scholarly journals Quantitative phosphoproteomics reveals regulatory state dependent efficacy of chemical Cdk1 inhibition and distinct Cdk5 complexes as novel Wee1 substrates

2021 ◽  
Author(s):  
Andrew V. Grassetti ◽  
Rufus Hards ◽  
Scott A. Gerber
Keyword(s):  
Phosphorylation Site ◽  
Eukaryotic Cell ◽  
Human Cells ◽  
Cyclin B ◽  
Regulatory State ◽  
State Dependent ◽  
Damage Repair ◽  
Signaling Axis ◽  
Wee1 Kinase ◽  
Chemical Inhibition

Wee1 kinase plays a central role in the eukaryotic cell cycle via its well-known negative regulation of Cdk1 activity at the G2/M transition, preventing progression into mitosis until DNA replication and/or DNA damage repair is complete. Recent genetic evidence in yeast, flies and human cells have suggested additional functions of Wee1 in mitosis and during mitotic exit, respectively. To discover new candidate substrates of Wee1 kinase, we used SILAC-based phosphoproteomics and selective chemical inhibition to quantitatively compare phosphorylation site abundances in the presence and absence of Wee1 activity. Unexpectedly, we uncovered a role for the Wee1-dependent phosphorylation of Cdk1-cyclin B at tyrosine 15 (Y15) in facilitating chemical inhibition of Cdk1-cyclin B by the inhibitor RO3306. Thermal shift stability assays demonstrated greater binding affinity of RO3306 for Y15-phosphorylated Cdk1-cyclin B versus unphosphorylated complex, providing an additional molecular basis for the observed Wee1 inhibitor-based toxicity in human cells. In addition, our experiments identified Cdk5-CABLES and Cdk5-cyclin B as novel substrates of Wee1 during chemically induced exit from mitosis. Collectively, these experiments facilitate a greater understanding of the Wee1-Cdk1 signaling axis and uncover new candidate substrates for Wee1.

scholarly journals Fine Tuning the Cell Cycle: Activation of the Cdk1 Inhibitory Phosphorylation Pathway during Mitotic Exit

2009 ◽  
Vol 20 (6) ◽  
pp. 1737-1748 ◽  
Author(s):  
Tamara A. Potapova ◽  
John R. Daum ◽  
Kendra S. Byrd ◽  
Gary J. Gorbsky
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Mitotic Exit ◽  
Fine Tuning ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Cell Cycle Activation ◽  
M Phase ◽  
Dependent Cell ◽  
Chemical Inhibition

Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle. We present evidence that down-regulation of Cdk1 activates Wee1 and Myt1 kinases and inhibits Cdc25 phosphatase during the M to G1 transition. If cyclin B/Cdk1 complex is present in G1, the inhibitory sites on Cdk1 become phosphorylated. Exit from mitosis induced by chemical Cdk inhibition can be reversed if cyclin B is preserved. However, this reversibility decreases with time after mitotic exit despite the continued presence of the cyclin. We show that this G1 block is due to phosphorylation of Cdk1 on inhibitory residues T14 and Y15. Chemical inhibition of Wee1 and Myt1 or expression of Cdk1 phosphorylation site mutants allows reversal to M phase even from late G1. This late Cdk1 reactivation often results in caspase-dependent cell death. Thus, in G1, the Cdk inhibitory phosphorylation pathway is functional and can lock Cdk1 in the inactive state.

scholarly journals PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

2020 ◽  
Author(s):  
James Bancroft ◽  
James Holder ◽  
Zoë Geraghty ◽  
Tatiana Alfonso-Pérez ◽  
Daniel Murphy ◽  
...  
Keyword(s):  
Sister Chromatid ◽  
Human Cells ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Checkpoint Response ◽  
Ubiquitin E3 Ligase ◽  
Chromatid Segregation ◽  
Chromosome Alignment ◽  
N Terminus ◽  
Chemical Inhibition

AbstractUbiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

Subcellular localisation of human wee1 kinase is regulated during the cell cycle

1995 ◽  
Vol 108 (6) ◽  
pp. 2425-2432
Author(s):  
V. Baldin ◽  
B. Ducommun
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Tyrosine Phosphatase ◽  
Cyclin B ◽  
Microtubule Assembly ◽  
Subcellular Localisation ◽  
Temporal Regulation ◽  
Wee1 Kinase ◽  
Cdc2 Activity ◽  
Intracellular Localisation

Wee1 kinase-dependent phosphorylation of cdc2 maintains the cdc2/cyclin B complex in an inert form until it is activated by the cdc25 tyrosine phosphatase at the end of G2. As described for cdc25, cell cycle-linked changes in the intracellular localisation of wee1 may constitute an important aspect of the temporal regulation of cdc2 activity. Here we report that the subcellular distribution of human wee1 changes during the cell cycle in HeLa and IMR90 cells. During interphase, wee1 is found almost exclusively in the nucleus. When the cell enters mitosis, wee1 is relocalised into the cytoplasm. During cell division, wee1 becomes restricted to the mitotic equator and by the end of mitosis it is found exclusively in association with midbody bridges, a phenomenon that is dependent on microtubule assembly. The relocalisations of wee1 and its association with subcellular structures may play key regulatory roles at different stages of the cell cycle and during mitosis.

