scholarly journals Distinct Phosphoisoforms of the XenopusMcm4 Protein Regulate the Function of the Mcm Complex

2000 ◽  
Vol 20 (10) ◽  
pp. 3667-3676 ◽  
Author(s):  
Inna Pereverzeva ◽  
Elizabeth Whitmire ◽  
Bettina Khan ◽  
Martine Coué
Keyword(s):  
Cell Cycle ◽  
Cyclin B ◽  
Detailed Characterization ◽  
Cyclin Dependent Kinases ◽  
Origins Of Replication ◽  
Mcm Complex ◽  
Initiation Of Dna Replication ◽  
And Function ◽  
Mitotic Phosphorylation

ABSTRACT Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.

Cdc6p establishes and maintains a state of replication competence during G1 phase

1997 ◽  
Vol 110 (6) ◽  
pp. 753-763 ◽  
Author(s):  
C.S. Detweiler ◽  
J.J. Li
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Transcriptional Repression ◽  
Prolonged Exposure ◽  
S Phase ◽  
Restrictive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Important Intermediate ◽  
Initiation Of Dna Replication ◽  
And Function

CDC6 is essential for the initiation of DNA replication in the budding yeast Saccharomyces cerevisiae. Here we examine the timing of Cdc6p expression and function during the cell cycle. Cdc6p is expressed primarily between mitosis and Start. This pattern of expression is due in part to posttranscriptional controls, since it is maintained when CDC6 is driven by a constitutively induced promoter. Transcriptional repression of CDC6 or exposure of cdc6-1(ts) cells to the restrictive temperature at mitosis blocks subsequent S phase, demonstrating that the activity of newly synthesized Cdc6p is required each cell cycle for DNA replication. In contrast, similar perturbations imposed on cells arrested in G(1) before Start have moderate or no effects on DNA replication. This suggests that, between mitosis and Start, Cdc6p functions in an early step of initiation, effectively making cells competent for replication. Prolonged exposure of cdc6-1(ts) cells to the restrictive temperature at the pre-Start arrest eventually does cripple S phase, indicating that Cdc6p also functions to maintain this initiation competence during G(1). The requirement for Cdc6p to establish and maintain initiation competence tightly correlates with the requirement for Cdc6p to establish and maintain the pre-replicative complex at a replication origin, strongly suggesting that the pre-replicative complex is an important intermediate for the initiation of DNA replication. Confining assembly of the complex to G(1) by restricting expression of Cdc6p to this period may be one way of ensuring precisely one round of replication per cell cycle.

scholarly journals Cell cycle analysis of the activity, subcellular localization, and subunit composition of human CAK (CDK-activating kinase).

1994 ◽  
Vol 127 (2) ◽  
pp. 467-478 ◽  
Author(s):  
J P Tassan ◽  
S J Schultz ◽  
J Bartek ◽  
E A Nigg
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Cell Cycle Control ◽  
Gel Filtration ◽  
Multiprotein Complex ◽  
Cyclin Dependent Kinases ◽  
Critical Residue ◽  
Cdk Activating Kinase

The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).

scholarly journals P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Elena Alcalde-Estévez ◽  
Ana Asenjo-Bueno ◽  
Patricia Sosa ◽  
Patricia Plaza ◽  
Diego Rodríguez-Puyol ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Muscle Regeneration ◽  
Uremic Toxins ◽  
C2c12 Cells ◽  
Cyclin B ◽  
Skeletal Muscle Regeneration ◽  
Regeneration Process ◽  
Cycle Arrest ◽  
Advanced Stages ◽  
And Function

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

Scanning Studies of Cell Surfaces

1973 ◽  
Vol 31 ◽  
pp. 608-609
Author(s):  
Virginia Fonte ◽  
Nancy Weller ◽  
Keith R. Porter
Keyword(s):  
Cell Cycle ◽  
Structural Organization ◽  
Cell Surfaces ◽  
Detailed Characterization ◽  
A Cell ◽  
Relationship Of ◽  
The Relationship ◽  
Scanning Electron

The surfaces of a cell in its topography and anti-genicity expresses subtle variations in the effective genome, as well as the physiology and structural organization of the underlying cytoplasm. Understanding the relationship of these various factors to the surface depends in part on obtaining a detailed characterization of the topography of cells and how this topography changes with phases in the cell cycle, with transformation to malignancy and with the cell's response to such physiologically active agents as cyclic AMP.We have therefore explored the usefulness of the scanning electron microscope in investigations of the cell's topography. Cells grown under favourable in vitro conditions have been fixed in glutaraldehyde, dehydrated in acetone and dried by the critical point method of Anderson.

scholarly journals Cell cycle regulation in Aspergillus by two protein kinases

1996 ◽  
Vol 317 (3) ◽  
pp. 633-641 ◽  
Author(s):  
Stephen A. OSMANI ◽  
Xiang S. YE
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Human Cells ◽  
Dominant Negative ◽  
Cyclin B ◽  
Cyclin Dependent Kinases ◽  
Cycle Regulation ◽  
Mitotic Regulation

Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.

scholarly journals Evolution of opposing regulatory interactions underlies the emergence of eukaryotic cell cycle checkpoints

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rosa D. Hernansaiz-Ballesteros ◽  
Csenge Földi ◽  
Luca Cardelli ◽  
László G. Nagy ◽  
Attila Csikász-Nagy
Keyword(s):  
Cell Cycle ◽  
General Rule ◽  
Eukaryotic Cell ◽  
Cell Cycle Checkpoints ◽  
General View ◽  
Cyclin Dependent Kinases ◽  
Phosphatase Regulation ◽  
Almost All ◽  
Mitotic Phosphorylation ◽  
Phosphate Groups

