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β-tubulin phosphorylation by Chk1 is required for normal spindle formation during cell division

ABSTRACT: The mitotic spindle is a microtubule-based apparatus that is responsible for accurate segregation of chromosomes into two daughter cells. In this study, we show that the DNA damage kinase Chk1 is required for optimal density and efficient polymerization of spindle microtubules during unperturbed mitosis in vertebrate cells. Chk1 phosphorylates -tubulin at the newly identified site threonine-285 (T285) at mitotic centrosomes. Impaired -tubulin-T285 phosphorylation correlates with improper spindles, delayed anaphase onset, erroneous chromosome alignment and segregation, unequal daughter cell-size and reduced cell proliferation, whereas expression of a phosphomimetic -tubulin rescues spindle formation and functions in Chk1-deficient cells. The ATR-interacting protein ATRIP promotes localization of the ATR kinase and mediator protein TopBP1 to mitotic centrosomes; furthermore, interaction of ATRIP with ATR and TopBP1 is required for Chk1 activation and - tubulin-T285 phosphorylation. Also, inhibition of T285 phosphorylation synergizes with microtubule poisons to inhibit cell proliferation. These results identify a novel mechanism that promotes normal spindle formation, through Chk1-mediated - tubulin-T285 phosphorylation.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Spodoptera Frugiperda

DISEASE(S): Disease Free

SUBMITTER: Sergio Lilla

LAB HEAD: Sara Rossana Zanivan

PROVIDER: PXD074543 | Pride | 2026-06-08

REPOSITORIES: Pride

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			Action	DRS
	20170303_SEL_HF_GEZ_EXT_020317_onB-AspN_beta_002.raw	Raw
	20170303_SEL_HF_GEZ_EXT_020317_onB-AspN_beta_002__F016412_.mzid	Mzid
	20170303_SEL_HF_GEZ_EXT_020317_onB-AspN_beta_002__F016412_.mzid_20170303_SEL_HF_GEZ_EXT_020317_onB-AspN_beta_002__F016412_.MGF	Mzid
	GEZ_EXT_180117_BetaTUB.sf3	Other
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β-tubulin phosphorylation by Chk1 is required for normal spindle formation during cell division.

Boutakoglou Nikos N Petsalaki Eleni E Balafouti Sofia S Efthymiou Dimitris D Lilla Sergio S Giatagana Eirini-Maria EM Zanivan Sara S Zachos George G

Communications biology 20260317 1

The mitotic spindle is a microtubule-based apparatus that is responsible for accurate segregation of chromosomes into two daughter cells. In this study, we show that the DNA damage kinase Chk1 is required for optimal density and efficient nucleation of spindle microtubules during unperturbed mitosis in vertebrate cells. Chk1 phosphorylates β-tubulin at the identified conserved site threonine-285 (T285) in vitro, and at mitotic centrosomes in prometaphase and metaphase. Impaired β-tubulin-T285 ph ...[more]

