scholarly journals Pseudosubstrate Inhibition of the Anaphase-Promoting Complex by Acm1: Regulation by Proteolysis and Cdc28 Phosphorylation

2008 ◽  
Vol 28 (15) ◽  
pp. 4653-4664 ◽  
Author(s):  
Denis Ostapenko ◽  
Janet L. Burton ◽  
Ruiwen Wang ◽  
Mark J. Solomon
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Anaphase Promoting Complex ◽  
Inhibitory Protein ◽  
Ubiquitin Ligase Activity ◽  
Dependent Protein ◽  
Apc Mutation

ABSTRACT The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.

scholarly journals Swm1/Apc13 Is an Evolutionarily Conserved Subunit of the Anaphase-Promoting Complex Stabilizing the Association of Cdc16 and Cdc27

2004 ◽  
Vol 24 (8) ◽  
pp. 3562-3576 ◽  
Author(s):  
Martin Schwickart ◽  
Jan Havlis ◽  
Bianca Habermann ◽  
Aliona Bogdanova ◽  
Alain Camasses ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Small Subunit ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Ligase Activity ◽  
Evolutionarily Conserved ◽  
Stable Association

ABSTRACT The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.

p21WAF1 is dynamically associated with JNK in human T-lymphocytes during cell cycle progression

1998 ◽  
Vol 111 (15) ◽  
pp. 2247-2255
Author(s):  
R. Patel ◽  
B. Bartosch ◽  
J.L. Blank
Keyword(s):  
Cell Cycle ◽  
T Lymphocytes ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Human T Lymphocytes ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Jnk Activation

We have examined the regulation of the c-Jun NH2-terminal kinase (JNK) subfamily of mitogen-activated protein kinases (MAPKs) in response to inhibition of DNA replication during the cell cycle of human T-lymphocytes. In this study, we demonstrate that JNK is rapidly activated following release of T-lymphocytes from G1/S-phase arrest and that this activation precedes resumption of DNA synthesis upon S-phase progression. We also show that activation of JNK correlates with dissociation of the cyclin-dependent protein kinase (CDK) inhibitor, p21WAF1, from JNK1. Since JNK1 isolated from T-lymphocytes by immunoprecipitation can be inhibited by recombinant p21WAF1 in vitro, these data suggest that JNK activation may be regulated in part by its dissociation from p21WAF1. The observation of a dynamic, physical association of native JNK1 and p21WAF1 in vivo has not previously been described and suggests a novel mechanism for JNK-mediated regulation of the cell cycle of human T-lymphocytes.

scholarly journals The Cak1p Protein Kinase Is Required at G1/S and G2/M in the Budding Yeast Cell Cycle

Genetics ◽  
1997 ◽  
Vol 147 (1) ◽  
pp. 57-71 ◽  
Author(s):  
Ann Sutton ◽  
Richard Freiman
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Protein Kinase Activity ◽  
Synthetic Lethal ◽  
Protein Levels ◽  
M Phase

Abstract The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase. Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase. Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase. Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription. Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function.

scholarly journals A Conserved Cyclin-Binding Domain Determines Functional Interplay between Anaphase-Promoting Complex–Cdh1 and Cyclin A-Cdk2 during Cell Cycle Progression

2001 ◽  
Vol 21 (11) ◽  
pp. 3692-3703 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mechanistic Explanation ◽  
Cyclin A ◽  
Binding Motif ◽  
Cell Cycle Regulators ◽  
Anaphase Promoting Complex ◽  
Cycle Progression

ABSTRACT Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G1/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1–cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G1/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.

scholarly journals Akt-Dependent Phosphorylation of p21Cip1 Regulates PCNA Binding and Proliferation of Endothelial Cells

2001 ◽  
Vol 21 (16) ◽  
pp. 5644-5657 ◽  
Author(s):  
Lothar Rössig ◽  
Amir S. Jadidi ◽  
Carmen Urbich ◽  
Cornel Badorff ◽  
Andreas M. Zeiher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Endothelial Cell Proliferation ◽  
Inhibitory Protein ◽  
Intact Cells ◽  
Biochemical Fractionation ◽  
Protein Kinase Akt

ABSTRACT The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21Cip1 at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21Cip1 at Thr 145 prevents the complex formation of p21Cip1 with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21Cip1 at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21Cip1 and attenuates the Cdk2 inhibitory activity of p21Cip1. Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21Cip1 phosphorylation is not caused by altered intracellular localization of p21Cip1. As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21Cip1, whereas the nonphosphorylatable p21Cip1 T145A construct arrests cells in G0 phase. These data suggest that the modulation of p21Cip1 cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

scholarly journals ID: 1023 All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth

