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PubMed2004

Mitotic phosphorylation of the peripheral Golgi protein Nir2 by Cdk1 provides a docking mechanism for Plk1 and affects cytokinesis completion.

The rearrangement of the Golgi apparatus during mitosis is regulated by several protein kinases, including Cdk1 and Plk1. Several peripheral Golgi proteins that dissociate from the Golgi during mitosis are implicated in regulation of cytokinesis or chromosome segregation, thereby coordinating mitotic and cytokinetic events to Golgi rearrangement. Here we show that, at the onset of mitosis, Cdk1 phosphorylates the peripheral Golgi protein Nir2 at multiple sites; of these, S382 is the most prominent. Phosphorylation of Nir2 by Cdk1 facilitates its dissociation from the Golgi apparatus, and phospho-Nir2(pS382) is localized in the cleavage furrow and midbody during cytokinesis. Mitotic phosphorylation of Nir2 is required for docking of the phospho-Ser/Thr binding module, the Polo box domain of Plk1, and overexpression of a Nir2 mutant, which fails to interact with Plk1, affects the completion of cytokinesis. These results demonstrate a mechanism for coordinating mitotic and cytokinetic events with Golgi rearrangement during cell division.

CDC2 Protein Kinase↗
PubMed2006

Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins.

In yeast, separation of duplicated spindle pole bodies (SPBs) (centrosomes in higher eukaryotes) is an indispensable step in the assembly of mitotic spindle and is triggered by severing of the bridge that connects the sister SPBs. This process requires Cdk1 (Cdc28) activation by Tyrosine 19 dephosphorylation. We show that cells that fail to activate Cdk1 are devoid of spindles due to persistently active APCCdh1, which targets microtubule-associated proteins Cin8, Kip1 and Ase1 for degradation. Tyrosine 19 dephosphorylation of Cdk1 is necessary to specifically prevent proteolysis of these proteins. Interestingly, SPB separation is dependent on the microtubule-bundling activity of Cin8 but not on its motor function. Since ectopic expression of proteolysis-resistant Cin8, Kip1 or Ase1 is sufficient for SPB separation even in the absence of Cdc28-Clb activity, we suggest that stabilization of these mechanical force-generating proteins is the predominant role of Cdc28-Clb in centrosome separation.

CDC2 Protein Kinase↗
PubMed2005

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B-Cdk1 activation.

The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregation and cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notion that the oncogenic properties associated with alteration of Int-6 originate from chromosomal instability.

CDC2 Protein Kinase↗
PubMed2002

Differential effects of ergosterol and cholesterol on Cdk1 activation and SRE-driven transcription.

Cholesterol is essential for cell growth and division, but whether this is just a consequence of its use in membrane formation or whether it also elicits regulatory actions in cell cycle machinery remains to be established. Here, we report on the specificity of this action of cholesterol in human cells by comparing its effects with those of ergosterol, a yeast sterol structurally similar to cholesterol. Inhibition of cholesterol synthesis by means of SKF 104976 in cells incubated in a cholesterol-free medium resulted in cell proliferation inhibition and cell cycle arrest at G2/M phase. These effects were abrogated by cholesterol added to the medium but not by ergosterol, despite that the latter was used by human cells and exerted similar homeostatic actions, as the regulation of the transcription of an SRE-driven gene construct. In contrast to cholesterol, ergosterol was unable to induce cyclin B1 expression, to activate Cdk1 and to resume cell cycle in cells previously arrested at G2. This lack of effect was not due to cytotoxicity, as cells exposed to ergosterol remained viable and, upon supplementing with UCN-01, an activator of Cdk1, they progressed through mitosis. However, in the presence of suboptimal concentrations of cholesterol, ergosterol exerted synergistic effects on cell proliferation. This is interpreted on the basis of the differential action of these sterols, ergosterol contributing to cell membrane formation and cholesterol being required for Cdk1 activation. In summary, the action of cholesterol on G2 traversal is highly specific and exerted through a mechanism different to that used for cholesterol homeostasis, reinforcing the concept that cholesterol is a specific regulator of cell cycle progression in human cells.

Base Sequence↗
PubMed2001

Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells.

