Overexpression of Aurora-A bypasses cytokinesis through phosphorylation of suppressed in lung cancer

Mitosis is a complicated process by which eukaryotic cells segregate duplicated genomes into two daughter cells. To achieve the goal, numerous regulators have been revealed to control mitosis. The oncogenic Aurora-A is a versatile kinase responsible for the regulation of mitosis including chromosome condensation, spindle assembly, and centrosome maturation through phosphorylating a range of substrates. However, overexpression of Aurora-A bypasses cytokinesis, thereby generating multiple nuclei by unknown the mechanisms. To explore the underlying mechanisms, we found that SLAN, a potential tumor suppressor, served as a substrate of Aurora-A and knockdown of SLAN induced immature cytokinesis. Aurora-A phosphorylates SLAN at T573 under the help of the scaffold protein 14-3-3η. The SLAN phosphorylation-mimicking mutants T573D or T573E, in contrast to the phosphorylation-deficiency mutant T573A, induced higher level of multinucleated cells, and the endogenous SLAN p573 resided at spindle midzone and midbody with the help of the microtubule motor MKLP1. The Aurora-A- or SLAN-induced multiple nuclei was prevented by the knockdown of 14-3-3η or Aurora-A respectively, thereby revealing a 14-3-3η/Aurora-A/SLAN cascade negatively controlling cytokinesis. Intriguingly, SLAN T573D or T573E inactivated and T573A activated the key cytokinesis regulator RhoA. RhoA interacted with SLAN np573, i.e., the nonphosphorylated form of SLAN at T573, which localized to the spindle midzone dictated by RhoA and ECT2. Therefore, we report here that SLAN mediates the Aurora-A-triggered cytokinesis bypass and SLAN plays dual roles in that process depending on its phosphorylation status.

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The mitosis-regulating and protein-protein interaction activities of Astrin are controlled by Aurora-A-induced phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.00164.2014 ◽

2014 ◽

Vol 307 (5) ◽

pp. C466-C478 ◽

Cited By ~ 10

Author(s):

Shao-Chih Chiu ◽

Jo-Mei Maureen Chen ◽

Tong-You Wade Wei ◽

Tai-Shan Cheng ◽

Ya-Hui Candice Wang ◽

...

Keyword(s):

Regulatory Networks ◽

Spindle Assembly Checkpoint ◽

Morphological Changes ◽

Spindle Assembly ◽

Aurora A ◽

Protein Protein Interaction ◽

Daughter Cells ◽

Sister Chromatids ◽

Complicated Process ◽

Spindle Stability

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser115. The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

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Difference of MreBCD Complex Interactome in Salmonella Typhimurium ST19 and ST213 Genotypes on Pathogenesis and Stress Response Pathways

Current Protein and Peptide Science ◽

10.2174/1389203722666210921111214 ◽

2021 ◽

Vol 22 ◽

Author(s):

Reyna Cristina Zepeda-Gurrola ◽

Gerardo Vázquez-Marrufo ◽

Xianwu Guo ◽

Isabel Cristina Rodríguez-Luna ◽

Alejandro Sánchez-Varela ◽

...

Keyword(s):

Stress Response ◽

Bacterial Virulence ◽

Eukaryotic Cells ◽

Interaction Patterns ◽

High Infection Rate ◽

Differential Interaction ◽

Sequence Variations ◽

High Infection ◽

Underlying Mechanisms

: Salmonella enterica is the etiological agent of salmonellosis, with a high infection rate worldwide. In Mexico, ST213 genotype of S. enterica ser. Typhimurium is displacing the ancestral ST19 genotype. Bacterial cytoskeleton protein complex MreBCD play an important role in S. enterica pathogenesis, but underlying mechanisms are unknown. In this study, 106 interactions among MreBCD and 15 proteins from S. Typhimurium Pathogenicity Islands 1 (SP-I) and 2 (SP-2) involved in both bacterial virulence and stress response were predicted in ST213 and ST19 genotypes, of which 12 interactions were confirmed in vitro. In addition, gene cluster analysis in 100 S. Typhimurium genomes was performed for these genes. The in silico and in vitro results showed a novel MreBCD interactome involved in the regulation of pathogenesis and stress response through interactions with virulence factors located at SPI-1 and SPI-2. Furthermore, both pseudogene presence and sequence variations in four tested proteins between genotypes resulted in differential interaction patterns that are involved in Salmonella motility and survival in eukaryotic cells, which could explain replacement of ST19 by ST213 in Mexico.