AbstractIn eukaryotes the entry into mitosis is initiated by activation of cyclin-dependent kinases (CDKs), which in turn activate a large number of protein kinases to induce all mitotic processes. The general view is that kinases are active in mitosis and phosphatases turn them off in interphase. Kinases activate each other by cross- and self-phosphorylation, while phosphatases remove these phosphate groups to inactivate kinases. Crucial exceptions to this general rule are the interphase kinase Wee1 and the mitotic phosphatase Cdc25. Together they directly control CDK in an opposite way of the general rule of mitotic phosphorylation and interphase dephosphorylation. Here we investigate why this opposite system emerged and got fixed in almost all eukaryotes. Our results show that this reversed action of a kinase-phosphatase pair, Wee1 and Cdc25, on CDK is particularly suited to establish a stable G2 phase and to add checkpoints to the cell cycle. We show that all these regulators appeared together in LECA (Last Eukaryote Common Ancestor) and co-evolved in eukaryotes, suggesting that this twist in kinase-phosphatase regulation was a crucial step happening at the emergence of eukaryotes.

scholarly journals Lineage Contribution of PDGFRα-Expressing Cells in the Developing Mouse Eye

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Deyi Zhuo ◽  
Yumei Diao ◽  
Xiaoqi Li ◽  
Yifei Huang ◽  
Liqiang Wang
Keyword(s):  
Stromal Cells ◽  
Eye Development ◽  
Early Embryogenesis ◽  
Detailed Characterization ◽  
Ocular Development ◽  
Fiber Cells ◽  
Retinal Astrocytes ◽  
Reporter Mice ◽  
And Function

PDGFRα signaling is critically important in ocular development. Previous data on PDGFRα lacks an expression map with high spatial and temporal resolution and lineage information. In this study, we aim to present a detailed PDGFRα expression and lineage map from early embryogenesis to adulthood. PDGFRα-CreER; mT/mG reporter mice were analyzed. mEGFP-positive cells contributed to multiple ocular lineages in a spatiotemporally regulated manner. A dynamic PDGFRα expression was identified in corneal stromal cells, lens epithelial cells, lens fiber cells, and retinal astrocytes during the entire period of eye development, while PDGFRα expression in retinal astrocytes from E17.5 onwards and in Müller glial cells was identified within two weeks after birth. By revealing detailed characterization of gene expression and function, we present a comprehensive map of PDGFRα-expressing cells in the eye for a better understanding of PDGFRα signaling’s role during eye development.

scholarly journals Excitation-contraction coupling and its relation to synaptic dysfunction in Drosophila

2020 ◽  
Author(s):  
Kiel G. Ormerod ◽  
Anthony E. Scibelli ◽  
J. Troy Littleton
Keyword(s):  
Muscle Performance ◽  
Synaptic Dysfunction ◽  
Muscle Contractility ◽  
Detailed Characterization ◽  
Synaptic Development ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
And Function ◽  
Identification And Characterization

AbstractThe Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic deficits alter neuromuscular transduction and muscle contractility that ultimately dictate behavioural output. Here we develop a system for detailed characterization of excitation-contraction coupling at Drosophila larval NMJs and demonstrate how specific synaptic and neuronal manipulations disrupt muscle contractility. Muscle contraction force increases with motoneuron stimulation frequency and duration, showing considerable plasticity between 5-40 Hz, while saturating above 50 Hz. Temperature is negatively correlated with muscle performance and enhanced at lower temperatures. A screen for modulators of muscle contractility led to the identification and characterization of the molecular and cellular pathway by which a specific FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to relate synaptic dysfunction to alterations in excitation-contraction coupling.

scholarly journals Deacetylation of catalytic lysine in CDK1 is essential for Cyclin-B binding and cell cycle

2018 ◽  
Author(s):  
Shaunak Deota ◽  
Sivasudhan Rathnachalam ◽  
Kanojia Namrata ◽  
Mayank Boob ◽  
Amit Fulzele ◽  
...  
Keyword(s):  
Cell Cycle ◽  
High Resolution ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Current Model ◽  
Cyclin B ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Evolutionarily Conserved ◽  
Cycle Dependent

AbstractCyclin-dependent-kinases (CDKs) are essential for cell cycle progression. While dependence of CDK activity on Cyclin levels is established, molecular mechanisms that regulate their binding are less studied. Here, we show that CDKl:Cyclin-B interactions are regulated by acetylation, which was hitherto unknown. We demonstrate that cell cycle dependent acetylation of the evolutionarily conserved catalytic lysine in CDK1 or eliminating its charge state abrogates Cyclin-B binding. Opposing activities of SIRT1 and P300 regulate acetylation, which marks a reserved pool of CDK1. Our high resolution structural analyses into the formation of kinase competent CDK1: Cyclin-B complex have unveiled long-range effects of catalytic lysine in configuring the CDK1 interface for Cyclin-B binding. Cells expressing acetylation mimic mutant of Cdc2 in yeast are arrested in G2 and fail to divide. Thus, by illustrating cell cycle dependent deacetylation as a determinant of CDK1:Cyclin-B interaction, our results redefine the current model of CDK1 activation and cell cycle progression.

scholarly journals p31comet acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment

2011 ◽  
Vol 22 (22) ◽  
pp. 4236-4246 ◽  
Author(s):  
Robert S. Hagan ◽  
Michael S. Manak ◽  
Håkon Kirkeby Buch ◽  
Michelle G. Meier ◽  
Patrick Meraldi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Mitotic Exit ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Microtubule Attachment ◽  
And Function ◽  
High Cyclin

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a “wait anaphase” signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31comet, a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31comet during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31comet traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31comet arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31comet is required for timely mitotic exit. We propose that p31comet is an essential component of the machinery that silences the checkpoint during each cell cycle.

Sign in / Sign up

Export Citation Format

Share Document