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G(1)/G(0)phase of the cell cycle in the presence of 2 microM indirubin-3'-monoxime. At higher drug concentrations (> or = 5 microM) an increase of the cell population in the G(2)/M phase is additionally observed. Cells synchronized in G(2)/M phase by nocodazole remain arrested in the G(2)/M phase after release, in the presence of indirubin-3'-monoxime (> or =5 microM). After 24 h treatment with 10 microM indirubin-3'-monoxime a sub-G(2)peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy.

Antibiotics, Antineoplastic↗
PubMed2005

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

CDC2 Protein Kinase↗
PubMed1997

A dependent pathway of cytoplasmic polyadenylation reactions linked to cell cycle control by c-mos and CDK1 activation.

During oocyte maturation and early development, mRNAs receive poly(A) in the cytoplasm at distinct times relative to one another and to the cell cycle. These cytoplasmic polyadenylation reactions do not occur during oogenesis, but begin during oocyte maturation and continue throughout early development. In this report, we focus on the link between cytoplasmic polyadenylation and control of the cell cycle during meiotic maturation. Activation of maturation promoting factor, a complex of CDK1 and cyclin, is required for maturation and dependent on c-mos protein kinase. We demonstrate here that two classes of polyadenylation exist during oocyte maturation, defined by their dependence of c-mos and CDK1 protein kinases. Polyadenylation of the first class of mRNAs (class I) is independent of c-mos and CDK1 kinase activities, whereas polyadenylation of the second class (class II) requires both of these activities. Class I polyadenylation, through its effects on c-mos mRNA, is required for class II polyadenylation. cis-acting elements responsible for this distinction reside in the 3'-untranslated region, upstream of the polyadenylation signal AAUAAA. Cytoplasmic polyadenylation elements (CPEs) are sufficient to specify class I polyadenylation, and subtle changes in the CPE can substantially, though not entirely, shift an RNA from class I to class II. Activation of class I polyadenylation events is independent of hyperphosphorylation of CPE-binding protein or poly(A) polymerase, and requires cellular protein synthesis. The two classes of polyadenylation and of mRNA define a dependent pathway, in which polyadenylation of certain mRNAs requires the prior polyadenylation of another. We propose that this provides one method of regulating the temporal order of polyadenylation events, and links polyadenylation to the control of the meiotic cell cycle.

Animals↗
PubMed2005

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Animals↗
PubMed2002

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope.

Syncytia arising from the fusion of cells expressing the HIV-1-encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR-mediated phosphorylation of p53 on Ser15 (p53(S15)), p53-dependent upregulation of Bax and activation of the mitochondrial death pathway. p53(S15) phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase-like dismantling of the nuclear envelope. Inhibition of cyclin-dependent kinase-1 (Cdk1) prevents karyogamy, mTOR activation, p53(S15) phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV-1-infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53(S15) phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV-1 infection of primary CD4 lymphoblasts. Thus, HIV-1 elicits a pro-apoptotic signal transduction pathway relying on the sequential action of cyclin B-Cdk1, mTOR and p53.

Adult↗
PubMed1999

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

Animals↗
PubMed2000

Antitumor imidazolyl disulfide IV-2 causes irreversible G(2)/M cell cycle arrest without hyperphosphorylation of cyclin-dependent kinase Cdk1.

Aberrant function of redox-regulated proteins is a possible cause for cellular transformation and loss of cell cycle control. The small protein thioredoxin has oncogenic properties and controls cell cycle movement through G(1), S, and G(2)/M phases. The redox-active, asymmetrical 1-methylpropyl-2-imidazolyl disulfide (IV-2) has previously been shown to react with and inhibit thioredoxin activity in vitro, the proliferation of human tumor cells in culture, and the growth of tumors in mice. We now examined the effects of IV-2 on cell cycle progression. In synchronized tsFT210 mouse mammary carcinoma cells, IV-2 halted cells in mitosis. In asynchronously growing MCF-7 human breast cancer cells, IV-2 exclusively and irreversibly blocked cells in G(2)/M at concentrations that correlated with its growth inhibitory activity. Neither the closely related, less redox active 2-hydroxy-1-methylpropyl-2-imidazolyl disulfide (AIV-2), which differs from IV-2 only by an additional hydroxyl group, nor the symmetrical diallyl disulfide caused a G(2)/M arrest under these conditions. Furthermore, MCF-7 cells treated with IV-2 showed increased Cdk1 kinase activity and a decrease in Cdk1 tyrosine phosphorylation, indicating that IV-2 did not directly inhibit Cdk1 or Cdc25 activities. IV-2 did, however, increase Bcl-2 phosphorylation. These data suggest that the thioredoxin inhibitor IV-2, despite its simple structure, is able to target redox-sensitive processes that are critical for cell cycle progression through mitosis. The results are also consistent with a role of thioredoxin regulating cell cycle progression through G(2)/M.

