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PubMed2003

Active cyclin B1-Cdk1 first appears on centrosomes in prophase.

Cyclin B1-Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against cyclin B1 that primarily recognise the active form of the complex. We show that cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. We conclude that cyclin B1-Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.

Animals↗

PubMed2005

Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling.

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.

Amino Acid Sequence↗

PubMed2005

Paclitaxel induces the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 through a Cdk1-dependent mechanism.

Initial chemotherapeutic treatment triggers a stress-related response, which can lead to an increase in the expression of survival proteins. In this study we examine whether paclitaxel (PTX) alters the expression and/or phosphorylation of the translation initiation proteins, eukaryotic initiation factor 4E (eIF-4E) and 4E-binding protein (4E-BP1), a suppressor of eIF-4E in the dephosphorylated state. We found that PTX induced the hyperphosphorylation of 4E-BP1 in the breast cancer cell line, MDA MB 231, which reduced its association with eIF-4E, but did not alter the expression and phosphorylation of eIF-4E. The hyperphosphorylation of 4E-BP1 correlated with G2/M accumulation and with an increase in the phosphorylation of cdk1 substrates. Cotreatment with a histone deacetylase inhibitor (an indirect inhibitor of cdk activity), purvalanol A and roscovitine (direct cdk inhibitors), and the reduction of cyclin B expression using RNA interference decreased the hyperphosphorylation of 4E-BP1 in PTX treated cells. The hyperphosphorylation of 4E-BP1 by PTX increased the association of eIF-4E with eIF-4G, whereas cotreatment with purvalanol A inhibited the association of eIF-4E with eIF-4G in PTX treated cells. Taken together, our data suggest that PTX-increases the functional level of eIF-4E by promoting the hyperphosphorylation and release of 4E-BP1 through a cdk1-dependent mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2003

RLIP, an effector of the Ral GTPases, is a platform for Cdk1 to phosphorylate epsin during the switch off of endocytosis in mitosis.

The Ral signaling pathway is critically involved in Ras-dependent oncogenesis. One of its key actors, RLIP/RalBP1, which participates in receptor endocytosis during interphase, is also involved in mitotic processes when endocytosis is switched off. During mitosis, RLIP76 is located on the duplicated centrosomes and is required for their proper separation and movement to the poles. We have looked for actors that associate with RLIP during mitosis. We show here that RLIP/RalBP1 interacts with an active p34cdc2.cyclinB1 (cdk1) enzyme and that this interaction is crucial for the mitotic phosphorylation of Epsin that, once phosphorylated, is no longer competent for endocytosis. We show also that this latter phosphorylation is dependent on Ral signaling. We propose that RLIP/RalBP1 is used as a platform by the mitotic cdk1 to facilitate the phosphorylation of Epsin, which makes Epsin incompetent for endocytosis during mitosis, when endocytosis is switched off.

ATP-Binding Cassette Transporters↗

PubMed2004

Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region.

F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCF(Fbx7) complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes.

Amino Acid Motifs↗

PubMed2001

The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus.

In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.

Animals↗

PubMed2026

Cdk1 and PP2A constitute a molecular switch controlling orderly degradation of atypical E2Fs.

Dynamic oscillations in the phosphorylation and ubiquitination of key proliferative regulators are defining features of the eukaryotic cell cycle. Resetting the cell cycle at the mitosis-to-G1 transition requires activation of the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which ensures cell cycle irreversibility by targeting dozens of substrates for degradation, safeguarding genome integrity. However, the overall coupling of substrate phosphorylation with target recognition and degradation by the APC/C remains relatively unexplored. As a paradigm for further defining these rules, we focused on E2F7 and E2F8-atypical E2F-family proteins that coordinate cell cycle gene expression by restraining the pro-proliferative transcriptional activity of E2F1. Leveraging complementary cell and cell-free systems, we demonstrate that flexible domains at the amino-termini of E2F7 and E2F8 contain APC/C recognition motifs adjacent to critical Thr residues, whose phosphorylation by Cdk1 is rate-limiting for degradation. The removal of this phosphorylation by PP2A serves as a molecular switch, coupling the degradation of E2F7 and E2F8 to the G1 phase, coinciding with the rise of E2F1. Collectively, these findings highlight a critical role for Cdk1-PP2A signaling in controlling the orderly degradation of APC/C substrates, ensuring precisely timed assembly of the transcriptional infrastructure that coordinates cell cycle commitment and progression.

Protein Phosphatase 2↗

PubMed2000

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity.

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.

Blotting, Western↗

PubMed2025

Cdk1 and PP2A constitute a molecular switch controlling orderly degradation of atypical E2Fs.

