Biology subjects

Ikura, M.

Publications and source records attributed to Ikura, M..

8 recordsLinked to original sources

bioRxiv2025

eIF2B Selectively Anchors and Activates Mutant KRAS

Much is known about how RAS oncoproteins regulate mRNA translation factors, but the reverse relationship, how translation factors influence RAS activity, has remained largely unexplored. At the plasma membrane (PM), Son of Sevenless (SOS) acts as the canonical guanine nucleotide exchange factor (GEF) for RAS proteins, yet mechanisms governing its specificity for individual RAS isoforms remain unknown. Here, we show that the translation initiation factor eIF2B, best known for its GEF function in translation initiation, forms a distinct complex with SOS and mutant KRAS at the PM, but not with other oncogenic RAS variants. Mechanistically, eIF2B acts as an allosteric regulator of SOS, selectively enhancing GDP-GTP exchange on mutant KRAS. This specificity arises from the translational activity of eIF2B, which upregulates glycosphingolipid (GSL) biosynthesis to remodel PM lipids and preferentially anchor mutant KRAS. Together, our results uncover an unexpected moonlighting function of eIF2B: acting both as a direct activator of SOS and as a regulator of GSL pathway that shapes the membrane landscape, both required for mutant KRAS activation. These insights redefine our understanding of eIF2B and mutant KRAS functions in cancer and have profound implications for KRAS-driven oncogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/686860v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f50934org.highwire.dtl.DTLVardef@2f55b9org.highwire.dtl.DTLVardef@1a51e39org.highwire.dtl.DTLVardef@1634aac_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIeIF2B interacts with mutant KRAS and SOS at the plasma membrane (PM). C_LIO_LIThe eIF2B:SOS complex promotes the GTP-bound active state of mutant KRAS. C_LIO_LIeIF2B enhances the translation of B4GALT5 mRNA, encoding a key enzyme of glycosphingolipid (GSL) biosynthesis. C_LIO_LIUpregulation of the GSL metabolites, ganglioside GM3 and sulfatide SM4, remodels PM lipid composition to facilitate eIF2B:SOS:KRAS complex formation and mutant KRAS nanoclustering. C_LIO_LIThrough its interaction with SOS and stimulation of GSL synthesis, eIF2B selectively activates mutant KRAS at the PM among RAS isoforms. C_LIO_LIeIF2B is required for the development of mKRAS-driven lung adenocarcinoma in mice. C_LIO_LIeIF2B is a marker of poor prognosis in mutant KRAS-driven cancers. C_LI

cancer biology↗

bioRxiv2025

α-catenin phosphorylation is actomyosin-sensitive and required for epithelial barrier functions through Afadin

Zonula adherens junctions (zAJ) are spatially proximal to tight junctions (TJ), in a superstructure known as the apical junctional complex (AJC). A key component of the AJC is a circumferential ring of filamentous (F)-actin, but how actomyosin contractility drives AJC structure and epithelial barrier function is incompletely understood. Here, we show that a central mechanosensitive component of zAJ, -catenin (-cat), undergoes force-dependent phosphorylation in an unstructured linker region. This modification in turn primes the -cat mechanosensitive Middle-region for effector-binding. We credential Afadin, a multi-domain TJ/AJ scaffold protein, as mechano-chemical binding partner of -cat, identifying residues in -cat required for this interaction. -cat phosphorylation and Afadin-binding are required for their co-enrichment at zAJ and epithelial barrier function. A mouse model that prevents -cat phosphorylation is particularly detrimental to post-natal brain development. These data support a stepwise model where -cat integrates mechanical and chemical signals to progressively promote zAJ enrichment, effector recruitment and epithelial barrier function.

cell biology↗

bioRxiv2025

CD33 and Clusterin Interact Biophysically and Genetically to Modulate Alzheimer Risk

We report the results of structural, functional and genetic studies on the CD33 sialic acid- binding receptor that reveal how non-coding variants in CD33 alter risk for Alzheimers disease (AD). The full-length CD33M isoform, whose expression is upregulated by non-coding AD-risk alleles, preferentially forms dimers at the cell surface, where they interact with AD-related proteins (clusterin and A{beta}). This interaction induces CD33M inhibitory signalling and downregulates protective microglial functions including phagocytic removal of amyloid plaques. Human brain expression quantitative trait loci (eQTL) and causal mediation analyses confirm that quantitative interactions between CLU and CD33 genotypes modulate AD phenotypes and suggest that genotypes at these loci might be used to personalise future therapeutic approaches. Our work also highlights several other unexpected aspects of CD33 biology, including a soluble shed extracellular fragment of CD33M and a similar soluble secreted product arising from a truncating mutation in the CD33 extracellular domain (CD33M{Delta}4bp).

neuroscience↗

bioRxiv2025

Erythrocytosis-inducing PHD2 mutations implicate biological role for N-terminal prolyl-hydroxylation in HIF1α oxygen-dependent degradation domain

