CDK1 and PLK1 cooperate to regulate BICD2, dynein activation and recruitment to the nucleus as well as centrosome separation in G2/M

The activity of dynein is regulated by a number of adaptors that mediate its interaction with dynactin, effectively activating the motor complex while also connecting it to different cargos. The regulation of adapters is consequently central to dynein physiology, but remains largely unexplored. We now describe that one of the best-known dynein adaptors, BICD2, is effectively activated through phosphorylation. In G2 phosphorylation of BICD2 by CDK1 promotes its interaction with PLK1. In turn, PLK1 phosphorylation of a single residue in the N-terminus of BICD2 results in a conformational change that facilitates interaction with dynein and dynactin, allowing the formation of active motor complexes. BICD2 phosphorylation is central for dynein recruitment to the nuclear envelope, centrosome tethering to the nucleus and centrosome separation in G2/M. This work reveals adapter activation through phosphorylation as crucial for the spatiotemporal regulation of dynein activity.

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Degradation of ornithine decarboxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2377-2383.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2377-2383

Author(s):

X Li ◽

P Coffino

Keyword(s):

Conformational Change ◽

Ornithine Decarboxylase ◽

Binding Protein ◽

Active Form ◽

Feedback Regulation ◽

Degradation Process ◽

Protein Inhibitor ◽

Inhibitory Protein ◽

C Terminus ◽

N Terminus

Polyamine-mediated degradation of vertebrate ornithine decarboxylase (ODC) is associated with the production of antizyme, a reversible tightly binding protein inhibitor of ODC activity. The interaction of antizyme with a binding element near the N terminus of ODC is essential but not sufficient for regulation of the enzyme by polyamines (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-2562, 1992). We now show that a second element present at the C terminus is required for the degradation process. Antizyme caused a conformational change in ODC, which made the C terminus of ODC more accessible. Blocking the C terminus with antibody prevented degradation. Tethering the C terminus by creating a circularly permuted, enzymatically active form of ODC prevented antizyme-mediated degradation. These data elucidate a form of feedback regulation whereby excess polyamines induce destruction of ODC, the enzyme that initiates their biosynthesis.

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The Vaccinia-related Kinases Phosphorylate the N′ Terminus of BAF, Regulating Its Interaction with DNA and Its Retention in the Nucleus

Molecular Biology of the Cell ◽

10.1091/mbc.e05-12-1179 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2451-2464 ◽

Cited By ~ 145

Author(s):

R. Jeremy Nichols ◽

Matthew S. Wiebe ◽

Paula Traktman

Keyword(s):

Drosophila Melanogaster ◽

Cell Division ◽

Nuclear Envelope ◽

Temperature Sensitivity ◽

Nuclear Membrane ◽

Essential Role ◽

Nuclear Architecture ◽

Nuclear Chromatin ◽

N Terminus ◽

Protein Barrier

The vaccinia-related kinases (VRKs) comprise a branch of the casein kinase family whose members are characterized by homology to the vaccinia virus B1 kinase. The VRK orthologues encoded by Caenorhabditis elegans and Drosophila melanogaster play an essential role in cell division; however, substrates that mediate this role have yet to be elucidated. VRK1 can complement the temperature sensitivity of a vaccinia B1 mutant, implying that VRK1 and B1 have overlapping substrate specificity. Herein, we demonstrate that B1, VRK1, and VRK2 efficiently phosphorylate the extreme N′ terminus of the BAF protein (Barrier to Autointegration Factor). BAF binds to both DNA and LEM domain-containing proteins of the inner nuclear membrane; in lower eukaryotes, BAF has been shown to play an important role during the reassembly of the nuclear envelope at the end of mitosis. We demonstrate that phosphorylation of ser4 and/or thr2/thr3 abrogates the interaction of BAF with DNA and reduces its interaction with the LEM domain. Coexpression of VRK1 and GFP-BAF greatly diminishes the association of BAF with the nuclear chromatin/matrix and leads to its dispersal throughout the cell. Cumulatively, our data suggest that the VRKs may modulate the association of BAF with nuclear components and hence play a role in maintaining appropriate nuclear architecture.

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N-terminal regions of Mps1 kinase determine functional bifurcation

The Journal of Cell Biology ◽

10.1083/jcb.200910027 ◽

2010 ◽

Vol 189 (1) ◽

pp. 41-56 ◽

Cited By ~ 38

Author(s):

Yasuhiro Araki ◽

Linda Gombos ◽

Suellen P.S. Migueleti ◽

Lavanya Sivashanmugam ◽

Claude Antony ◽

...

Keyword(s):

Nuclear Envelope ◽

Chemical Biology ◽

Molecular Level ◽

Budding Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Deletion Analysis ◽

Checkpoint Activation ◽

Pole Body ◽

N Terminus

Mps1 is a conserved kinase that in budding yeast functions in duplication of the spindle pole body (SPB), spindle checkpoint activation, and kinetochore biorientation. The identity of Mps1 targets and the subdomains that convey specificity remain largely unexplored. Using a novel combination of systematic deletion analysis and chemical biology, we identified two regions within the N terminus of Mps1 that are essential for either SPB duplication or kinetochore biorientation. Suppression analysis of the MPS1 mutants defective in SPB duplication and biochemical enrichment of Mps1 identified the essential SPB components Spc29 and the yeast centrin Cdc31 as Mps1 targets in SPB duplication. Our data suggest that phosphorylation of Spc29 by Mps1 in G1/S recruits the Mps2–Bbp1 complex to the newly formed SPB to facilitate its insertion into the nuclear envelope. Mps1 phosphorylation of Cdc31 at the conserved T110 residue controls substrate binding to Kar1 protein. These findings explain the multiple SPB duplication defects of mps1 mutants on a molecular level.