PMID: 41844775

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Project description:Comprehensive Model Description Overview This model simulates the cellular response to DNA damage under conditions of ATM/ATR deficiency—such as in ataxia telangiectasia—and predicts the potential benefits of drug repositioning with Omaveloxolone and spermidine. It integrates several interconnected modules: DNA damage production and repair, ATM/ATR–p53 signaling, NRF2/KEAP1 regulation, spermidine-induced autophagy (with additional positive modulation of ATR signaling), and downstream p53 effectors involved in cell fate decisions. 1. DNA Damage Production and Repair Module Basal DNA Damage Production: DNA damage is continuously generated at a constant rate, representing both endogenous processes and low-level environmental stress. This reaction acts as a source term for the DNA_damage species. Repair Pathways: Several mechanisms work to reduce DNA damage: ATM-Mediated Repair: Active ATM facilitates the direct repair of DNA damage. ATR-Mediated Repair: Active ATR, in cooperation with CHK1, repairs DNA damage. NRF2-Mediated Repair: Active NRF2 triggers antioxidant responses that reduce DNA damage. Autophagy-Mediated Repair: The autophagy pathway, induced by spermidine, repairs DNA damage in a saturable manner (modeled using Michaelis–Menten kinetics). This ensures that even a significant pulse of damage is only partially repaired, leaving a nonzero steady-state level. 2. ATM/ATR–p53 Signaling Module ATM and ATR Activation: DNA damage, along with a positive input from TOPBP1, converts inactive ATM and ATR to their active forms. Under ATM/ATR-deficient conditions (as in ataxia telangiectasia), the activation is severely reduced, resulting in higher levels of unrepaired DNA damage. p53 Activation: Both ATM_active and ATR_active phosphorylate and activate p53. Active p53 then acts as a central integrator of the DNA damage signal. Regulatory Proteins: HDAC4: HDAC4 cycles between inactive and active states. Its active form can attenuate p53 activity. TOPBP1: TOPBP1 is activated in a DNA damage–dependent manner and helps promote ATR activation. CK2: CK2 is a constitutively active kinase that supports TOPBP1 activation. 3. NRF2/KEAP1 Module and Omaveloxolone NRF2 Activation: DNA damage stimulates the conversion of NRF2_inactive to NRF2_active. Active NRF2 enhances antioxidant defenses and promotes DNA repair. KEAP1-Mediated Degradation: Under normal conditions, KEAP1 targets NRF2_inactive for degradation, keeping NRF2 activity low. Modulation by Omaveloxolone: Omaveloxolone is modeled as a constant (boundary) species. It inhibits KEAP1’s activity, thereby reducing NRF2 degradation. This shifts the balance in favor of increased NRF2_active, which boosts the cell’s antioxidant and repair responses. 4. Spermidine-Induced Autophagy and Positive Modulation of ATR Signaling Spermidine is modeled as a constant species and has two major effects: Induction of Autophagy: Spermidine promotes the conversion of Autophagy_inactive to Autophagy_active. The active autophagy machinery repairs DNA damage using a saturable (Michaelis–Menten) mechanism. This pathway helps clear DNA damage but is limited by its maximum capacity. Enhancement of ATR Signaling: Spermidine also positively modulates ATR signaling by enhancing the CK2-mediated activation of TOPBP1. In the model, the effective rate of TOPBP1 activation is increased proportionally to the concentration of spermidine. This is represented by a modulation factor, where the effective rate constant for TOPBP1 activation is multiplied by a term that increases with spermidine concentration. The boosted TOPBP1_active level in turn enhances ATR activation, partially compensating for the loss of ATM/ATR function. 5. Downstream p53 Targets: Cell Fate Decisions Activated p53 regulates cell fate by inducing the production of several downstream effectors: p21: p21 is produced in response to p53 activation and is associated with cell cycle arrest and senescence. Its production is balanced by a degradation reaction. BAX and PUMA: Both BAX and PUMA are pro-apoptotic proteins. They are produced in proportion to p53_active and are subsequently degraded. Their relative levels help determine whether the cell will undergo apoptosis. 6. Reaction Kinetics (Qualitative Overview) Mass-Action and Michaelis–Menten Kinetics: Most reactions in the model are governed by mass-action kinetics. For example, activation and deactivation reactions (such as ATM activation by DNA damage or p53 activation by ATM/ATR) follow simple proportional relationships. 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Drug Repositioning Predictions: The model can be used to predict the outcomes of repositioning drugs: Omaveloxolone is expected to stabilize NRF2, enhancing antioxidant defenses and reducing DNA damage. Spermidine acts dually by promoting autophagy (with a saturable repair capacity) and by boosting ATR signaling via TOPBP1 activation. Cell Fate Outcomes: The downstream p53 targets (p21, BAX, and PUMA) serve as markers for cell cycle arrest/senescence and apoptosis. By simulating the dynamics of these proteins, the model can predict whether a cell is likely to survive, arrest its cycle, or undergo apoptosis based on the integrated response to DNA damage and drug interventions. Conclusion This detailed and comprehensive model integrates multiple layers of the cellular response to DNA damage in ATM/ATR-deficient conditions. 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Project description:This model is from the article: A dynamical model of the spindle position checkpoint Ayse Koca Caydasi, Maiko Lohel, Gerd Grünert, Peter Dittrich, Gislene Pereira, Bashar Ibrahim Molecular Systems Biology 2012; 582 doi: 10.1038/msb.2012.15 Abstract: The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-to-daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase-activating protein (GAP) complex Bfa1–Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1–Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1–Bub2 and Tem1 localization at the SPBs. Based on the measured SPB-bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.