for 24 hours and then transfected with β-catenin mutants. Then, cells were treated with ctrl/CM or 90K/CM, followed by immunoblotting with antibodies against β-catenin, Herc5, and actin. The exogenous and endogenous β-catenin levels were measured by densitometry in triplicate experiments, and the mean fold changes of relative β-catenin level (exo. and endo. β-ctn/actin) compared with actin between ctrl/CM and 90K/CM groups were indicated below each gel lane.
Mentions: Next, we assumed that 90K-induced β-catenin degradation would occur if there was a lysine or serine residue at the N-terminus of β-catenin and asked whether the N-terminal 19-75 aa domain, which contains mostly lysine, serine, and threonine residues, is required for 90K-induced β-catenin degradation. To examine this, we generated deletion mutants lacking 19-75 aa (Δ19-75), Δ19-74 (harboring Thr-75), and Δ20-75 (harboring Lys-19). All the deletion mutants were degraded by 90K (Figure 4A). These mutants contained the Thr-3 residue; therefore, we introduced the T3A mutation. Surprisingly, all the β-catenin mutants were still degraded by 90K (Figure 4B). Thus, even though the N-terminus of β-catenin plays an essential role in 90K-induced β-catenin degradation, the specific residues responsible for this activity remain unknown. At least from Figure 4C, the results suggest that degradations of these β-catenin mutants are dependent on Herc5 and implicate that β-catenin N-terminus is not responsible for Herc5 interaction.

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