Recent insights into the therapeutic strategies targeting the pseudokinase PTK7 in cancer

Oncogene, Год журнала: 2024, Номер 43(26), С. 1973 - 1984

Опубликована: Май 21, 2024

Abstract The generation of drugs counteracting deregulated protein kinases has been a major focus in cancer therapy development. Breakthroughs this effort have produced many therapeutic agents to the benefit patients, mostly through development chemical or antibody-based targeting active kinases. These strategies are challenged when considering catalytically inactive (or pseudokinases), which represent 10% human kinome with relevance cancer. Among so-called pseudotyrosine kinases, PTK7 receptor tyrosine kinase (RTK) stands as bona fide target overexpressed several solid tumors and hematological malignancies linked metastasis, poor prognosis, resistance treatment. Despite lack catalytic activity, signaling capacities heterodimerization RTKs offers pharmacological opportunities its domain. Moreover, PTK7-targeting based on antibody-drug conjugates, aptamers, CAR-T cell-based therapies demonstrated encouraging results preclinical clinical settings. We review most recent data assigning prominent role progression well current against RTK family pseudokinases including PTK7.

Язык: Английский

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Trans-activating mutations of the pseudokinase ERBB3

Oncogene, Год журнала: 2024, Номер 43(29), С. 2253 - 2265

Опубликована: Май 28, 2024

Abstract Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for inhibitor drugs. ERBB3 is a pseudokinase member that, although lacking fully active kinase domain, well known its potent signaling activity heterodimeric complex with ERBB2. Previous studies have identified few transforming mutations while great majority hundreds different somatic variants observed cancer types remain unknown significance. Here, we describe an unbiased functional genetics screen potential thousands parallel. The based on previously described iSCREAM (in vitro activating mutations) platform, addressing context ERBB3/ERBB2 heterodimers, 18 hit mutations. Validation experiments Ba/F3, NIH 3T3, MCF10A cell backgrounds demonstrated presence both missense functioning either single or cis pairwise combination. Drug sensitivity assays trastuzumab, pertuzumab neratinib indicated actionability variants.

Язык: Английский

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Allosteric activation of the co-receptor BAK1 by the EFR receptor kinase initiates immune signaling

eLife, Год журнала: 2023, Номер 12

Опубликована: Ноя. 23, 2023

Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many these are pseudokinase domains, however, suggests additional mechanisms must govern RK activation. Non-catalytic protein domains have described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed regulate RKs with non-RD Here, using as model, we describe activation mechanism LRR-RKs an active kinase, kinase-dead variant retains ability enhance catalytic activity its co-receptor BAK1/SERK3 (brassinosteroid insensitive 1-associated 1/somatic embryogenesis 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis designing homology-based intragenic suppressor mutations, provide evidence domain adopt conformation order activate BAK1 allosterically, likely supporting αC-helix positioning BAK1. Our results suggest toggle model signaling, which first phosphorylates loop stabilize conformation, allowing turn allosterically

Язык: Английский

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Atlas of the Bacterial Serine-Threonine Kinases expands the functional diversity of the kinome

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 15, 2025

Bacterial serine-threonine protein kinases (STKs) regulate diverse cellular processes associated with cell growth, virulence, and pathogenicity. They are evolutionarily related to the druggable eukaryotic STKs. However, an incomplete knowledge of how bacterial STKs differ from their counterparts they have diverged signaling functions presents a bottleneck in targeting them for drug discovery efforts. Here, we classified over 300,000 STK sequences NCBI RefSeq non-redundant UniProt databases into 35 canonical seven non-canonical (pseudokinase) families based on patterns evolutionary constraints conserved catalytic domain flanking regulatory domains. Through statistical comparisons, identified distinguishing features STKs, including distinctive arginine residue helix (C-Helix) that dynamically couples ATP substrate binding lobes kinase domain. Biochemical peptide-library screens demonstrated constrained residues contribute specificity activation Mycobacterium tuberculosis PknB. Collectively, these findings open new avenues investigating development selective inhibitors.

