Biology subjects

Gybel, T.

Publications and source records attributed to Gybel, T..

5 recordsLinked to original sources

bioRxiv2026

Molecular mechanisms behind the functional (non)redundancy of CK1 paralogs in the Wnt pathway

The casein kinase 1 (CK1) family of serine/threonine protein kinases consists of seven isoforms in humans. CK1 family members are important regulators of the Wnt/{beta}-catenin signaling pathway. Using a comprehensive panel of CRISPR/Cas9-generated knockout cell lines, we demonstrated the opposing roles of endogenous CK1 (negative, via phosphorylation of {beta}-catenin in the destruction complex) and CK1{delta}/{epsilon} (positive, via phosphorylation of DVL in the signalosome), while no phenotype was observed for CK1{gamma}1/2/3 triple knockout cells. Using in vitro kinase assays and TurboID-based interactomics, we revealed that this functional divergence between CK1 and CK1{epsilon} is not due to the intrinsically different capacity to phosphorylate {beta}-catenin or DVL but rather due to different affinities for the destruction complex and signalosome in the cellular environment. Through functional analysis of CK1-CK1{epsilon} chimeras containing both N-terminal and C-terminal domain swaps, we identified the N-terminal lobe of CK1 and the C-terminus of CK1{epsilon} as determinants mediating increased affinity towards the degradasome and signalosome, respectively. We further show that the CK1 N-lobe not only drives cellular activity toward {beta}-catenin but also underlies the CK1-specific interaction with the scaffolding protein SACK1G (also known as FAM83G and PAWS1). Additionally, despite clearly distinct physiological roles of CK1 and CK1{delta}/{epsilon}, we provide evidence that, in the absence of CK1, CK1{delta} and CK1{epsilon} can physically and functionally substitute for CK1 in the {beta}-catenin destruction complex. This rewires CK1{delta} and CK1{epsilon} as negative regulators acting via phosphorylation of {beta}-catenin in the destruction complex. These findings resolve prior contradictions by (i) clarifying context-dependent CK1 roles, (ii) identifying mechanistic determinants navigating CK1 and CK1{epsilon} to different substrates, and (iii) defining the limitations of these affinity-based subcellular distributions that become apparent especially in the physical absence of the physiological, high-affinity kinase. An important implication of our findings is the identification of a mechanism that changes the ultimate outcome of CK1{delta}/{epsilon} inhibitor treatment from Wnt/{beta}-catenin pathway inhibition to its robust activation.

cell biology↗

bioRxiv2026

NOTCH1-specific phosphorylation of S1970 by Casein Kinase 1 is required for NOTCH1 transcriptional competence and signaling activity in vivo

The Notch and Wnt/{beta}-catenin signaling pathways are essential regulators for cell-fate decisions, cellular patterning, and tissue homeostasis. Multiple studies point to their orchestrated role during development, but the molecular mechanism of the protein-protein crosstalk is largely unknown. Here, after screening effects of Wnt/{beta}-catenin component loss on NOTCH1 protein, we identify Casein Kinase 1 (CK1) as a positive regulator of NOTCH1 activity in vitro and in vivo. We demonstrate that CK1 associates with NOTCH1 and that its kinase activity is required to sustain Notch-driven transcription. Using UltraID proximity-assay, we revealed that CK1 is required for the NOTCH1 interactivity with transporter proteins, and MAML1 both prior and after ligand-induced activation. Combining structural modelling, NMR, and mass spectrometry, we identified Serine 1970 (S1970) as a previously unreported residue within the Notch1 Intracellular Domain (N1ICD) essential for its signaling competence. Our modeling predicts that the phosphorylation of S1970 facilitates an intra-domain conformational switch with R1937 and R1962 residues altering the assembly of the N1ICD-MAML1-RBPJk transcriptional complex. Finally, we demonstrate the biological significance of N1ICD S1970 in vivo using Xenopus laevis axis-duplication rescue assay. Our results establish CK1 as a key positive mediator of the Notch receptor transcriptional activity.

cell biology↗

bioRxiv2025

A Wnt-induced conformational phospho-switch in DVL3 controls interaction with Frizzled receptors and Wnt/β-catenin signaling

When Wnt ligands bind to Frizzled (FZD) receptors, Dishevelled protein (DVL) gets multiphosphorylated by Casein kinase 1 (CK1). Although, it is well known that DVL phosphorylation relays Wnt signals from receptors to downstream effectors, any mechanistic aspects of DVL function related to phosphorylation remain unresolved. Here, we uncovered a Wnt-induced DVL phospho-switch which is mutually exclusive with FZD engagement. CK1 multiphosphorylation changes dramatically the bulk electrostatics to promote DVL intramolecular coupling between the DEP domain and the adjacent disordered region. A panel of phospho-switch mutants demonstrated a switch-like coupling at the molecular level when a charge threshold is reached. Charge accumulation proximal to DEP proved to be a key functional event required, but not sufficient, for Wnt/{beta}-catenin signaling. Interestingly, the charge-dependent switch-like character of DVL controlled its association with FZD. By integrating findings at different levels, we propose a universal mechanism in which Wnt-induced DVL conformational phospho-switch outcompetes FZD binding and triggers DVL detachment from FZD.

molecular biology↗

bioRxiv2022

Dual Specificity Phosphatase 7 Drives the Formation of Cardiac Mesoderm in Mouse Embryonic Stem Cells

Dual specificity phosphatase 7 (DUSP7) is a protein belonging to a broad group of phosphatases that 14 can dephosphorylate phosphoserine/phosphothreonine as well as phosphotyrosine residues within the 15 same substrate. DUSP7 has been linked to the negative regulation of mitogen activated protein kinases 16 (MAPK), and in particular to the regulation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). 17 MAPKs play an important role in embryonic development, where their duration, magnitude, and 18 spatiotemporal activity must be strictly controlled by other proteins, among others by DUSPs. In this 19 study, we focused on the effect of DUSP7 depletion on the in vitro differentiation of mouse embryonic 20 stem (ES) cells. We showed that even though DUSP7 knock-out ES cells do retain some of their basic 21 characteristics, when it comes to differentiation, they preferentially differentiate towards neural cells, 22 while the formation of early cardiac mesoderm is repressed. Therefore, our data indicate that DUSP7 23 is necessary for the correct formation of neuroectoderm and cardiac mesoderm during the in vitro 24 differentiation of ES cells.

cell biology↗

bioRxiv2021

RNF43 inhibits WNT5A driven signaling and suppresses melanoma invasion

RNF43 is a E3 ubiquitin ligase and known negative regulator of WNT/{beta}-catenin signaling. We demonstrate that RNF43 is also regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1 and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor. This activity of RNF43 is physiologically relevant and blocks pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E inhibitor. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

cancer biology↗