scholarly journals Activating Phosphorylation of theSaccharomyces cerevisiaeCyclin-dependent Kinase, Cdc28p, Precedes Cyclin Binding

2000 ◽  
Vol 11 (5) ◽  
pp. 1597-1609 ◽  
Author(s):  
Karen E. Ross ◽  
Philipp Kaldis ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Eukaryotic Cell ◽  
Regulatory Mechanisms ◽  
Wild Type ◽  
Cycle Progression ◽  
Threonine Residue ◽  
Cyclin Dependent Kinases ◽  
Higher Eukaryotes

Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiaeCdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28pT169Amutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation.

scholarly journals Core Control Principles of the Eukaryotic Cell Cycle

2021 ◽  
Author(s):  
Souradeep Basu ◽  
Paul Nurse ◽  
Andrew Jones
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Cycle Control ◽  
Eukaryotic Cell ◽  
Protein Phosphatase 1 ◽  
Cycle Progression ◽  
Substrate Specificities ◽  
Cyclin Dependent Kinases ◽  
Do So

Abstract Cyclin dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different Cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs). However, the principles on which Cyclin-CDKs organise the temporal order of cell cycle events are contentious. The currently most widely accepted model, is that the S-CDKs and M-CDKs are functionally specialised, with significant different substrate specificities to execute different cell cycle events. A second model is that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall cellular CDK activity. Here we reconcile these two views of core cell cycle control. Using a multiplexed phosphoproteomics assay of in vivo S-CDK and M-CDK activities in fission yeast, we show that S-CDK and M-CDK substrate specificities are very similar, showing that S-CDKs are not completely specialised for S-phase alone. Normally S-CDK cannot undergo mitosis, but is able to do so when Protein Phosphatase 1 (PP1) is removed from the centrosome, allowing several mitotic substrates to be better phosphorylated by S-CDK in vivo. Thus, an increase in S-CDK activity in vivo is sufficient to allow S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine which temporally orders cell cycle progression is largely based upon a quantitative increase of CDK activity through the cell cycle, combined with minor qualitative differences in catalytic specialisation of S-CDKs and M-CDKs.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

1996 ◽  
Vol 16 (12) ◽  
pp. 6634-6643 ◽  
Author(s):  
N Mathias ◽  
S L Johnson ◽  
M Winey ◽  
A E Adams ◽  
L Goetsch ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Large Family ◽  
S Phase ◽  
Genetic Interactions ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Ubiquitin Conjugating Enzyme ◽  
Regulation Of Cell Cycle

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

scholarly journals Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

1996 ◽  
Vol 16 (8) ◽  
pp. 4445-4455 ◽  
Author(s):  
K M Latham ◽  
S W Eastman ◽  
A Wong ◽  
P W Hinds
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Growth Arrest ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Aberrant Expression ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Rat Fibroblasts ◽  
Wild Type P53

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.

A Novel p27kip1-Smad3 Regulated Pathway Is Involved in Induction of T Cell Tolerance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 867-867
Author(s):  
Lequn Li ◽  
Yoshiko Iwamoto ◽  
Alla Berezovskaya ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Cell Cycle Progression ◽  
T Cell Tolerance ◽  
Cell Tolerance ◽  
Wild Type ◽  
Cycle Progression ◽  
Induction Of Tolerance

Abstract Induction and maintenance of peripheral tolerance is essential for homeostasis of the immune system. In vivo studies demonstrate the significance of tolerance induction in preventing autoimmunity, graft rejection and GVHD. Upregulation of the cyclin-dependent kinase inhhibitor, p27, correlates with induction of T cell tolerance in vitro and in vivo. p27 interacts with cdk2, cdc2, grb2, and Rho family GTPases. Extensive studies support an essential role of cdks, particularly cdk2, in cell cycle re-entry. Cdk2 promotes cell cycle progression in part by phosphorylating Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Recent work indicates that cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits progression from G1 to S phase, and impaired phosphorylation on the cdk-specific sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. In spite the strong correlation between p27 expression level and T cell tolerance, it remains unclear whether p27 has a causative role in induction of tolerance. Here, we examined the role of p27 during induction of tolerance of naïve T cells in vivo, using RAG2 deficient, DO11.10 TCR-transgenic T cells that lack the cyclin-cdk-binding domain of p27 (p27Δ) thereby disrupting only the interactions of p27 with cyclin-cdk complexes. We adoptively transferred CD4+ T cells from RAG2−/−DO11.10 TCR-transgenic mice (DO11.10) or RAG2−/−DO11.10 TCR-transgenic p27Δ mice (DO11.10/p27Δ) into syngeneic wild-type recipients and compared the development of immune responses to immunogenic or tolerizing stimulus in vivo. Following exposure to immunogenic or tolerizing stimulus, DO11.10 and DO11.10/p27Δ CD4+ T cells underwent equal numbers of divisions in vivo, and both cell types exhibited reduced number of divisions in response to tolerizing stimulus. Strikingly, only wild-type DO11.10 TCR-transgenic T cells were tolerized as determined by impaired cyclin E activation, proliferation, and IL-2 production upon antigen-specific rechallenge. Compared to primed wild-type DO11.10 cells, tolerized wild-type DO11.10 cells exhibited impaired cdk2 and cdc2 activity, reduced levels of Smad3 phosphorylation on cdk-specific sites, and increased Smad3-transactivation leading to upregulation of the cdk4/6-specific cdk inhibitor p15. In contrast, after either priming or tolerizing stimulus, DO11.10/p27Δ cells exhibited comparable cdk2 and cdc2 activity, cdk-mediated phosphorylation of Smad3, low-level Smad3 transactivation, and no upregulation of p15. Furthermore, knockdown of Smad3 by expression of Smad3 shRNA in wild-type DO11.10 T cells recapitulated the functional and molecular findings observed in DO11.10/p27Δ cells, preventing induction of tolerance and upregulation of p15, and resulting in production of IL-2 and cell cycle progression. In contrast, expression of Smad3 mutant resistant to cdk-mediated phosphorylation in DO11.10/p27Δ cells recapitulated the molecular and functional effects of tolerance and resulted in inhibition of IL-2 production, upregulation of p15 and blockade of cell cycle progression. These results show that p27 plays a causative role in the induction of tolerance of naïve T cells and Smad3 is a critical component of a pathway downstream of p27 regulating the induction of tolerance in vivo.