Язык: Английский

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TMK interacting network of receptor like kinases for auxin canalization and beyond

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Март 2, 2025

SUMMARY Receptor-like kinases (RLKs), particularly the Transmembrane Kinase (TMK) family, play essential roles in signaling and development, with TMKs being key components of auxin perception downstream phosphorylation events. While TMKs’ involvement canalization, a process for vasculature formation regeneration, has been established, nonetheless, additional regulatory partners remain poorly understood. In this study, we identify characterize seven leucine-rich repeat RLKs (TINT1–TINT7) as novel interactors TMK1, revealing their diverse evolutionary, structural, functional characteristics. Our results show that TINTs interact TMK1 highlight regulating various developmental processes. Majority contributes, together to TINT5 linking other canalization component CAMEL. Beyond also establish role TINT-TMK1 interactions processes such stomatal movement hypocotyl’s gravitropic response. These findings suggest TINTs, through interaction are integral networks, contributing both broader plant development.

Язык: Английский

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The Medicago truncatulaLYR4 intracellular domain serves as a scaffold in immunity signaling independent of its phosphorylation activity

New Phytologist, Год журнала: 2025, Номер unknown

Опубликована: Март 10, 2025

Plants engage in a wealth of interactions with beneficial and pathogenic bacteria fungi therefore need to monitor their surroundings. To this end, cell-surface receptors, such as lysin motif (LysM) perceive microbe-associated molecular patterns (MAMPs) elicit immunity responses or initiate symbiotic associations (Buendia et al., 2018). LysM receptors have an ectodomain built three interconnected domains most feature single transmembrane helix either active kinase pseudokinase intracellular domain (Gust 2012). The main ligands are carbohydrates containing N-acetylglucosamine moieties, namely chitooligosaccharides (CO; chitin), lipochitooligosaccharides (LCO), peptidoglycan (Willmann 2011; Gust 2012; Bozsoki 2017, 2020; Buendia 2018; Gysel 2021). In the model legume Lotus japonicus (Lotus), receptor CERK6 (previously called LYS6) tandem duplicated LYS13 LYS14 involved chitin-triggered (Bozsoki 2017). classified pseudokinases, expression increases roots shoots upon chitin treatment plant (Lohmann 2010; Ruman & Kawaharada, 2023). Single mutants lys13 lys14 respond similarly wild-type, suggesting functional redundancy between LYS14, whereas cerk6 cannot produce reactive oxygen species (ROS) upregulation defense-response genes phosphorylation MAPK3/6 (mitogen-activated protein kinases) impaired Further investigation signaling is difficult, double mutant not available. Medicago truncatula (Medicago), CERK1 LYK9) homolog LYR4 2017; Feng 2019). (CERK1 hereafter) (LYR4 been reported be indispensable for both cerk1 lyr4 having increased lesion size infection leaves Botrytis cinerea addition, ROS were absent MAPK3 MAPK6 decreased octamer (CO8) compared wild-type Zhang 2024). has all canonical motifs eukaryotic kinase, while due degeneration several key motifs: truncated glycine-rich loop, DFG-motif NFG, HRD-motif HKN 2023; Fig. 1a). Canonically, loop regulatory lysine on β3-strand stabilize phosphates bound ATP. Aspartate from important positioning phosphate transfer arginine aspartate contribute ordering activation incoming substrate, respectively (Taylor Kornev, Taylor study, we investigate how mediates downstream immunity. We determine crystal structure