Cdk1 phosphorylation of fission yeast paxillin inhibits its cytokinetic ring localization

Divisions of the genetic material and cytoplasm are coordinated spatially and temporally to ensure genome integrity. This coordination is mediated in part by the major cell cycle regulator cyclin-dependent kinase (Cdk1). Cdk1 activity peaks during mitosis, but during mitotic exit/cytokinesis Cdk1 activity is reduced, and phosphorylation of its substrates is reversed by various phosphatases including Cdc14, PP1, PP2A and PP2B. Cdk1 is known to phosphorylate several components of the actin- and myosin-based cytokinetic ring (CR) that mediates division of yeast and animal cells. Here we show that Cdk1 also phosphorylates the Schizosaccharomyces pombe CR-component paxillin Pxl1. We determined that both the Cdc14 phosphatase Clp1 and the PP1 phosphatase Dis2 contribute to Pxl1 dephosphorylation at mitotic exit, but PP2B/calcineurin does not. Preventing Pxl1 phosphorylation by Cdk1 results in increased Pxl1 levels, precocious Pxl1 recruitment to the division site, and increases the duration of CR constriction. In vitro, Cdk1-mediated phosphorylation of Pxl1 inhibits its interaction with the F-BAR domain of the cytokinetic scaffold Cdc15, thereby disrupting a major mechanism of Pxl1 recruitment. Thus, Pxl1 is a novel substrate through which S. pombe Cdk1 and opposing phosphatases coordinate mitosis and cytokinesis.

Download Full-text

Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

Molecular Biology of the Cell ◽

10.1091/mbc.01-11-0535 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2289-2300 ◽

Cited By ~ 92

Author(s):

Brett K. Kaiser ◽

Zachary A. Zimmerman ◽

Harry Charbonneau ◽

Peter K. Jackson

Keyword(s):

Cell Cycle ◽

Phosphatase Activity ◽

Chromosome Segregation ◽

Mitotic Spindle ◽

Genomic Stability ◽

Mitotic Exit ◽

Protein Abundance ◽

Cyclin Dependent Kinase ◽

Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

Download Full-text

Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e12-08-0608 ◽

2013 ◽

Vol 24 (9) ◽

pp. 1290-1304 ◽

Cited By ~ 31

Author(s):

Franz Meitinger ◽

Saravanan Palani ◽

Birgit Hub ◽

Gislene Pereira

Keyword(s):

Critical Role ◽

Coiled Coil ◽

Dual Function ◽

Serine Residue ◽

Bar Domain ◽

Division Site ◽

Coiled Coil Region ◽

Ndr Kinase

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR). Neither the function of Hof1 at the septin complex nor the mechanism by which Hof1 supports AMR constriction is understood. Here we establish that Dbf2 has a dual function in Hof1 regulation. First, we show that the coiled-coil region, which is adjacent to the conserved F-BAR domain, is required for the binding of Hof1 to septins. The Dbf2-dependent phosphorylation of Hof1 at a single serine residue (serine 313) in this region diminishes the recruitment of Hof1 to septins both in vitro and in vivo. Genetic and functional analysis indicates that the binding of Hof1 to septins is important for septin rearrangement and integrity during cytokinesis. Furthermore, Dbf2 phosphorylation of Hof1 at serines 533 and 563 promotes AMR constriction most likely by inhibiting the SH3-domain–dependent interactions of Hof1. Thus our data show that Dbf2 coordinates septin and AMR functions during cytokinesis through the regulation/control of Hof1.

Download Full-text

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

Protein and Peptide Letters ◽

10.2174/0929866526666191025102902 ◽

2020 ◽

Vol 27 (5) ◽

pp. 432-446

Author(s):

Akiko Yamamoto ◽

Ken-ichiro Matsunaga ◽

Toyoaki Anai ◽

Hitoshi Kawano ◽

Toshihisa Ueda ◽

...

