Poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis

The timing of centrosome separation and the distance moved apart influence the formation of the bipolar spindle, affecting chromosome stability. Epidermal growth factor receptor (EGFR) signaling induces early centrosome separation through downstream G protein-coupled receptor kinase GRK2, which phosphorylates the Hippo pathway component MST2 (Mammalian STE20-like protein kinase 2), in turn allowing NIMA kinase Nek2A activation for centrosomal linker disassembly. However, the mechanisms that counterbalance centrosome disjunction and separation remain poorly understood. We unveil that timely degradation of GRK2 by the E3 ligase Mdm2 limits centrosome separation in the G2. Both knockout expression and catalytic inhibition of Mdm2 result in GRK2 accumulation and enhanced centrosome separation before mitosis onset. Phosphorylation of GRK2 on residue S670 enables a complex pattern of non-K48-linked polyubiquitin chains assembled by Mdm2, which correlate with kinase protein degradation. Remarkably, GRK2-S670A protein fails to phosphorylate MST2 despite overcoming Mdm2-dependent degradation, which results in defective centrosome separation, shorter spindles, and abnormal chromosome congression. Conversely, extra levels of wild-type kinase in the G2 cause increased inter-centrosome distances with longer spindles, also converging in congression issues. Our findings show that the signals enabling activity of the GRK2/MST2/Nek2A axis for separation also switches on Mdm2 degradation of GRK2 to ensure accurate centrosome dynamics and proper mitotic spindle functionality.

Download Full-text

Depletion of Centromeric MCAK Leads to Chromosome Congression and Segregation Defects Due to Improper Kinetochore Attachments

Molecular Biology of the Cell ◽

10.1091/mbc.e03-08-0581 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1146-1159 ◽

Cited By ~ 192

Author(s):

Susan L. Kline-Smith ◽

Alexey Khodjakov ◽

Polla Hergert ◽

Claire E. Walczak

Keyword(s):

Essential Role ◽

Dominant Negative ◽

Primary Role ◽

Complex Behavior ◽

Chromosome Movement ◽

Immunofluorescence Localization ◽

Microtubule Attachment ◽

Chromosome Congression

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore–microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore–microtubule attachments.

Download Full-text

RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0511 ◽

2009 ◽

Vol 20 (1) ◽

pp. 410-418 ◽

Cited By ~ 81

Author(s):

Ulf R. Klein ◽

Markus Haindl ◽

Erich A. Nigg ◽

Stefan Muller

Keyword(s):

Mammalian Cells ◽

Spindle Assembly Checkpoint ◽

Critical Role ◽

Specific Interaction ◽

Regulatory Function ◽

Mitotic Progression ◽

Chromosome Congression ◽

Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

Download Full-text

BUB-1 targets PP2A:B56 to regulate chromosome congression during meiosis I in C. elegans oocytes

10.1101/2020.06.12.148254 ◽

2020 ◽

Author(s):

Laura Bel Borja ◽

Flavie Soubigou ◽

Samuel J.P. Taylor ◽

Conchita Fraguas Bringas ◽

Jacqueline Budrewicz ◽

...

Keyword(s):