scholarly journals Requirements for Cdk7 in the Assembly of Cdk1/Cyclin B and Activation of Cdk2 Revealed by Chemical Genetics in Human Cells

2007 ◽  
Vol 25 (6) ◽  
pp. 839-850 ◽  
Author(s):  
Stéphane Larochelle ◽  
Karl A. Merrick ◽  
Marie-Emilie Terret ◽  
Lara Wohlbold ◽  
Nora M. Barboza ◽  
...  
Keyword(s):  
Chemical Genetics ◽  
Human Cells ◽  
Cyclin B

scholarly journals Nucleolar Localization and Dynamic Roles of Flap Endonuclease 1 in Ribosomal DNA Replication and Damage Repair

2008 ◽  
Vol 28 (13) ◽  
pp. 4310-4319 ◽  
Author(s):  
Zhigang Guo ◽  
Limin Qian ◽  
Ren Liu ◽  
Huifang Dai ◽  
Mian Zhou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Phosphorylation Site ◽  
Replication Forks ◽  
Repair Capacity ◽  
Flap Endonuclease 1 ◽  
Damage Repair ◽  
Dependent Endonuclease ◽  
Cross Links ◽  
Cellular Compartmentalization

ABSTRACT Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser187Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser187 by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.

scholarly journals Arp2/3 complex-mediated actin assembly drives microtubule-independent motility and phagocytosis in the evolutionarily divergent amoeba Naegleria

2020 ◽  
Author(s):  
Katrina B. Velle ◽  
Lillian K. Fritz-Laylin
Keyword(s):  
Actin Cytoskeleton ◽  
Common Ancestor ◽  
Genomic Analysis ◽  
Eukaryotic Cell ◽  
Model System ◽  
Human Cells ◽  
Actin Assembly ◽  
Cell Type ◽  
Cytoplasmic Microtubules ◽  
Work Supports

ABSTRACTMuch of our current understanding of actin-driven phenotypes in eukaryotes has come from the “yeast to human” opisthokont lineage, as well as the related amoebozoa. Outside of these groups lies the genus Naegleria, which shared a common ancestor with humans over a billion years ago, and includes the deadly “brain-eating amoeba.” Unlike nearly every other known eukaryotic cell type, Naegleria amoebae are thought to lack cytoplasmic microtubules. The absence of microtubules suggests that these amoebae rapidly crawl and phagocytose bacteria using actin alone. Although this makes Naegleria a powerful system to probe actin-driven functions in the absence of microtubules, surprisingly little is known about Naegleria’s actin cytoskeleton. Here, we use microscopy and genomic analysis to show that Naegleria amoebae have an extensive actin cytoskeletal repertoire, complete with nucleators and nucleation promoting factors. Naegleria use this cytoskeletal machinery to generate Arp2/3-dependent lamellar protrusions, which correlate with the capacity to migrate and phagocytose bacteria. Because human cells also use Arp2/3-dependent lamellar protrusions for motility and phagocytosis, this work supports an evolutionarily ancient origin for these actin-driven processes and establishes Naegleria as a natural model system for studying microtubule-independent cytoskeletal phenotypes.

scholarly journals Plasma membrane damage removal by F-actin-mediated shedding from repurposed filopodia

2019 ◽  
Author(s):  
Shrawan Kumar Mageswaran ◽  
Wei Y. Yang ◽  
Yogaditya Chakrabarty ◽  
Catherine M. Oikonomou ◽  
Grant J. Jensen
Keyword(s):  
Plasma Membrane ◽  
Membrane Damage ◽  
Repair Process ◽  
Eukaryotic Cell ◽  
Actin Dynamics ◽  
Membrane Domains ◽  
Plasma Membrane Damage ◽  
Damage Repair ◽  
Electron Cryotomography ◽  
Membrane Shedding

AbstractRepairing plasma membrane damage is vital to eukaryotic cell survival. Membrane shedding is thought to be key to this repair process, but a detailed view of how the process occurs is still missing. Here we used electron cryotomography to image the ultrastructural details of plasma membrane wound healing. We found that filopodia-like protrusions are built at damage sites, accompanied by retraction of neighboring filopodia, and that these repurposed protrusions act as scaffolds for membrane shedding. This suggests a new role for filopodia as reservoirs of membrane and actin for plasma membrane damage repair. Damage-induced shedding was dependent on F-actin dynamics and Myo1a, as well as Vps4B, an important component of the ESCRT machinery. Thus we find that damage shedding is more complex than current models of simple vesiculation from flat membrane domains. Rather, we observe structural similarities between damage-mediated shedding and constitutive shedding from enterocytes that argue for conservation of a general membrane shedding mechanism.

scholarly journals Cyclin B/cdc2 Induces c-Mos Stability by Direct Phosphorylation in Xenopus Oocytes

2001 ◽  
Vol 12 (9) ◽  
pp. 2660-2671 ◽  
Author(s):  
Anna Castro ◽  
Marion Peter ◽  
Laura Magnaghi-Jaulin ◽  
Suzanne Vigneron ◽  
Simon Galas ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Phosphorylation Site ◽  
Cyclin B ◽  
Mobility Shift ◽  
Cdc2 Kinase ◽  
Phosphopeptide Analysis ◽  
Vertebrate Eggs ◽  
The One

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described ( Freeman et al., 1992 ). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.

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