Dynamic oscillations in the phosphorylation and ubiquitination of key proliferative regulators are defining features of the eukaryotic cell cycle. Resetting the cell cycle at the mitosis-to-G1 transition requires activation of the E3 ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which ensures cell cycle irreversibility by targeting dozens of substrates for degradation, safeguarding genome integrity. However, the overall coupling of substrate phosphorylation with target recognition and degradation by the APC/C remains relatively unexplored. As a paradigm for further defining these rules, we focused on E2F7 and E2F8 - atypical E2F-family proteins that coordinate cell cycle gene expression by restraining the pro-proliferative transcriptional activity of E2F1. Leveraging complementary cell and cell-free systems, we demonstrate that flexible domains in the amino-termini of E2F7 and E2F8 contain APC/C recognition motifs adjacent to critical Thr residues, whose phosphorylation by Cdk1 is rate limiting for degradation. The removal of this phosphorylation by PP2A phosphatase serves as a molecular switch, coupling the degradation of E2F7 and E2F8 to the G1 phase, coinciding with the rise of E2F1. Collectively, these findings highlight a critical role for Cdk1-PP2A signaling in controlling the orderly degradation of APC/C substrates, ensuring precisely timed assembly of the transcriptional infrastructure that coordinates cell cycle commitment and progression.

APC/C substrate↗

PubMed2005

Srs2 and Sgs1 DNA helicases associate with Mre11 in different subcomplexes following checkpoint activation and CDK1-mediated Srs2 phosphorylation.

Mutations in the genes encoding the BLM and WRN RecQ DNA helicases and the MRE11-RAD50-NBS1 complex lead to genome instability and cancer predisposition syndromes. The Saccharomyces cerevisiae Sgs1 RecQ helicase and the Mre11 protein, together with the Srs2 DNA helicase, prevent chromosome rearrangements and are implicated in the DNA damage checkpoint response and in DNA recombination. By searching for Srs2 physical interactors, we have identified Sgs1 and Mre11. We show that Srs2, Sgs1, and Mre11 form a large complex, likely together with yet unidentified proteins. This complex reorganizes into Srs2-Mre11 and Sgs1-Mre11 subcomplexes following DNA damage-induced activation of the Mec1 and Tel1 checkpoint kinases. The defects in subcomplex formation observed in mec1 and tel1 cells can be recapitulated in srs2-7AV mutants that are hypersensitive to intra-S DNA damage and are altered in the DNA damage-induced and Cdk1-dependent phosphorylation of Srs2. Altogether our observations indicate that Mec1- and Tel1-dependent checkpoint pathways modulate the functional interactions between Srs2, Sgs1, and Mre11 and that the Srs2 DNA helicase represents an important target of the Cdk1-mediated cellular response induced by DNA damage.

Blotting, Western↗

PubMed2006

Discovery and evaluation of dual CDK1 and CDK2 inhibitors.

In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis.

Animals↗

PubMed2003

Chemical and biological profile of dual Cdk1 and Cdk2 inhibitors.

The importance of Cdks in cell cycle regulation, their interaction with oncogenes and tumor suppressors, and their frequent deregulation in human tumors, has encouraged an active search for agents capable of perturbing the function of Cdks. In our laboratories, a variety of selective and potent low molecular weight inhibitors directed against the ATP binding sites of the Cdk1, Cdk2 have been developed. Extensive biological profiling of two distinct classes of Cdk inhibitors - the phenylamino pyrimidines (PAPs) and trisubstituted purines has revealed distinct differences in their cellular effects in normal cells compared to tumor cells. Due to their intact G1/S checkpoints, normal cells are shown to be reversibly blocked by these Cdk inhibitors in either the G1/S-phase or at the G2/M boarder. In transformed cells these control points are either absent or defective and treatment with the compounds resulted in pronounced proliferation block at the G2/M transition. Furthermore, there is strong evidence that this G2/M arrest is less well tolerated by the cells and consequently, they undergo apoptotic cell death. Finally, these dual Cdk1/ Cdk2 inhibitors are also found to be significantly more active on proliferating cells compared to quiescent cells reflecting their specific activity. Despite these encouraging results demonstrating a distinct outcome after treatment with such dual Cdk inhibitors in normal compared to de-regulated tumor cells, it remains to be determined whether a comparable therapeutic window might be observed in vivo experiments. Furthermore the intracellular kinase selectivity of inhibitors which are putatively selective in vitro remains a complicating feature that is only recently begun to be addressed by affinity chromatography and phosphoproteomics techniques. Once efficacy can be demonstrated in animal models at well-tolerated doses, there will be strong evidence for the development of cell cycle antagonists for cancer therapy.

Animals↗

PubMed2006

Anaphase topsy-turvy: Cdk1 a securin, separase a CKI.

Chromosome segregation in mitosis and meiosis is triggered by activation of a large protease, separase. While it has been known for some time that the anaphase inhibitor securin regulates separase activity recent work shows that this is only half the story. In vertebrates Cdk1-dependent inhibition of separase represents a second, securin-independent branch of anaphase regulation. Furthermore, an unanticipated ability of separase to inhibit Cdk1 suggests additional, nonproteolytic functions of separase.

Anaphase↗

PubMed2006

Cell cycle synchronization at the G2/M phase border by reversible inhibition of CDK1.

