The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance.
WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1...
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Public Library of Science (PLoS)
2025-05-01
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| Series: | PLoS Genetics |
| Online Access: | https://doi.org/10.1371/journal.pgen.1011677 |
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| author | Joseph K McKenna Yalan Wu Praveen Sonkusre Caleb K Sinclear Raj Chari Andres M Lebensohn |
| author_facet | Joseph K McKenna Yalan Wu Praveen Sonkusre Caleb K Sinclear Raj Chari Andres M Lebensohn |
| author_sort | Joseph K McKenna |
| collection | DOAJ |
| description | WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway. |
| format | Article |
| id | doaj-art-5ef0b2dae7cf48da9d20981c222cc26a |
| institution | DOAJ |
| issn | 1553-7390 1553-7404 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Public Library of Science (PLoS) |
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| spelling | doaj-art-5ef0b2dae7cf48da9d20981c222cc26a2025-08-20T02:39:44ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042025-05-01215e101167710.1371/journal.pgen.1011677The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance.Joseph K McKennaYalan WuPraveen SonkusreCaleb K SinclearRaj ChariAndres M LebensohnWNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.https://doi.org/10.1371/journal.pgen.1011677 |
| spellingShingle | Joseph K McKenna Yalan Wu Praveen Sonkusre Caleb K Sinclear Raj Chari Andres M Lebensohn The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. PLoS Genetics |
| title | The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. |
| title_full | The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. |
| title_fullStr | The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. |
| title_full_unstemmed | The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. |
| title_short | The ubiquitin ligase HUWE1 enhances WNT signaling by antagonizing destruction complex-mediated β-catenin degradation and through a mechanism independent of changes in β-catenin abundance. |
| title_sort | ubiquitin ligase huwe1 enhances wnt signaling by antagonizing destruction complex mediated β catenin degradation and through a mechanism independent of changes in β catenin abundance |
| url | https://doi.org/10.1371/journal.pgen.1011677 |
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