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Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during ingression into Drosophila flight muscles

10.1101/669028 ◽

2019 ◽

Cited By ~ 1

Author(s):

Julia Sauerwald ◽

Wilko Backer ◽

Till Matzat ◽

Frank Schnorrer ◽

Stefan Luschnig

Keyword(s):

Matrix Metalloproteinase ◽

Growth Cone ◽

Cultured Cells ◽

Collagen Iv ◽

Dual Roles ◽

Invasion Speed ◽

Invasive Behavior ◽

Matrix Metalloproteinase 1 ◽

Flight Muscles ◽

Underlying Mechanisms

SUMMARYTubular networks like the vasculature extend branches throughout the bodies of animals, but how developing vessels interact with and invade tissues is not well understood. We investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the Collagen IV-containing matrix around branch tips and promotes degradation of Branchless FGF in cultured cells. Thus, tracheal-derived MMP1 may play dual roles in sustaining branch invasion by modulating ECM properties as well as by shaping the distribution of the FGF chemoattractant.

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Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Journal of Experimental Medicine ◽

10.1084/jem.20062324 ◽

2007 ◽

Vol 204 (9) ◽

pp. 2213-2224 ◽

Cited By ~ 123

Author(s):

Dale A. Moulding ◽

Michael P. Blundell ◽

David G. Spiller ◽

Michael R.H. White ◽

Giles O. Cory ◽

...

Keyword(s):

Live Cell Imaging ◽

Actin Polymerization ◽

Cell Imaging ◽

Human Gene ◽

Filamentous Actin ◽

Gene Encoding ◽

Activating Mutations ◽

Spindle Midzone ◽

Underlying Mechanisms ◽

Syndrome Protein

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

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Dual functions of Rack1 in regulating Hedgehog pathway

Cell Death and Differentiation ◽

10.1038/s41418-020-0563-7 ◽

2020 ◽

Vol 27 (11) ◽

pp. 3082-3096 ◽

Cited By ~ 1

Author(s):

Yan Li ◽

Xiaohan Sun ◽

Dongqing Gao ◽

Yan Ding ◽

Jinxiao Liu ◽

...

Keyword(s):

Mammalian Cells ◽

Cellular Localization ◽

Multiple Roles ◽

Dual Roles ◽

Cubitus Interruptus ◽

Trimeric Complex ◽

Physiological Processes ◽

Underlying Mechanisms ◽

The Stability ◽

Fine Tune

Abstract Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.

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WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

The Journal of Cell Biology ◽

10.1083/jcb.201701074 ◽

2017 ◽

Vol 216 (6) ◽

pp. 1673-1688 ◽

Cited By ~ 51

Author(s):

Lillian K. Fritz-Laylin ◽

Samuel J. Lord ◽

R. Dyche Mullins

Keyword(s):

Actin Polymerization ◽

Eukaryotic Cells ◽

Human Neutrophils ◽

Evolutionary Relationships ◽

Actin Assembly ◽

Complex Environments ◽

Clear Trend ◽

Cell Crawling ◽

Evolutionarily Conserved ◽

Underlying Mechanisms

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.

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PLK1 plays dual roles in centralspindlin regulation during cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201805036 ◽

2019 ◽

Vol 218 (4) ◽

pp. 1250-1264 ◽

Cited By ~ 10

Author(s):

Ingrid E. Adriaans ◽

Angika Basant ◽

Bas Ponsioen ◽

Michael Glotzer ◽

Susanne M.A. Lens

Keyword(s):

Small Gtpase ◽

The Other ◽

Aurora B ◽

Cleavage Furrow ◽

Contractile Ring ◽

Dual Roles ◽

Early Step ◽

Rhoa Activation ◽

Anaphase Onset ◽

Spindle Midzone

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells. Knock-down of PRC1, which disrupts the spindle midzone, revealed the existence of two pathways that can initiate cleavage furrow ingression. One pathway depends on a well-organized spindle midzone and PLK1, while the other depends on Aurora B activity and centralspindlin at the equatorial cortex and can operate independently of PLK1. We further show that PLK1 inhibition sequesters centralspindlin onto the spindle midzone, making it unavailable for Aurora B at the equatorial cortex. We propose that PLK1 activity promotes the release of centralspindlin from the spindle midzone through inhibition of PRC1, allowing centralspindlin to function as a regulator of spindle midzone formation and as an activator of RhoA at the equatorial cortex.