scholarly journals Dp1 Is Largely Dispensable for Embryonic Development

2004 ◽  
Vol 24 (16) ◽  
pp. 7197-7205 ◽  
Author(s):  
Matthew J. Kohn ◽  
Sandra W. Leung ◽  
Vittoria Criniti ◽  
Monica Agromayor ◽  
Lili Yamasaki
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Cycle Progression ◽  
Cell Cycle Genes

ABSTRACT E2F/DP complexes activate or repress the transcription of E2F target genes, depending on the association of a pRB family member, thereby regulating cell cycle progression. Whereas the E2F family consists of seven members, the DP family contains only two (Dp1 and Dp2), Dp1 being the more highly expressed member. In contrast to the inactivation of individual E2F family members, we have recently demonstrated that loss of Dp1 results in embryonic lethality by embryonic day 12.5 (E12.5) due to the failure of extraembryonic lineages to develop and replicate DNA properly. To bypass this placental requirement and search for roles of Dp1 in the embryo proper, we generated Dp1-deficient embryonic stem (ES) cells that carry the ROSA26-LacZ marker and injected them into wild-type blastocysts to construct Dp1-deficient chimeras. Surprisingly, we recovered mid- to late gestational embryos (E12.5 to E17.5), in which the Dp1-deficient ES cells contributed strongly to most chimeric tissues as judged by X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining and Western blotting. Importantly, the abundance of DP2 protein does not increase and the expression of an array of cell cycle genes is virtually unchanged in Dp1-deficient ES cells or chimeric E15.5 tissues with the absence of Dp1. Thus, Dp1 is largely dispensable for embryonic development, despite the absolute extraembryonic requirement for Dp1, which is highly reminiscent of the restricted roles for Rb and cyclins E1/E2 in vivo.

p38α Activates Purine Metabolism to Initiate Hematopoietic Stem Cell Cycling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 776-776
Author(s):  
Daiki Karigane ◽  
Shinichiro Okamoto ◽  
Toshio Suda ◽  
Keiyo Takubo
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Purine Metabolism ◽  
Stress Conditions ◽  
Wild Type ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. However, how stress hematopoiesis, including bone marrow transplantation (BMT), induces metabolic changes in HSCs remains unclear. Here, we report a critical role for the p38MAPK family isoform p38α in initiating HSC proliferation during stress hematopoiesis in mouse. First, we identified p38α as the major p38MAPK isozyme highly expressed in HSCs and we also performed conditional knockout of p38α in mice. This mouse showed no overt difference relative to wild type mouse. However, treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type mice (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type mice, N=6, p=0.03) and loss of p38α in HSCs showed a defective transplantation capacity in primary and secondary transplantation. To gain further insight into p38MAPK function during hematological stress, we evaluated the time course of p38MAPK activation in stressful contexts by intracellular flow cytometry. We found that p38MAPK was immediately phosphorylated in HSCs after hematological stress and returned to normal in a short period, suggesting that p38α functions rapidly after hematological stresses to activate downstream events. To identify events downstream of p38α after hematological stress, we initially evaluated mechanisms such as homing, apoptosis, and ROS generation immediately after BMT. However, defects seen in p38α-deficient HSCs after hematological stress could not be explained by these mechanisms. Therefore we next focused on cell cycle. In CFSE assay, p38α loss resulted in defective recovery from hematological stress and a delay in initiating cycling of HSCs. In addition, p38α-deficient HSCs showed lower BrdU incorporation in vivo (p=0.045) and EdU incorporation in vitro (p=0.003). Transcriptome analysis of transplanted wild-type or p38α-deficient HSCs suggested that p38α-deficient HSCs showed lower enrichment of genes related to HSC-related markers and proliferation. Taken together, loss of p38α resulted in defective HSC cell cycle progression in stressed settings such as transplantation. Given that altered metabolic activities can change cell cycle status, we asked whether p38α regulation of a particular metabolic pathway could initiate HSC cycling under stress conditions. To do so, we collected p38α-deficient or wild-type LSK cells either at steady state or after BMT and extracted metabolites for metabolome analysis using mass spectrometry. Among metabolites surveyed, we focused on changes in glycine and aspartic acid, which are required for purine biosynthesis. Levels of both increased in p38α-deficient as compared with wild-type LSK cells after BMT. Also, mice transplanted with p38α-deficient compared with wild-type LSK cells showed lower levels of allantoin, a product of purine catabolism. These findings suggest that p38α loss suppresses purine metabolism during stress hematopoiesis. Next, we evaluated mRNAs encoding key enzymes functioning in purine metabolism by qPCR. Expression of both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), and guanosine monophosphate synthetase (GMPS) was significantly decreased in p38α-deficient HSCs relative to wild-type HSCs on day 1 after BMT. To assess how changes in purine metabolism could affect the HSC response to stress, we treated HSCs with cytokines in the presence or absence of mycophenolic acid (MPA), an IMPDH2 inhibitor. MPA treatment significantly suppressed colony formation capacity of HSCs in a dose-dependent manner. Also, EdU incorporation into HSCs was reduced by MPA dose-dependently. Finally, isolated HSCs were cultured with or without MPA for 3 days and then transplanted into recipients along with competitor cells. PB chimerism was dose-dependently decreased in recipients of MPA-treated cells. These findings suggest that purine metabolism directly maintains proliferation capacity of HSCs in stress conditions. In summary, expression of purine-synthesizing enzymes decreased in p38α-deficient HSCs after transplantation, an activity correlated with defective cell cycle progression in vitro and in vivo. Overall, this is the first report of p38α-regulated changes in purine metabolism associated with HSC stress and cell cycle initiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of the Bur1-Bur2 Cyclin-Dependent Kinase Complex by Cak1