ATP-analog noncanonical manner show that it catalytically despite its lack features. However, planta experiments demonstrate ability necessary function production, but presence indispensable. Thus, serves scaffold independent catalytic activity. G. cv R108 was used wild-type. lyr4-2 (NF15280) (NF16753) previously (Tadege 2008; lyr4-1 generated by crossing (NF10265) plants. exhibit similiar CO8 described Escherichia coli TOP10 (Thermo Fisher Scientific, Waltham, MA, USA) cloning grown LB medium at 37°C. Chemically competent E. Rosetta 2 cells (Sigma-Aldrich) specified below. Agrobacterium rhizogenes strain AR1193 hairy root transformations, tumefaciens AGL1 transient transformation Nicotiana benthamiana. strains 28°C. cerk1, lyr4, seeds treated sulfuric acid 3 min, washed ddH2O, surface-sterilized 3% sodium hypochlorite, again incubated ddH2O h. Swollen transferred square plates wet filter paper 21°C under 16 h : 8 h, light dark conditions d. Seedlings then slope ¼ B&D (Broughton Dilworth, 1971) solidified 1.4% Agar Noble (Difco, Sparks, MD, USA). agar covered (AGF 651; Frisenette ApS, Knebel, Denmark) metal bar 10 holes placed top slope. Plates boxes, excluding below bars. After d plates, seedlings cut hypocotyl, one full each well white 96-well flat-bottomed polystyrene plate (Greiner Bio-One, Kremsmünster, Austria) kept overnight sterile water. water replaced reaction mixture consisting 0.5 mM L-012 (Wako Chemicals, Neuss, Germany), 5 μg ml−1 horseradish peroxidase (Sigma-Aldrich), 1 μM octa-N-acetyl-chitooctaose (CO8, obtained Sébastien Fort; Masselin 2021) 0.1 mg shrimp-cell (Sigma-Aldrich). Luminescence recorded Varioskan LUX multimode microplate reader Scientific) 30 min. For generation M. vectors, sequences variants assembled native promoter 35S terminator via Golden Gate (Weber 2011). constructs thereafter cloned into pIV10 vector alongside construct YFP fluorophores fused nuclear localization signal (tYFP-NLS), which marker. experiment N. benthamiana, sequence pICH An overview provided Supporting Information Table S1. 0.8% Gelrite (Duchefa Biochemie, Haarlem, Netherlands) supplemented ½ Gamborg's B5 nutrient solution Biochemie). Transconjugant A. carrying interest ampicillin, rifampicin, spectinomycin, final concentration 100 ml−1. Cells resuspended 4 ml YMB (5 g l−1 mannitol, yeast extract, K2HPO4, 0.2 MgSO4 × 7 H2O, NaCl; pH 6.8). Four-day-old transformed using syringe needle (Sterican Ø 0.40 20 mm), wounding hypocotyl placing droplet bacterial suspension wound. conditions. wk, primary removed emerging Magenta boxes filled clay aggregate (LECA, 2–4 mm; Saint-Gobain Weber A/S, Copenhagen, 80 KNO3. Variants tested complement mutant. wk growth expressing tYFP-NLS marker small pieces c. mm length. Pieces 1.5 cm total length Bio-One) cuttings left room temperature overnight. Afterward, Chemicals), (Sigma), added. Values derived curves without clear peak within min excluded. Agrobacterium-mediated benthamiana performed (Ochoa-Fernandez 2020). short, Lyr4-ΔIC-GFP (green fluorescent protein) driven infiltration (10 MgCl2, MES, 150 acetosyringone; 5.6) OD600 = 0.025, infiltrated abaxial side blunt end syringe. imaged after infiltration. GFP-tagged LYR4-ΔIC 488 nm excitation 491–544 emission Zeiss LSM 780 confocal microscope. (residues 298–660 residues 340–636) 307–598) codon optimized (Genscript, Leiden, N-terminally histidine tag, SUMO 3C protease cleavage site. 298–660) A377F, L392D, (307–598) K349A made site-directed mutagenesis aforementioned constructs. S2. expression, 0.6 relevant antibiotics 37°C shaking. culture chilled ice induced adding IPTG, 19°C shaking harvested 4400 15 medium, pelleted 3050 pellet lysis buffer (50 Tris–HCl 8.0, 