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Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport

Molecular Biology of the Cell ◽

10.1091/mbc.e15-07-0493 ◽

2016 ◽

Vol 27 (6) ◽

pp. 897-906 ◽

Cited By ~ 16

Author(s):

Xiaobo Bai ◽

Eva P. Karasmanis ◽

Elias T. Spiliotis

Keyword(s):

Nmda Receptor ◽

Hippocampal Neurons ◽

Intracellular Transport ◽

Tail Domain ◽

Septin 9 ◽

Microtubule Motor ◽

Underlying Mechanisms ◽

Dependent Transport ◽

Developing Rat ◽

Cargo Binding

Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.

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A Small C-Terminal Sequence of Aurora B Is Responsible for Localization and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0447 ◽

2005 ◽

Vol 16 (1) ◽

pp. 292-305 ◽

Cited By ~ 27

Author(s):

Laetitia Scrittori ◽

Dimitrios A. Skoufias ◽

Fabienne Hans ◽

Véronique Gerson ◽

Paolo Sassone-Corsi ◽

...

Keyword(s):

Aurora B ◽

Aurora A ◽

Terminal Sequence ◽

Function Test ◽

Spindle Midzone ◽

Spindle Microtubules ◽

And Function ◽

Interfering Rna ◽

Sequence Requirements

Aurora B, a protein kinase required in mitosis, localizes to inner centromeres at metaphase and the spindle midzone in anaphase and is required for proper chromosome segregation and cytokinesis. Aurora A, a paralogue of Aurora B, localizes instead to centrosomes and spindle microtubules. Except for distinct N termini, Aurora B and Aurora A have highly similar sequences. We have combined small interfering RNA (siRNA) ablation of Aurora B with overexpression of truncation mutants to investigate the role of Aurora B sequence in its function. Reintroduction of Aurora B during siRNA treatment restored its localization and function. This permitted a restoration of function test to determine the sequence requirements for Aurora B targeting and function. Using this rescue protocol, neither N-terminal truncation of Aurora B unique sequence nor substitution with Aurora A N-terminal sequence affected Aurora B localization or function. Truncation of unique Aurora B C-terminal sequence from terminal residue 344 to residue 333 was without effect, but truncation to 326 abolished localization and function. Deletion of residues 326-333 completely abolished localization and blocked cells at prometaphase, establishing this sequence as critical to Aurora B function. Our findings thus establish a small sequence as essential for the distinct localization and function of Aurora B.

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Recruitment of MKLP1 to the spindle midzone/midbody by INCENP is essential for midbody formation and completion of cytokinesis in human cells

Biochemical Journal ◽

10.1042/bj20050097 ◽

2005 ◽

Vol 389 (2) ◽

pp. 373-381 ◽

Cited By ~ 49

Author(s):

Changjun Zhu ◽

Ella Bossy-Wetzel ◽

Wei Jiang

Keyword(s):

Chromosome Segregation ◽

Mammalian Cells ◽

Three Dimensional ◽

Human Cells ◽

Multiple Roles ◽

Multinucleated Cells ◽

Chromosomal Passenger ◽

Dimensional Reconstruction ◽

Spindle Midzone ◽

Interfering Rna

The INCENP (inner centromere protein) is a chromosomal passenger protein that plays multiple roles in regulating mitosis and cytokinesis. The MKLP1 (mitotic kinesin-like protein) is a component of centralspindlin complex that has been implicated in assembly of midzone/midbody during mitosis and is essential for cytokinesis. In the present study, we investigated functions of INCNEP and MKLP1 and their interplay in regulating spindle midzone/midbody formation and cytokinesis in human cells. Immunofluorescence and live-cell imaging analyses have shown that, in addition to multiple chromosome segregation defects, cells that lacked INCENP by RNAi (RNA interference) exhibit abnormal spindle midzone/midbody formation, resulting in formation of binucleated/multinucleated cells. Suppression of MKLP1 expression by siRNA (small interfering RNA) did not cause any abnormality of chromosome segregation and midzone formation, but abrogated midbody formation and completion of cytokinesis. Furthermore, we show that INCENP is required for recruiting MKLP1 to the spindle midzone/midbody. Three-dimensional reconstruction imaging analysis suggests that recruitment of MKLP1 to the midzone/midbody by INCENP is a crucial step for the midbody formation and completion of cytokinesis in mammalian cells.

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