2002 ◽  
Vol 22 (19) ◽  
pp. 6750-6758 ◽  
Author(s):  
Sheng Yao ◽  
Gregory Prelich
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Single Gene ◽  
Eukaryotic Cell ◽  
Temperature Sensitive ◽  
Cycle Progression ◽  
Carboxy Terminal

ABSTRACT Cyclin-dependent kinases (Cdks) were originally identified as regulators of eukaryotic cell cycle progression, but several Cdks were subsequently shown to perform important roles as transcriptional regulators. While the mechanisms regulating the Cdks involved in cell cycle progression are well documented, much less is known regarding how the Cdks that are involved in transcription are regulated. In Saccharomyces cerevisiae, Bur1 and Bur2 comprise a Cdk complex that is involved in transcriptional regulation, presumably mediated by its phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. To investigate the regulation of Bur1 in vivo, we searched for high-copy-number suppressors of a bur1 temperature-sensitive mutation, identifying a single gene, CAK1. Cak1 is known to activate two other Cdks in yeast by phosphorylating a threonine within their conserved T-loop domains. Bur1 also has the conserved threonine within its T loop and is therefore a potential direct target of Cak1. Additional tests establish a direct functional interaction between Cak1 and the Bur1-Bur2 Cdk complex: Bur1 is phosphorylated in vivo, both the conserved Bur1 T-loop threonine and Cak1 are required for phosphorylation and Bur1 function in vivo, and recombinant Cak1 stimulates CTD kinase activity of the purified Bur1-Bur2 complex in vitro. Thus, both genetic and biochemical evidence demonstrate that Cak1 is a physiological regulator of the Bur1 kinase.

scholarly journals To Divide or Not to Divide? How Deuterium Affects Growth and Division of Chlamydomonas reinhardtii

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 861
Author(s):  
Veronika Kselíková ◽  
Vilém Zachleder ◽  
Kateřina Bišová
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Cell Cycle Progression ◽  
Model Organism ◽  
Cycle Progression ◽  
Synchronous Cultures ◽  
Multiple Fission ◽  
First Choice ◽  
High Doses

Extensive in vivo replacement of hydrogen by deuterium, a stable isotope of hydrogen, induces a distinct stress response, reduces cell growth and impairs cell division in various organisms. Microalgae, including Chlamydomonas reinhardtii, a well-established model organism in cell cycle studies, are no exception. Chlamydomonas reinhardtii, a green unicellular alga of the Chlorophyceae class, divides by multiple fission, grows autotrophically and can be synchronized by alternating light/dark regimes; this makes it a model of first choice to discriminate the effect of deuterium on growth and/or division. Here, we investigate the effects of high doses of deuterium on cell cycle progression in C. reinhardtii. Synchronous cultures of C. reinhardtii were cultivated in growth medium containing 70 or 90% D2O. We characterize specific deuterium-induced shifts in attainment of commitment points during growth and/or division of C. reinhardtii, contradicting the role of the “sizer” in regulating the cell cycle. Consequently, impaired cell cycle progression in deuterated cultures causes (over)accumulation of starch and lipids, suggesting a promising potential for microalgae to produce deuterated organic compounds.

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