500 NaCl, 10% v/v glycerol, imidazole, β-mercaptoethanol (BME)) lysed sonication. cleared supernatant subjected nickel-immobilized metal-affinity chromatography (Ni-IMAC) Protino Ni-NTA column (Macherey-Nagel, Düren, Germany) equilibrated buffer. sample loaded onto peristaltic pump, 20-column volumes A BME) before eluted 12 B 200 BME). added eluate 50 molar ratio digestion dialyzed against l dialysis 250 5% 4°C. product another Ni-IMAC remove digested tags purified gel filtration step Superdex75 increase 10/300 GL Superdex200 (GE Healthcare, Chicago, IL, (25 All purification steps analyzed dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) elution fractions pooled accordingly. crystallized sitting drop vapor diffusion setup 24-well (Molecular Dimensions, Rotherham, UK). Crystal formation condition M ammonium sulfate, cacodylate trihydrate 6.5 30% w/v PEG 8000 gave rise seed stock microseeding drops adenylyl-imidodiphosphate (AMP-PNP) 0.3 6.5, 25% 8000. Needle-like crystals cryoprotected supplementing crystallization glycerol fished flash-cooled liquid nitrogen. data set collected Diamond Light Source beamline I04. diffraction processed scaled XDS (Kabsch, 2010), xtriage Phenix program suite. Due high anisotropy, STARANISO server (Global Phasing Ltd (Tickle 2016)) along anisotropic resolution limit surface (CC(1/2) ≥ 0.3, I/sigI 2.0, Rpim ≤ 0.6). crystallographic phase problem solved replacement Phaser NFR5 (PDB-ID 8S79) search (McCoy 2007). Refinement (Adams manual building carried out Coot (Emsley 2010). Data collection statistics spherical ellipsoidal datasets refinement S3. atomic coordinates factors deposited Protein Bank, PDB ID: 8PS7. Structural analyses figures PyMol 2.3.2 (Schrödinger LLC, New York, NY, Binding nucleotide indirectly assayed nano differential scanning fluorimetry (nano-DSF) Tycho-NT.6 (Nanotemper, Munich, Germany). 298–660), L392D ATP, AMP-PNP, MgCl2 each. Four micrograms nCi [γ32-P] ATP (PerkinElmer, cold samples separated 15% SDS-PAGE stained InstantBlue Coomassie Stain (Abcam, Cambridge, UK) exposed Autoradiography Hypercassette (Amersham/Cytiva, Amersham, Radiographs developed Typhoon FLA 9500 (Amersham/Cytiva). individual contributions signaling, response when shrimp-shell (Fig. 1b). While see almost no mutant, CO8. production is, however, attenuated curve broadened peaks later than From conclude does fully depend can also individually signaling. understand juxtamembrane part predicted flexible C-terminal tail S1a). analog AMP-PNP. Diffraction resulted 3.1 Å anisotropically 2.1 8PS7) 1c; S3). Based amino sequence, defined pseudokinase. electron density revealed clearly AMP-PNP molecule binding site 1d), showing retained abilities. Closer examination binds way compensates traditional S1b). Pseudokinases display compensatory mechanisms retain degraded motifs, proposed (Zeqiraj Van Aalten, Murphy 2014; Sheetz Lemmon, 2022). coordinated lysine, indeed employ (K379) coordinate α-phosphate, too short reach Instead, N494 modified α- β-phosphate, R514 β- γ-phosphate. Active kinases two hydrophobic spines going through core – (R-) spine (C-) spine. cycle, assemble break these they toggle inactive states. C-spine nucleotide, R-spine αC moves in-position forms salt bridge lysine. Both able phosphorylate substrate (Kornev 2006; conformation, intact C-spine, broken being out-position S1c,d). Furthermore, shows C-terminus 1c, labeled helix). This blocks access bringing position coordinating γ-phosphate representing undescribed conformation. Guided structure, designed whether LYR4. LYR4-A377F phenylalanine site, predict will abolish ATP-binding 1e). LYR4-L392D introduces negative charge helix, make unable move R-spine, keeping