2017 ◽  
Vol 4 (S) ◽  
pp. 98
Author(s):  
P H Nguyen ◽  
J Giraud ◽  
C Staedel ◽  
L Chambonnier ◽  
P Dubus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Gastric Carcinoma ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
3D Culture ◽  
All Trans Retinoic Acid

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-transretinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

scholarly journals CDK4/6 inhibitors: a novel strategy for tumor radiosensitization

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Yilan Yang ◽  
Jurui Luo ◽  
Xingxing Chen ◽  
Zhaozhi Yang ◽  
Xin Mei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Studies ◽  
Cell Cycle Progression ◽  
Combined Therapy ◽  
Small Sample ◽  
Combination Strategies ◽  
Novel Strategy ◽  
Underlying Mechanisms

Abstract Recently, the focus of enhancing tumor radiosensitivity has shifted from chemotherapeutics to targeted therapies. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are a novel class of selective cell cycle therapeutics that target the cyclin D-CDK4/6 complex and induce G1 phase arrest. These agents have demonstrated favorable effects when used as monotherapy or combined with endocrine therapy and targeted inhibitors, stimulating further explorations of other combination strategies. Multiple preclinical studies have indicated that CDK4/6 inhibitors exhibit a synergistic effect with radiotherapy both in vitro and in vivo. The principal mechanisms of radiosensitization effects include inhibition of DNA damage repair, enhancement of apoptosis, and blockade of cell cycle progression, which provide the rationale for clinical use. CDK4/6 inhibitors also induce cellular senescence and promote anti-tumor immunity, which might represent potential mechanisms for radiosensitization. Several small sample clinical studies have preliminarily indicated that the combination of CDK4/6 inhibitors and radiotherapy exhibited well-tolerated toxicity and promising efficacy. However, most clinical trials in combined therapy remain in the recruitment stage. Further work is required to seek optimal radiotherapy-drug combinations. In this review, we describe the effects and underlying mechanisms of CDK4/6 inhibitors as a radiosensitizer and discuss previous clinical studies to evaluate the prospects and challenges of this combination.

scholarly journals Regulation of Focal Adhesion Kinase by a Novel Protein Inhibitor FIP200

2002 ◽  
Vol 13 (9) ◽  
pp. 3178-3191 ◽  
Author(s):  
Smita Abbi ◽  
Hiroki Ueda ◽  
Chuanhai Zheng ◽  
Lee Ann Cooper ◽  
Jihe Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cell Cycle Progression ◽  
Protein Inhibitor ◽  
Cellular Functions ◽  
Cycle Progression ◽  
Novel Protein

Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-l-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.

scholarly journals Anticancer Activity of Cynomorium coccineum

Cancers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 354 ◽  
Author(s):  
Mouna Sdiri ◽  
Xiangmin Li ◽  
William Du ◽  
Safia El-Bok ◽  
Yi-Zhen Xie ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Anticancer Activity ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cycle Progression ◽  
Cycle Progression

The extensive applications of Cynomorium species and their rich bioactive secondary metabolites have inspired many pharmacological investigations. Previous research has been conducted to examine the biological activities and numerous interesting pharmaceutical activities have been reported. However, the antitumor activities of these species are unclear. To understand the potential anticancer activity, we screened Cynomorium coccineum and Cynomorium songaricum using three different extracts of each species. In this study, the selected extracts were evaluated for their ability to decrease survival rates of five different cancer cell lines. We compared the cytotoxicity of the three different extracts to the anticancer drug vinblastine and one of the most well-known medicinal mushrooms Amaurederma rude. We found that the water and alcohol extracts of C. coccineum at the very low concentrations possessed very high capacity in decreasing the cancer cells viability with a potential inhibition of tumorigenesis. Based on these primitive data, we subsequently tested the ethanol and the water extracts of C. coccineum, respectively in in vitro and in vivo assays. Cell cycle progression and induction of programmed cell death were investigated at both biological and molecular levels to understand the mechanism of the antitumor inhibitory action of the C. coccineum. The in vitro experiments showed that the treated cancer cells formed fewer and smaller colonies than the untreated cells. Cell cycle progression was inhibited, and the ethanol extract of C. coccineum at a low concentration induced accumulation of cells in the G1 phase. We also found that the C. coccineum’s extracts suppressed viability of two murine cancer cell lines. In the in vivo experiments, we injected mice with murine cancer cell line B16, followed by peritoneal injection of the water extract. The treatment prolonged mouse survival significantly. The tumors grew at a slower rate than the control. Down-regulation of c-myc expression appeared to be associated with these effects. Further investigation showed that treatment with C. coccineum induced the overexpression of the tumor suppressor Foxo3 and other molecules involved in inducing autophagy. These results showed that the C. coccineum extract exerts its antiproliferative activity through the induction of cell death pathway. Thus, the Cynomorium plants appear to be a promising source of new antineoplastic compounds.

Sign in / Sign up

Export Citation Format

Share Document