Tubulin Tyrosine Ligase and Stathmin Compete for Tubulin Binding In Vitro

Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.

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Multimerization and tubulin binding are required for the SPIRAL2 protein to localize to and stabilize microtubule minus ends

10.1101/2021.10.05.463238 ◽

2021 ◽

Author(s):

YUANWEI FAN ◽

Natasha Bilkey ◽

Ram Dixit

Keyword(s):

Coiled Coil ◽

Spatial Arrangement ◽

In Planta ◽

Structural Determinants ◽

Coiled Coil Domain ◽

Tubulin Binding ◽

And Function ◽

Necessary And Sufficient ◽

Basic Region

Accruing evidence points to the control of microtubule minus-end dynamics as being crucial for the spatial arrangement and function of the microtubule cytoskeleton. In plants, the SPIRAL2 (SPR2) protein has emerged as a microtubule minus-end regulator that is structurally distinct from the animal minus-end regulators. Previously, SPR2 was shown to autonomously localize to microtubule minus ends and decrease their depolymerization rate. Here, we used in vitro and in planta experiments to identify the structural determinants required for SPR2 to recognize and stabilize microtubule minus ends. We show that SPR2 contains a single N-terminal TOG domain that binds to soluble tubulin. The TOG domain, a basic region, and coiled-coil domain are necessary and sufficient to target and stabilize microtubule minus ends. We demonstrate that the coiled-coil domain mediates multimerization of SPR2 that provides avidity for microtubule binding and is essential for binding to soluble tubulin. While TOG domain-containing proteins are traditionally thought to function as microtubule plus-end regulators, our results reveal that nature has repurposed the TOG domain of SPR2 to regulate microtubule minus ends.

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Inhalable nanostructured lipid particles of 9-bromo-noscapine, a tubulin-binding cytotoxic agent: In vitro and in vivo studies

Journal of Colloid and Interface Science ◽

10.1016/j.jcis.2014.12.092 ◽

2015 ◽

Vol 445 ◽

pp. 219-230 ◽

Cited By ~ 31

Author(s):

Kiran Jyoti ◽

Karanvir Kaur ◽

Ravi Shankar Pandey ◽

Upendra Kumar Jain ◽

Ramesh Chandra ◽

...

Keyword(s):

Cytotoxic Agent ◽

In Vivo Studies ◽

Tubulin Binding ◽

Lipid Particles

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Insights into the Molecular Mechanisms of Eg5 Inhibition by (+)-Morelloflavone

Pharmaceuticals ◽

10.3390/ph12020058 ◽

2019 ◽

Vol 12 (2) ◽

pp. 58 ◽

Cited By ~ 1

Author(s):

Tomisin Happy Ogunwa ◽

Emiliano Laudadio ◽

Roberta Galeazzi ◽

Takayuki Miyanishi

Keyword(s):

Molecular Dynamics ◽

Molecular Mechanisms ◽

Adenosine Diphosphate ◽

Umbrella Sampling ◽

Inhibitory Mechanisms ◽

Tubulin Binding ◽

Poisson Boltzmann ◽

Kinesin Spindle Protein ◽

Dynamics Simulations

(+)-Morelloflavone (MF) is an antitumor biflavonoid that is found in the Garcinia species. Recently, we reported MF as a novel inhibitor of ATPase and microtubules-gliding activities of the kinesin spindle protein (Eg5) in vitro. Herein, we provide dynamical insights into the inhibitory mechanisms of MF against Eg5, which involves binding of the inhibitor to the loop5/α2/α3 allosteric pocket. Molecular dynamics simulations were carried out for 100 ns on eight complexes: Eg5-Adenosine diphosphate (Eg5-ADP), Eg5-ADP-S-trityl-l-cysteine (Eg5-ADP-STLC), Eg5-ADP-ispinesib, Eg5-ADP-MF, Eg5-Adenosine triphosphate (Eg5-ATP), Eg5-ATP-STLC, Eg5-ATP-ispinesib, and Eg5-ATP-MF complexes. Structural and energetic analyses were done using Umbrella sampling, Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) method, GROMACS analysis toolkit, and virtual molecular dynamics (VMD) utilities. The results were compared with those of the known Eg5 inhibitors; ispinesib, and STLC. Our data strongly support a stable Eg5-MF complex, with significantly low binding energy and reduced flexibility of Eg5 in some regions, including loop5 and switch I. Furthermore, the loop5 Trp127 was trapped in a downward position to keep the allosteric pocket of Eg5 in the so-called “closed conformation”, comparable to observations for STLC. Altered structural conformations were also visible within various regions of Eg5, including switch I, switch II, α2/α3 helices, and the tubulin-binding region, indicating that MF might induce modifications in the Eg5 structure to compromise its ATP/ADP binding and conversion process as well as its interaction with microtubules. The described mechanisms are crucial for understanding Eg5 inhibition by MF.

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Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.755 ◽

1985 ◽

Vol 101 (3) ◽

pp. 755-765 ◽

Cited By ~ 69

Author(s):

T J Mitchison ◽

M W Kirschner

Keyword(s):

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Lag Phase ◽

Ovary Cells ◽

Tubulin Binding ◽

Mixed Polarity ◽

Tubulin Immunofluorescence

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.