Keyword(s):

In Situ Hybridization ◽

Immunohistochemical Analysis ◽

Protein Characterization ◽

Intermediate Filament Protein ◽

Tail Domain ◽

Animal Cells ◽

Dugesia Japonica ◽

Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

Download Full-text

Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical Communications ◽

10.1039/c4cc08862j ◽

2015 ◽

Vol 51 (37) ◽

pp. 7887-7890 ◽

Cited By ~ 17

Author(s):

Hideto Maruyama ◽

Kazuhiro Furukawa ◽

Hiroyuki Kamiya ◽

Noriaki Minakawa ◽

Akira Matsuda

Keyword(s):

Nucleic Acids ◽

Mammalian Cells ◽

Genetic Material ◽

Rna Polymerases ◽

Chemically Modified ◽

Biological Studies ◽

Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

Download Full-text

Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽

10.3390/cancers13092116 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2116

Author(s):

Xiaoyong Wang ◽

Lijuan Zhang ◽

Qi Dai ◽

Hongzong Si ◽

Longyun Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cell ◽

Breast Cancer Cells ◽

Cultured Cells ◽

Inhibitory Effect ◽

Cyclin Dependent Kinase ◽

Xenograft Mice ◽

The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

Download Full-text

DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽

10.3390/cells10061357 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1357

Author(s):

Rubén Torres ◽

Carolina Gándara ◽

Begoña Carrasco ◽

Ignacio Baquedano ◽

Silvia Ayora ◽

...

Keyword(s):

Atp Hydrolysis ◽

Holliday Junctions ◽

Genome Integrity ◽

Stable Complex ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Structures ◽

Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

Download Full-text

Mitochondrial enrichment in infertile patients: a review of different mitochondrial replacement therapies

Therapeutic Advances in Reproductive Health ◽

10.1177/26334941211023544 ◽

2021 ◽

Vol 15 ◽

pp. 263349412110235

Author(s):

Cristina Rodríguez-Varela ◽

Sonia Herraiz ◽

Elena Labarta

Keyword(s):

Stem Cells ◽

Genetic Material ◽

External Source ◽

Oocyte Quality ◽

Third Party ◽

In Vitro Fertilisation ◽

Mitochondrial Activity ◽

Mitochondrial Transfer ◽

Infertile Patients

Poor ovarian responders exhibit a quantitative reduction in their follicular pool, and most cases are also associated with poor oocyte quality due to patient’s age, which leads to impaired in vitro fertilisation outcomes. In particular, poor oocyte quality has been related to mitochondrial dysfunction and/or low mitochondrial count as these organelles are crucial in many essential oocyte processes. Therefore, mitochondrial enrichment has been proposed as a potential therapy option in infertile patients to improve oocyte quality and subsequent in vitro fertilisation outcomes. Nowadays, different options are available for mitochondrial enrichment treatments that are encompassed in two main approaches: heterologous and autologous. In the heterologous approach, mitochondria come from an external source, which is an oocyte donor. These techniques include transferring either a portion of the donor’s oocyte cytoplasm to the recipient oocyte or nuclear material from the patient to the donor’s oocyte. In any case, this approach entails many ethical and safety concerns that mainly arise from the uncertain degree of mitochondrial heteroplasmy deriving from it. Thus the autologous approach is considered a suitable potential tool to improve oocyte quality by overcoming the heteroplasmy issue. Autologous mitochondrial transfer, however, has not yielded as many beneficial outcomes as initially expected. Proposed mitochondrial autologous sources include immature oocytes, granulosa cells, germline stem cells, and adipose-derived stem cells. Presently, it would seem that these autologous techniques do not improve clinical outcomes in human infertile patients. However, further trials still need to be performed to confirm these results. Besides these two main categories, new strategies have arisen for oocyte rejuvenation by improving patient’s own mitochondrial function and avoiding the unknown consequences of third-party genetic material. This is the case of antioxidants, which may enhance mitochondrial activity by counteracting and/or preventing oxidative stress damage. Among others, coenzyme-Q10 and melatonin have shown promising results in low-prognosis infertile patients, although further randomised clinical trials are still necessary.