retaining 1f). expressed S2) determined bind testing thermostability nano-DSF magnesium 2a). together stability 4.1°C, indicating likewise stabilized 3.7°C, present, only marginally 1.5°C, no, very weak, ATP-binding. test potential activity, vitro assays 32P-labeled observed autophosphorylate transphosphorylate myelin basic (MBP), making 2b, lanes 3, 4). As expected, neither nor showed activity (Figs had autophosphorylation lower 5). Next, investigated other. created kinase-inactive version lacked (CERK1-K349A) 9, 10, lane 11). Conversely, 12). dependent subsequent conformational changes, plants Lyr4, Lyr4-A377F (no binding), Lyr4-L392D (kinase inactive, binding). Lyr4-A377F, Lyr4-L392D, produced equal empty 2c), required all, constructed variant lacking entire domain, Lyr4-ΔIC, roots. When trafficked plasma membrane properly folded S4a). could, mediate 2c, S4b,c), crucial immune Together, our results chitin-induced comprise up 17% kinome numerous pathways, example, those (Kwon understanding remains limited. perception absence CERK1, although resulting levels attenuated. Previously, shown completely could pairing less efficient abundant partner. LYK subfamily receptor-like harboring potentially kinases, 11 members Medicago, LYR LYR7 phylogenetically closest Recently, al. LYK8 some extent functionally redundant arbuscular mycorrhizal symbiosis. Some detail yet, might overlapping capacities take over functions. better mediated pseudokinase, ATP-analog. density, prompted us relevance phosphotransfer ability. Thermostability additionally confirmed closer inspection manner. Despite pseudokinases demonstrated nucleotides various event itself lead regulation Mace Murphy, 2021; auto- transphosphorylation general, many experimentally, employing Dar, 2013). changes Complementation studies production. It intriguing apparent relevance, exclude possibility plays role other pathways. elicitation 2d). noncatalytic responsible putative CERK1–LYR4 complex. broader view, scaffolding like correctly arrange co-receptors, partners. often dynamic typical allows them act switches allosteric regulators acting scaffolds complex assembly (Sheetz 2022; Mühlenbeck Future elucidate complexes formed thank Majken Kiel Sørensen care glasshouse, Sara Bährentz help work, Leila Margot Henkes proofreading manuscript. acknowledge provision experimental facilities staff I04 assistance collection. work funded Novo Nordisk Foundation (NNF18OC0052855), Danish Council Independent Research (3103-00137B), Carlsberg (CF21-0139), Agence Nationale de la Recherche Labex ARCANE CBH-EUR-GS (ANR-17-EURE-0003), Glyco@Alps (ANR-15-IDEX-02), ICMG (UAR 2607) Grenoble Chemistry Nanobio mass spectrometry platform, USDA-NIFA grant (2022-67014-38607), project Enabling Nutrient Symbioses Agriculture (ENSA), Bill Melinda Gates Agricultural Innovations (INV-57461), Foreign, Commonwealth Development Office (INV-55767). None declared. BS, HR, MVK, MML, CK GK investigation. KG, ML, JS, SR KRA formal analysis. SF FF resources. BS HR visualization. conceptualization. supervision. administration. GEDO, funding acquisition. writing original draft preparation. authors review editing. contributed equally work. Bank (PDB code (8PS7)). S1 Purification detailed structural S2 Gel profiles corresponding SDS-PAGEs purifications recombinant proteins. S3 assay variants. S4 including versions LYR4-ΔIC. assays. Construct purification. statistics. Please note: Wiley content functionality any supplied authors. Any queries (other missing material) should directed Phytologist Central Office. publisher supporting information content) author article. neutral regard jurisdictional claims maps institutional affiliations.

