Biology subjects

Tripsianes, K.

Publications and source records attributed to Tripsianes, K..

7 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↗

bioRxiv2024

Cell attachment and tail contraction of S. aureus phage phi812

Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.

microbiology↗

bioRxiv2023

A new mechanism of posttranslational polyglutamylation regulates phase separation and signaling of the Wnt pathway protein Dishevelled.

Polyglutamylation is a reversible post-translational modification that is catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Disheveled 3 (DVL3), thereby changing the interactome of DVL3, as well as it increases its capacity to get phosphorylated, to undergo liquid-liquid phase separation (LLPS), and to act in the non-canonical Wnt pathway. Both carboxyterminal polyglutamylation and the resulting reduction in LLPS capacity of DVL3 were reverted by the deglutamylating enzyme CCP6, which demonstrates the causal relationship between TTLL11-mediated polyglutamylation and LLPS. We thus discovered a novel type of posttranslational modification, which significantly broadens the range of proteins that can be modified by polyglutamylation and provides the first evidence that polyglutamylation can act as a regulator of protein LLPS.

cell biology↗

bioRxiv2022

Structural basis for the mechanism and antagonism of receptor signaling mediated by Interleukin-9 (IL-9)

Interleukin-9 (IL-9) is the hallmark cytokine in Th9 immunity and is also central to Innate Lymphocyte 2 (ILC2) biology. Furthermore, receptor signaling mediated by IL-9 has been linked to inflammatory and autoimmune diseases, and cancer. Despite its functional pleiotropy, the structure-function landscape of IL-9 had remained enigmatic. Here, we show via a combination of X-ray crystallography and NMR that human IL-9 adopts a helical bundle fold with unprecedented structural features among helical cytokines, including five disulfide bridges. Binding of IL-9 to the interdomain junction of IL-9R results in marked structural changes on the opposite face of IL-9 that prime the binary complex for recruiting the common gamma chain ({gamma}c) for signaling. Surprisingly, this tripartite cytokine-receptor assembly displays a markedly lower affinity than the IL-9: IL-9R complex, which we trace to distinct features of IL-9R that might destabilize the ternary complex. Furthermore, we developed monoclonal antibodies that antagonize IL-9 activity by sterically competing for the binding footprint of IL-9R. Collectively, we here provide a structural and mechanistic blueprint to facilitate interrogation and modulation of pleiotropic signaling outputs of IL-9 in physiology and disease.

immunology↗

bioRxiv2020

Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding.

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Whereas one EH domain binds negatively charged PI(4,5)P2 lipids, unbiased peptidome profiling by mass-spectrometry revealed that the other EH domain interacts with the double N-terminal NPF motif of a novel TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 have in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

plant biology↗