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Mechanism of action of rigosertib does not involve tubulin binding

10.1101/2019.12.12.874719 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stacey J. Baker ◽

Stephen C. Cosenza ◽

Saikrishna Athuluri-Divakar ◽

M.V. Ramana Reddy ◽

Rodrigo Vasquez-Del Carpio ◽

...

Keyword(s):

Mechanism Of Action ◽

Human Tumor ◽

Binding Activity ◽

Phase Iii ◽

Clinical Grade ◽

Wild Type ◽

Binding Domains ◽

Tubulin Binding

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

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α-Tubulin detyrosination impairs mitotic error correction by suppressing MCAK centromeric activity

The Journal of Cell Biology ◽

10.1083/jcb.201910064 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 1

Author(s):

Luísa T. Ferreira ◽

Bernardo Orr ◽

Girish Rajendraprasad ◽

António J. Pereira ◽

Carolina Lemos ◽

...

Keyword(s):

Error Correction ◽

Posttranslational Modification ◽

Chromosomal Instability ◽

Experimental Manipulation ◽

Microtubule Dynamics ◽

Global Impact ◽

Human Cancers ◽

Microtubule Stability ◽

Tubulin Tyrosine Ligase

Incorrect kinetochore–microtubule attachments during mitosis can lead to chromosomal instability, a hallmark of human cancers. Mitotic error correction relies on the kinesin-13 MCAK, a microtubule depolymerase whose activity in vitro is suppressed by α-tubulin detyrosination—a posttranslational modification enriched on long-lived microtubules. However, whether and how MCAK activity required for mitotic error correction is regulated by α-tubulin detyrosination remains unknown. Here we found that detyrosinated α-tubulin accumulates on correct, more stable, kinetochore–microtubule attachments. Experimental manipulation of tubulin tyrosine ligase (TTL) or carboxypeptidase (Vasohibins-SVBP) activities to constitutively increase α-tubulin detyrosination near kinetochores compromised efficient error correction, without affecting overall kinetochore microtubule stability. Rescue experiments indicate that MCAK centromeric activity was required and sufficient to correct the mitotic errors caused by excessive α-tubulin detyrosination independently of its global impact on microtubule dynamics. Thus, microtubules are not just passive elements during mitotic error correction, and the extent of α-tubulin detyrosination allows centromeric MCAK to discriminate correct vs. incorrect kinetochore–microtubule attachments, thereby promoting mitotic fidelity.

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The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle

Journal of Cell Science ◽

10.1242/jcs.112.8.1257 ◽

1999 ◽

Vol 112 (8) ◽

pp. 1257-1271 ◽

Cited By ~ 2

Author(s):

Y. Gachet ◽

S. Tournier ◽

M. Lee ◽

A. Lazaris-Karatzas ◽

T. Poulton ◽

...

Keyword(s):

Cell Cycle ◽

Mutational Analysis ◽

Binding Protein ◽

Binding Domain ◽

Dependent Manner ◽

Cell Extracts ◽

Tubulin Binding ◽

Cycle Dependent ◽

Almost All

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100–150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

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Abstract A183: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo

10.1158/1535-7163.targ-15-a183 ◽

2015 ◽

Author(s):

Raphael Berges ◽

Aurélie Tchoghandjian ◽

Stephane Honore ◽

Dominique Figarella-Branger ◽

Felix Bachmann ◽

...

Keyword(s):

The Novel ◽

Antitumor Effects ◽

Tubulin Binding

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Crescerin uses a TOG domain array to regulate microtubules in the primary cilium

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0603 ◽

2015 ◽

Vol 26 (23) ◽

pp. 4248-4264 ◽

Cited By ~ 21

Author(s):

Alakananda Das ◽

Daniel J. Dickinson ◽

Cameron C. Wood ◽

Bob Goldstein ◽

Kevin C. Slep

Keyword(s):

Crystal Structure ◽

Binding Activity ◽

Structure And Function ◽

Sensory Disorders ◽

Tubulin Binding ◽

Sensory Cilia ◽

And Function ◽

Binding Determinants ◽

Extracellular Environment

Eukaryotic cilia are cell-surface projections critical for sensing the extracellular environment. Defects in cilia structure and function result in a broad range of developmental and sensory disorders. However, mechanisms that regulate the microtubule (MT)-based scaffold forming the cilia core are poorly understood. TOG domain array–containing proteins ch-TOG and CLASP are key regulators of cytoplasmic MTs. Whether TOG array proteins also regulate ciliary MTs is unknown. Here we identify the conserved Crescerin protein family as a cilia-specific, TOG array-containing MT regulator. We present the crystal structure of mammalian Crescerin1 TOG2, revealing a canonical TOG fold with conserved tubulin-binding determinants. Crescerin1's TOG domains possess inherent MT-binding activity and promote MT polymerization in vitro. Using Cas9-triggered homologous recombination in Caenorhabditis elegans, we demonstrate that the worm Crescerin family member CHE-12 requires TOG domain–dependent tubulin-binding activity for sensory cilia development. Thus, Crescerin expands the TOG domain array–based MT regulatory paradigm beyond ch-TOG and CLASP, representing a distinct regulator of cilia structure.

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