Mutations in EGLN1, the gene encoding for hypoxia-inducible factor (HIF) prolyl-4-hydroxylase 2 (PHD2), cause erythrocytosis and in rare cases the development of neuroendocrine tumors. In the presence of oxygen, PHD2 hydroxylates one or both conserved prolines in the oxygen-dependent degradation domain (ODD) of HIF subunits, sufficiently marking HIF for binding and ubiquitylation via the von Hippel-Lindau (VHL) tumor suppressor protein-containing E3 ubiquitin ligase and subsequent degradation by the 26S proteasome. However, prolyl-hydroxylation in the C-terminal ODD appears to be the predominant and sufficient event in triggering the oxygen-dependent destruction of HIF, rendering the biological significance of N-terminal ODD proline unclear. Here, we examined 7 disease-associated EGLN1 mutations scattered across the catalytic core and show definitively that all PHD2 mutants have a structural and/or catalytic activity defect as measured by time-resolved nuclear magnetic resonance. Notably, we identified one of the PHD2 mutants, P317R, to retain comparably wild-type capacity to hydroxylate the predominant proline in the C-terminal ODD but had uniquely compromised ability to hydroxylate the N-terminal ODD proline. These findings support the notion that deregulation of HIF ultimately underlies PHD2-driven erythrocytosis and challenge the currently held uncertainty that the N-terminal ODD prolyl-hydroxylation event is dispensable in normal hypoxic signaling pathway.

biochemistry↗

bioRxiv2023

α-catenin mechanosensitivity as a route to cytokinesis failure through sequestration of LZTS2

Epithelial cells can become polyploid upon tissue injury, but mechanosensitive cues that trigger this state are poorly understood. Using -catenin (-cat) knock-out Madin Darby Canine Kidney (MDCK) cells reconstituted with wild-type and mutant forms of -cat as a model system, we find that an established -cat actin-binding domain unfolding mutant designed to reduce force-sensitive binding to F-actin (-cat-H0-FABD+) can promote cytokinesis failure, particularly along epithelial wound-fronts. Enhanced -cat coupling to cortical actin is neither sufficient nor mitotic cell-autonomous for cytokinesis failure, but critically requires the mechanosensitive Middle-domain (M1-M2-M3) and neighboring cells. Disease relevant -cat M-domain missense mutations known to cause a form of retinal pattern dystrophy (-cat E307K or L436P) are associated with elevated binucleation rates via cytokinesis failure. Similar binucleation rates are seen in cells expressing an -cat salt-bridge destabilizing mutant (R551A) designed to promote M2-M3 domain unfurling at lower force thresholds. Since binucleation is strongly enhanced by removal of the M1 as opposed to M2-M3 domains, cytokinetic fidelity is most sensitive to -cat M2-M3 domain opening. To identify -cat conformation-dependent proximity partners that contribute to cytokinesis, we used a biotin-ligase approach to distinguished proximity partners that show enhanced recruitment upon -cat M-domain unfurling (R551A). We identified Leucine Zipper Tumor Suppressor 2 (LZTS2), an abscission factor previously implicated in cytokinesis. We confirm that LZTS2 enriches at the midbody, but discover it also localizes to tight and tricellular junctions. LZTS2 knock-down promotes binucleation in both MDCK and Retinal Pigmented Epithelial (RPE) cells. -cat mutants with persistent M2-M3 domain opening showed elevated junctional enrichment of LZTS2 from the cytosol compared -cat wild-type cells. These data implicate LZTS2 as a mechanosensitive effector of -cat that is critical for cytokinetic fidelity. This model rationalizes how persistent mechano-activation of -cat may drive tension-induced polyploidization of epithelia post-injury and suggests an underlying mechanism for how pathogenic -cat mutations drive macular dystrophy.

cell biology↗

bioRxiv2021

Regulation of GTPase function by autophosphorylation

A unifying feature of the RAS superfamily is a functionally conserved GTPase cycle that proteins use to transition between active and inactive states. Here, we demonstrate that active site autophosphorylation of some small GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, thus altering the on/off switch that forms the basis for their signaling functions. Using x-ray crystallography, nuclear magnetic resonance spectroscopy, biolayer interferometry binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II domain and that autophosphorylation promotes nucleotide exchange by opening of the active site and extraction of the stabilizing Mg. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from the non-phosphorylated counterpart.

biochemistry↗

bioRxiv2020

p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile -motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation. Summary statementp.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

cell biology↗

bioRxiv2018

Plasma redox imbalance caused by albumin oxidation promotes lung-predominant NETosis and pulmonary cancer metastasis

Neutrophil extracellular traps (NETs) entrap circulating tumor cells (CTCs) and promote metastasis within distant organs in preclinical models1,2. In these models, NETosis is triggered by exogenous massive inflammatory stimuli, and thus it remains unknown whether cancer hosts under physiologic inflammation-free conditions experience NETosis and consequent cancer metastasis. Here we show that plasma redox imbalance caused by albumin oxidation promotes inflammation-independent NETosis and cancer metastasis specifically in the lungs. Albumin is the major source of free thiol that maintains redox balance in vitro and in vivo. Oxidation of albumin-derived free thiol is sufficient to trigger NETosis via accumulation of reactive oxygen species within neutrophils. The resultant NETs are found predominantly within lungs where they contribute to the colonization of CTCs leading to pulmonary metastases in mouse models. These effects are abrogated by pharmacologic inhibition of NET formation. Moreover, albumin oxidation and the resultant decline of plasma free thiol are associated with pulmonary metastasis in a cohort of head and neck cancer patients. These results implicate plasma redox balance as an endogenous and physiologic regulator of NETosis and pulmonary cancer metastasis, providing new therapeutic and diagnostic opportunities for combatting cancer progression.

cancer biology↗