Download Full-text

Cyclin-Dependent Kinase Regulatory Subunit 2 Indicated Poor Prognosis and Facilitated Aggressive Phenotype of Hepatocellular Carcinoma

Disease Markers ◽

10.1155/2019/8964015 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13

Author(s):

Jie Zhang ◽

Qianqian Song ◽

Jinxia Liu ◽

Lina Lu ◽

Yuqing Xu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Poor Prognosis ◽

Disease Free Survival ◽

Normal Liver ◽

Regulatory Subunit ◽

Mrna Levels ◽

Cyclin Dependent Kinase ◽

Normal Tissues ◽

Hcc Cells

Cyclin-dependent kinase regulatory subunit 2 (CKS2) is a member of the cell cycle-dependent protein kinase subunit family, which is implicated as an oncogene in various malignancies. However, the clinical significance, oncogenic functions, and related mechanisms of CKS2 in hepatocellular carcinoma (HCC) remain largely unclear. In the present study, expression features and prognostic value of CKS2 were evaluated in the bioinformatic databases and HCC tissues. The effects of CKS2 on the malignant phenotypes of HCC cells were explored in vitro. According to the analyses of three bioinformatic databases, mRNA levels of CKS2 were elevated in HCC tissues compared with the normal tissues. Immunohistochemical assays found that high CKS2 expression was closely associated with liver cirrhosis (P=0.019), poor differentiation (P=0.02), portal vein invasion (P<0.001), TNM stage (P=0.019), tumor metastasis (P=0.008), and recurrence (P=0.003). The multivariate regression analyses suggested that CKS2 was an independent prognostic factor for overall survival (HR=2.088, P=0.014) and disease-free survival (HR=2.511, P=0.002) of HCC patients. Moreover, the bioinformatic analyses indicated that CKS2 might be associated with the malignant phenotypes in HCC progression. In addition, in vitro assays showed that CKS2 expression was higher in HCC cell lines than in normal liver cells. Knockdown of CKS2 remarkably repressed the proliferation, colony formation (P=0.0003), chemoresistance, migration (P=0.0047), and invasion (P=0.0012) of HCC cells. Taken together, overexpression of CKS2 was significantly correlated with poor prognosis of HCC patients and the malignant phenotypes of HCC cells, suggesting that it was a novel prognostic biomarker and potential target of HCC.

Download Full-text

Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1524538113 ◽

2016 ◽

Vol 113 (17) ◽

pp. 4788-4793 ◽

Cited By ~ 21

Author(s):

Monica Markovski ◽

Jessica L. Bohrhunter ◽

Tania J. Lupoli ◽

Tsuyoshi Uehara ◽

Suzanne Walker ◽

...

Keyword(s):

Polymerase Activity ◽

Activation Mechanism ◽

Phenotypic Analysis ◽

Outer Membranes ◽

Division Site ◽

Physiological Relevance ◽

Membrane Lipoproteins ◽

Shed Light

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a ofEscherichia colirequire the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b–LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

Download Full-text

Effect of guaianolides in the meiosis reinitiation of amphibian oocytes

Zygote ◽

10.1017/s0967199416000265 ◽

2016 ◽

Vol 25 (1) ◽

pp. 10-16 ◽

Cited By ~ 2

Author(s):

J. Zapata-Martínez ◽

G. Sánchez-Toranzo ◽

F. Chaín ◽

C.A.N. Catalán ◽

M.I. Bühler

Keyword(s):

Biological Activities ◽

Wide Spectrum ◽

Sesquiterpene Lactones ◽

Inhibitory Effect ◽

Biological Molecules ◽

Plant Metabolites ◽

Major Mechanism ◽

Bufo Arenarum ◽

Asteraceae Family

SummarySesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure–activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β–α′,β′-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

Download Full-text