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.
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Basal pancreatic ductal adenocarcinoma (PDAC) has the worst overall survival and is the only subtype that serves as an independent poor prognostic factor. We identify elevated levels of LIN28B and its downstream target, HMGA2, in basal PDAC. Notably, LIN28B significantly accelerates KRAS-driven PDAC progression in a mouse model. Here, we show that HMGA2 promotes basal PDAC pathogenesis by enhancing mRNA translation downstream of LIN28B. Mechanistically, HMGA2 suppresses leucine carboxyl methyltransferase 1 (LCMT1) at the chromatin level, reducing PP2A methylation and activity. This leads to increased phosphorylation of S6K and eIF4B, boosting mRNA translation. Additionally, HMGA2 downregulates B56α (PPP2R5A), disrupting functional PP2A holoenzyme assembly and further sustaining phosphorylated S6K levels. Impaired PP2A function mimics HMGA2's effects, reinforcing increased mRNA translation and basal lineage features. This work uncovers a critical link between the LIN28B/HMGA2 axis, protein synthesis, and PDAC lineage specificity via LCMT1-mediated PP2A methylation and B56α-PP2A disruption.
© 2025. The Author(s).

The Wingless/Int-1 (WNT) signaling network is essential to orchestrate central physiological processes such as embryonic development and tissue homeostasis. In the currently held tenet, WNT/β-catenin signaling is initiated by WNT-induced recruitment of Frizzleds (FZDs) and LRP5/6 followed by the formation of a multiprotein signalosome complex. Here, we use bioluminescence resonance energy transfer (BRET) to show that different WNT paralogs dynamically trigger FZD-LRP6 association. While WNT-induced receptor interaction was independent of C-terminal LRP6 phosphorylation, it was allosterically modulated by binding of the phosphoprotein Dishevelled (DVL) to FZD. WNT-16B emerged as a ligand of particular interest, as it efficiently promoted FZD-LRP6 association but, unlike WNT-3A, did not lead to WNT/β-catenin signaling. Transcriptomic analysis further revealed distinct transcriptional fingerprints of WNT-3A and WNT-16B stimulation in HEK293 cells. Additionally, single-molecule tracking demonstrated that, despite increasing FZD5 and LRP6 confinement, WNT-16B stimulation did not result in formation of higher-order receptor clusters, in contrast to WNT-3A. Our results suggest that FZD-WNT-LRP5/6 complex formation alone is not sufficient for the initiation of WNT/β-catenin signaling. Instead, we propose a two-step model, where initial ligand-induced FZD-LRP6 association must be followed by receptor clustering into higher-order complexes and subsequent phosphorylation of LRP6 for efficient activation of WNT/β-catenin signaling.
© 2025. The Author(s).

The metabolic, immune, and endocrine systems show profound seasonal changes in animals, including humans. In addition, morbidity from cardiovascular and psychiatric diseases is more severe and mortality rate is higher in winter. However, their molecular mechanisms remain unknown. Here we report the seasonal transcriptome of 80 tissues collected over 1 year from male and female rhesus macaques kept in a semi-natural outdoor environment. We find seasonal changes in plasma metabolites and hormones. Transcriptome analysis identifies sex differences in seasonally oscillating genes (SOGs) in all tissues studied, and we generate the web database 'Non-Human Primate Seasonal Transcriptome Atlas (NHPSTA).' Transcriptional regulatory network analysis, siRNA knockdown, and mutant mouse analyses reveal regulation of SOGs by GA-binding protein (GABP). We also demonstrate seasonal oscillations in the expression of disease risk factor genes and drug interacting genes. NHPSTA provides a molecular resource for seasonally regulated physiology and targets for therapeutic interventions for seasonally regulated diseases.
© 2025. The Author(s).

Abstract VEcadherin is a major component of endothelial adherens junctions and pivotal to the regulation of vascular barrier integrity. Whereas two phosphorylation sites of VEcadherin (Y685 and Y731) are known to be relevant for the regulation of endothelial junctions in vivo, several others were suggested to be relevant based on in vitro studies. Here, we analyze for two of these, serine 665 (S665) and tyrosine 658 (Y658), whether they are relevant for the induction of vascular permeability in vivo. To this end, we generated and characterized two point-mutated VEcadherin knockin mouse lines where either S665 was replaced by valine (S665V) or Y658 by phenylalanine (Y658F). We found that the induction of vascular permeability by histamine or VEGF in the skin was clearly reduced in S665V mice, whereas Y658F mice showed a normal increase of permeability. In line with this, we found that histamine-induced endocytosis was impaired for the VEcadherin-S665V mutant, but not for the Y658F mutant. Comparing the regulation of VEcadherin phosphorylation at S665, Y658 and Y685, we found that only phosphorylation of S665 and Y685 were strongly induced by inflammatory mediators, while phosphorylation of Y658 increased weakly. Interestingly, phosphorylation of S665 and Y685 occurred with different kinetics, but independent of each other. Collectively, our results demonstrate that Y658 is irrelevant for vascular leak formation under inflammatory conditions and establish S665 of VEcadherin as an important phosphorylation site regulating the induction of endothelial permeability in vivo.