Kinase Domain ◽

Meiotic Spindle ◽

Dependent Manner ◽

Female Meiosis ◽

Linear Motif ◽

Interaction Domain ◽

C Elegans ◽

Chromosome Congression ◽

B Subunits ◽

Meiosis I

ABSTRACTProtein Phosphatase 2A (PP2A) is an heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits with various key roles during cell division. While A and C subunits form the core enzyme, the diversity generated by interchangeable B subunits dictates substrate specificity. Within the B subunits, B56-type subunits play important roles during meiosis in yeast and mice by protecting centromeric cohesion and stabilising the kinetochore-microtubule attachments. These functions are achieved through targeting of B56 subunits to centromere and kinetochore by Shugoshin and BUBR1. In the nematode Caenorhabditis elegans (C. elegans) the closest BUBR1 ortholog lacks the B56 interaction domain and the Shugoshin orthologue is not required for normal segregation during oocyte meiosis. Therefore, the role of PP2A in C. elegans female meiosis is not known. Here, we report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. Specifically, B56 subunits PPTR-1 and PPTR-2 associate with chromosomes during prometaphase I and regulate chromosome congression. The chromosome localization of B56 subunits does not require shugoshin orthologue SGO-1. Instead we have identified the kinase BUB-1 as the key B56 targeting factor to the chromosomes during meiosis. PP2A BUB-1 recruits PP2A:B56 to the chromosomes via dual mechanism: 1) PPTR-1/2 interacts with the newly identified LxxIxE short linear motif (SLiM) within a disordered region in BUB-1 in a phosphorylation-dependent manner; and 2) PPTR-2 can also be recruited to chromosomes in a BUB-1 kinase domain-dependent manner. Our results highlight a novel, BUB-1-dependent mechanism for B56 recruitment, essential for recruiting a pool of PP2A required for proper chromosome congression during meiosis I.

Download Full-text

Wnt signaling recruits KIF2A to the spindle to ensure chromosome congression and alignment during mitosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2108145118 ◽

2021 ◽

Vol 118 (34) ◽

pp. e2108145118

Author(s):

Anja Bufe ◽

Ana García del Arco ◽

Magdalena Hennecke ◽

Anchel de Jaime-Soguero ◽

Matthias Ostermaier ◽

...

Keyword(s):

Stem Cells ◽

Cell Division ◽

Wnt Signaling ◽

Pluripotent Stem Cells ◽

Target Genes ◽

Genome Maintenance ◽

Spindle Poles ◽

Chromosome Congression ◽

Chromosome Alignment ◽

Canonical Wnt

Canonical Wnt signaling plays critical roles in development and tissue renewal by regulating β-catenin target genes. Recent evidence showed that β-catenin–independent Wnt signaling is also required for faithful execution of mitosis. However, the targets and specific functions of mitotic Wnt signaling still remain uncharacterized. Using phosphoproteomics, we identified that Wnt signaling regulates the microtubule depolymerase KIF2A during mitosis. We found that Dishevelled recruits KIF2A via its N-terminal and motor domains, which is further promoted upon LRP6 signalosome formation during cell division. We show that Wnt signaling modulates KIF2A interaction with PLK1, which is critical for KIF2A localization at the spindle. Accordingly, inhibition of basal Wnt signaling leads to chromosome misalignment in somatic cells and pluripotent stem cells. We propose that Wnt signaling monitors KIF2A activity at the spindle poles during mitosis to ensure timely chromosome alignment. Our findings highlight a function of Wnt signaling during cell division, which could have important implications for genome maintenance, notably in stem cells.

Download Full-text

The Human Kinesin Kif18A Is a Motile Microtubule Depolymerase Essential for Chromosome Congression

Current Biology ◽

10.1016/j.cub.2007.02.036 ◽

2007 ◽

Vol 17 (6) ◽

pp. 488-498 ◽

Cited By ~ 186

Author(s):

Monika I. Mayr ◽

Stefan Hümmer ◽

Jenny Bormann ◽

Tamara Grüner ◽

Sarah Adio ◽

...

Keyword(s):

Chromosome Congression

Download Full-text

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

Science Signaling ◽

10.1126/scisignal.aaw2939 ◽

2019 ◽

Vol 12 (594) ◽

pp. eaaw2939 ◽

Cited By ~ 6

Author(s):

Rozita Adib ◽

Jessica M. Montgomery ◽

Joseph Atherton ◽

Laura O’Regan ◽

Mark W. Richards ◽

...

Keyword(s):

Double Mutant ◽

Constitutive Activation ◽

Mitotic Kinases ◽

Terminal Domain ◽

Microtubule Stability ◽

Cryo Electron Microscopy ◽

Chromosome Congression ◽

Spindle Microtubules ◽

Mitotic Phosphorylation

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

Download Full-text