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Membrane binding by CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation

10.1101/049221 ◽

2016 ◽

Cited By ~ 1

Author(s):

Yolanda Olmos ◽

Anna Perdrix ◽

Jeremy G Carlton

Keyword(s):

Nuclear Envelope ◽

Membrane Binding ◽

Point Mutations ◽

Binding Activity ◽

Mitotic Exit ◽

Exit Point ◽

Cellular Functions ◽

Endosomal Sorting ◽

N Terminus ◽

And Function

AbstractAmongst other cellular functions, the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) machinery controls nuclear envelope (NE) reformation during mitotic exit by sealing holes in the reforming NE. ESCRT-III also acts to repair this organelle upon migration-induced rupture. The ESCRT-III component CHMP7 is responsible for recruitment of ESCRT-III to the NE. Here, we show that the N-terminus of CHMP7, comprising tandem Winged Helix (WH)-domains, is a membrane-binding module. This activity allows CHMP7 to bind to the Endoplasmic Reticulum (ER), an organelle continuous with the NE, and provides a platform to direct NE-recruitment of ESCRT-III during mitotic exit. Point mutations that disrupt membrane-binding prevent CHMP7 localising to the ER and its subsequent enrichment at the reforming NE. These mutations prevent both assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalisation. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.One Sentence SummaryCHMP7’s atypical N-terminus is a membrane-binding module that allows assembly and function of ESCRT-III at the nuclear envelope during mitotic exit.

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Trackosome: a computational toolbox to study the spatiotemporal dynamics of centrosomes, nuclear envelope and cellular membrane

10.1101/2020.04.27.064204 ◽

2020 ◽

Author(s):

Domingos Castro ◽

Vanessa Nunes ◽

Joana T. Lima ◽

Jorge G. Ferreira ◽

Paulo Aguiar

Keyword(s):

Nuclear Envelope ◽

Cellular Membrane ◽

Spatiotemporal Dynamics ◽

Imaging Analysis ◽

Imaging Data ◽

Analysis Software ◽

Model Free ◽

Centrosome Separation ◽

Efficient Access ◽

Spatiotemporal Relations

AbstractDuring the initial stages of mitosis, multiple mechanisms drive centrosome separation and positioning. How they are functionally coordinated to promote centrosome migration to opposite sides of the nucleus remains unclear. Imaging analysis software has been used to quantitatively study centrosome dynamics at this stage. However, available tracking tools are generic and not fine-tuned for the constrains and motion dynamics of centrosome pairs. Such generality limits the tracking performance and may require exhaustive optimization of parameters. Here, we present Trackosome, a freely available open-source computational tool to track the centrosomes and reconstruct the nuclear and cellular membranes, based on volumetric live-imaging data. The toolbox runs in MATLAB and provides a graphical user interface for easy and efficient access to the tracking and analysis algorithms. It outputs key metrics describing the spatiotemporal relations between centrosomes, nucleus and cellular membrane. Trackosome can also be used to measure the dynamic fluctuations of the nuclear envelope. A fine description of these fluctuations is important because they are correlated with the mechanical forces exerted on the nucleus by its adjacent cytoskeletal structures. Unlike previous algorithms based on circular/elliptical approximations of the nucleus, Trackosome measures membrane movement in a model-free condition, making it viable for irregularly shaped nuclei. Using Trackosome, we demonstrate significant correlations between the movements of the two centrosomes, and identify specific modes of oscillation of the nuclear envelope. Overall, Trackosome is a powerful tool to help unravel new elements in the spatiotemporal dynamics of subcellular structures.

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A Conformational Change in the N Terminus of SLC38A9 Signals mTORC1 Activation

Structure ◽

10.1016/j.str.2020.11.014 ◽

2020 ◽

Author(s):

Hsiang-Ting Lei ◽

Xuelang Mu ◽

Johan Hattne ◽

Tamir Gonen

Keyword(s):

Conformational Change ◽

N Terminus ◽

Mtorc1 Activation

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Helical α/β-depsipeptides with alternating residue types: conformational change from the 11-helix to the 14/15-helix

Organic & Biomolecular Chemistry ◽

10.1039/c6ob01602b ◽

2016 ◽

Vol 14 (36) ◽

pp. 8438-8442 ◽

Cited By ~ 1

Author(s):

Jaeyeon Lee ◽

Geunhyeok Jang ◽

Philjae Kang ◽

Moon-Gun Choi ◽

Soo Hyuk Choi

Keyword(s):

Lactic Acid ◽

Conformational Change ◽

The Third ◽

N Terminus ◽

Α Helix

Short α/β-depsipeptides of which the third residue from the N-terminus is an (S)-lactic acid residue predominantly adopt 14/15-helical conformations analogous to the α-helix.

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Timing of centrosome separation is important for accurate chromosome segregation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0095 ◽

2012 ◽

Vol 23 (3) ◽

pp. 401-411 ◽

Cited By ~ 94

Author(s):

William T. Silkworth ◽

Isaac K. Nardi ◽

Raja Paul ◽

Alex Mogilner ◽

Daniela Cimini

Keyword(s):

Nuclear Envelope ◽

Chromosome Segregation ◽

Intermediate Stage ◽

Spindle Pole ◽

Cellular Space ◽

Nuclear Envelope Breakdown ◽

Spindle Poles ◽

Centrosome Separation ◽

Large Numbers ◽

Close Proximity

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not “search” the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.

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