Язык: Английский

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Decoding TBCK: from bioinformatic insights of domain architecture to disease implications

Frontiers in Biophysics, Год журнала: 2025, Номер 3

Опубликована: Апрель 4, 2025

TBCK is an essential protein in neurodevelopment. Mutations the gene are associated with Syndrome, a genetic neurological disorder characterized by global developmental delay. enigmatic multidomain that contains pseudokinase domain, TBC (Tre2-Bub2-Cdc16) and rhodanese-like domain. Emerging evidence increasingly links to multiple cellular processes, including mTOR signaling, autophagy, lysosomal function, mitochondrial maintenance. This review consolidates recent advances our understanding of TBCK, emphasizing comparative sequence analysis, structural modeling, its functions. Our analysis shows both kinase domain likely lack catalytic activity instead primarily function as scaffolds or regulatory domains. The all conserved residues, suggesting it may act GTPase-activating (GAP). These functional hypotheses provide foundation for further investigations into TBCK’s physiological pathological roles.

Язык: Английский

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An atlas of bacterial serine-threonine kinases reveals functional diversity and key distinctions from eukaryotic kinases

Science Signaling, Год журнала: 2025, Номер 18(885)

Опубликована: Май 6, 2025

Bacterial serine-threonine kinases (STKs) regulate diverse cellular processes associated with cell growth, virulence, and pathogenicity are evolutionarily related to the druggable eukaryotic STKs. A deeper understanding of how bacterial STKs differ from their counterparts they have evolved signaling functions is crucial for advancing discovery development new antibiotic therapies. Here, we classified more than 300,000 STK sequences NCBI RefSeq nonredundant UniProt protein databases into 35 canonical seven pseudokinase families on basis patterns evolutionary constraints in conserved catalytic domain flanking regulatory domains. Through statistical comparisons, identified features distinguishing STKs, including an arginine residue a helix (C helix) that dynamically couples ATP- substrate-binding lobes kinase domain. Biochemical peptide library screens demonstrated constrained residues contributed substrate specificity activation Mycobacterium tuberculosis PknB. Together, these findings open previously unidentified avenues investigating developing selective inhibitors.

Язык: Английский

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Progress on the Pharmacological Targeting of Janus Pseudokinases

Journal of Medicinal Chemistry, Год журнала: 2023, Номер 66(16), С. 10959 - 10990

Опубликована: Авг. 14, 2023

The Janus kinases (JAKs) are key components of the JAK-STAT signaling pathway and involved in myriad physiological processes. Though they molecular targets many FDA-approved drugs, these drugs manifest adverse effects due part to their inhibition requisite JAK kinase activity. However, JAKs uniquely possess an integrated pseudokinase domain (JH2) that regulates adjacent (JH1). therapeutic targeting JH2 domains has been less thoroughly explored may present avenue modulate without associated with JH1 domain. potential this strategy was recently demonstrated FDA approval TYK2 ligand deucravacitinib for treating plaque psoriasis. In light, structure targetability pseudokinases discussed, conjunction state development ligands bind domains.

Язык: Английский

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Kinome‐Wide Synthetic Lethal Screen Identifies PANK4 as a Modulator of Temozolomide Resistance in Glioblastoma

Advanced Science, Год журнала: 2024, Номер 11(15)

Опубликована: Фев. 14, 2024

Abstract Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition resistance limits its therapeutic potential. The human kinome is an undisputable source druggable targets, still, current knowledge remains confined to a limited fraction it, with multitude under‐investigated proteins yet be characterized. Here, following kinome‐wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as modulator TMZ in GBM. Validation PANK4 across various TMZ‐resistant GBM cell models, patient‐derived lines, tissue samples, well vivo studies, corroborates potential translational significance these findings. Moreover, expression induced during treatment, and associated worse clinical outcome. Furthermore, Tandem Mass Tag (TMT)‐based quantitative proteomic approach, reveals that abrogation leads significant downregulation host central roles cellular detoxification response oxidative stress. More specifically, cells undergo genotoxic stress exposure, depletion crucial event can lead accumulation intracellular reactive oxygen species (ROS) subsequent death. Collectively, previously unreported role mediating unveiled.

Язык: Английский

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