Chemical agents for cell cycle synchronization have greatly facilitated the study of biochemical events driving cell cycle progression. G1, S and M phase inhibitors have been developed and used widely in cell cycle research. However, currently there are no effective G2 phase inhibitors and synchronization of cultured cells in G2 phase has been challenging. Recently, a selective CDK1 inhibitor, RO-3306, has been identified that reversibly arrests proliferating human cells at the G2/M phase border and provides a novel means for cell cycle synchronization. A single-step protocol using RO-3306 permits the synchronization of >95% of cycling cancer cells in G2 phase. RO-3306 arrested cells enter mitosis rapidly after release from the G2 block thus allowing for isolation of mitotic cells without microtubule poisons. RO-3306 represents a new molecular tool for studying CDK1 function in human cells.

Animals↗

PubMed2002

Cyclin b1 promoter activity and functional cdk1 complex formation in G1 phase of human breast cancer cells.

Overexpression of cyclin B has been detected in various human breast cancer cell lines, breast tumor tissues, and immortalized but nontransformed breast cells. The cause of this overexpression has not been thoroughly investigated, nor is it known if cyclin B protein forms a functional complex with its partner, cdk1, at inappropriate cell cycle periods. In this study we examined the pattern of cyclin B1 promoter activity in three breast cancer cell lines, BT-549, MDA-MB-157, T-47D, and the immortalized breast cell line MCF-10F. Using cells stably transfected with a cyclin B1 promoter-luciferase reporter, luciferase activity was measured throughout the cell cycle in lovastatin synchronized cells and in G1 and S/G2 phases of asynchronized cells by flow cytometry. Results demonstrate that the cyclin B1 promoter activity increases, as expected, during the S/G2 period in all the cell lines. However, some promoter activity can be detected in G1 phase of the different cell line with BT-549 displaying the more altered pattern. Functional cyclin B1-cdk 1 protein complex was detected in G1 phase of BT-549 and T-47D cell lines. These results suggest that in a subset of transformed breast cancer cells altered cyclin B1 promoter activity may contribute to the misexpression of cyclin B protein.

Antineoplastic Agents↗

PubMed2002

Multiple Cdk1 inhibitory kinases regulate the cell cycle during development.

The Wee kinases block entry into mitosis by phosphorylating and inhibiting the activity of the mitotic cyclin-dependent kinase, Cdk1. We have found that the various Xenopus Wee kinases have unique temporal and spatial patterns of expression during development. In addition, we have isolated and characterized a new Wee1-like kinase, Xenopus Wee2. By both in vivo and in vitro tests, Xenopus Wee2 functions as a Wee1-like kinase. The previously isolated Wee1-like kinase, Xenopus Wee1, is expressed only as maternal gene product. In contrast, Xenopus Wee2 is predominantly a zygotic gene product, while the third Wee kinase, Xenopus Myt1, is both a maternal and zygotic gene product. Concurrent with the changing levels of these Cdk inhibitory kinases, the pattern of embryonic cell division becomes asynchronous and spatially restricted in the Xenopus embryo. Interestingly, once zygotic transcription begins, Xenopus Wee2 is expressed in regions of the embryo that are devoid of mitotic cells, such as the involuting mesoderm. In contrast, Xenopus Myt1 is expressed in regions of the embryo that have high levels of proliferation, such as the developing neural tissues. The existence of multiple Wee kinases may help explain how distinct patterns of cell division arise and are regulated during development.

Amino Acid Sequence↗

PubMed2006

A synthetic uracil derivative with antitumor activity through decreasing cyclin D1 and Cdk1, and increasing p21 and p27 in MCF-7 cells.

The anticarcinogenic potential of (RS)-1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)uracil (DBDU), with the naturally occurring pyrimidine base uracil, is reported against the MCF-7 cancer cell line. The arrest in the G0/G1 and G2/M cell cycle phases was accounted for by decrease in the expression of the cyclin D1 and Cdk1 proteins, and increase in p21 and p27 proteins. Using a reverse transcription-polymerase chain reaction-based assay at a dose of 5 muM of DBDU cyclin D1 mRNA was decreased, suggesting that DBDU exerts its regulatory action on cyclin D1 at the level of transcription. DNA fragmentation was performed and demonstrated that apoptosis occurred in the tumor cell line treated with DBDU. The G0/G1 arrest is an irreversible process and the cells undergo apoptosis in a p53-independent manner. DBDU administered intravenously twice a week (50 mg/kg dose each time) induced neither toxicity nor death in mice for 5 weeks.

Animals↗

PubMed2006

Identification of potent 5-pyrimidinyl-2-aminothiazole CDK4, 6 inhibitors with significant selectivity over CDK1, 2, 5, 7, and 9.

5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin-dependent kinases (CDKs) by a screening of the Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the gate keeper residue of CDK4 (Phe93), led to significant enhancement of selectivity for CDK4 over other CDKs. Compound 3 exhibited more than 300-fold selectivity for CDK4 over CDK1, 2, 5, 7, and 9. Subsequent improvements in aqueous solubility afforded compound 4, which is available for further in vivo studies and this compound inhibited pRb phosphorylation and BrdU incorporation in tumor models.

Bromodeoxyuridine↗