Allyl Compounds↗
PubMedDate not supplied

Induction of apoptosis in human leukemia cells by the CDK1 inhibitor CGP74514A.

We have examined the effects of the CDK1 inhibitor CGP74514A on cell cycle- and apoptosis-related events in human leukemia cells. An 18-hr exposure to 5 microM CGP74514A induced mitochondrial damage (i.e., loss of Delta psi(m)) and apoptosis in multiple human leukemia cell lines (e.g., U937, HL-60, KG-1, CCRF-CEM, Raji, and THP; range 30-95%). In U937 cells, CGP74514A- induced apoptosis (5 microM) became apparent within 4 hr and approached 100% by 24 hr. The pan- caspase inhibitor Boc-fmk and the caspase-8 inhibitor lETD-fmk opposed CGP74514A-induced caspase-9 activation and PARP degradation, but not cytochrome c or Smac/DIABLO release. CGP74514A-mediated apoptosis was substantially blocked by ectopic expression of full-length Bel- 2, a loop-deleted mutant Bcl-2, and Bcl-x(L). CGP74514A treatment (5 microM; 18 hr) resulted in increased p21(CIP1) expression, p27(KIP1) degradation, diminished E2F1 expression, and dephosphorylation of p34(CDC2). It also induced early (i.e., within 2 hr) inhibition of CDK1 activity and dephosphorylation of pRb, followed by pRb degradation, but did not block pRb phosphorylation at CDK2- and CDK4- specific sites. These findings indicate that the selective CDK1 inhibitor, CGP74514A, induces complex changes in cell cycle-related proteins in human leukemia cells accompanied by extensive mitochondrial damage, caspase activation, and apoptosis.

2-Aminopurine↗
PubMed2025

CRISPR/Cas9 screenings reveal the role of STX1A and CDK1 in Cathepsin G entering and killing colorectal cancer cells.

Neutrophils are the major populations of white blood cells and have been reported to facilitate cancer metastasis. Meanwhile, emerging evidence has recently suggested the anti-cancer role of neutrophils. Our previous study revealed that CB-839 and 5-FU-treated colorectal cancer (CRC) tumors recruited neutrophils and induced neutrophil extracellular traps (NETs). Cathepsin G (CTSG), which is released during NET formation, enters CRC cells through the receptor for advanced glycation end products (RAGE) and cleaves 14-3-3ε to promote apoptosis. However, the detailed mechanism underlying CTSG's anti-tumor function remains less studied. In this study, we report that CTSG enters CRC cells through RAGE-mediated endocytosis. Knocking out RAGE or inhibiting endocytosis blocks CTSG from entering CRC cells and attenuates CTSG-induced apoptosis. Furthermore, the clathrin coat assembly complex and SNARE proteins were enriched in an arrayed CRISPR/Cas9 screening targeting human membrane trafficking genes. Knocking out SNARE protein STX1A prevents the spread of CTSG in CRC cells and the induction of cleaved PARP. A pooled genome-wide CRISPR/Cas9 screening further identifies the role of CDK1 in the NET-induced killing of CRC cells. Inhibiting CDK1 protected CRC cells from killing by CTSG. Our study reveals novel mechanisms by which CTSG enters and kills CRC cells.

CDK1↗
PubMed1999

Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

Animals↗
PubMed2006

Structure-based drug design of a highly potent CDK1,2,4,6 inhibitor with novel macrocyclic quinoxalin-2-one structure.

The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.

Animals↗
PubMed2001

Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1.

We have investigated the effects of a series of flavonoids on cell proliferation and cell cycle distribution in human melanoma cells OCM-1. Among the compounds that potently inhibited OCM-1 cell proliferation, we show that the presence of a hydroxyl group at the 3'-position of the ring B in quercetin and luteolin, correlated to a G1 cell cycle arrest while its absence in kaempferol and apigenin correlated to a G2 block. Genistein with a hydroxyl at 5-position of the ring A arrested cells in G2 while daidzein which lacks it, induced an accumulation of cells in G1. We demonstrate that flavonoids, which induced a cell cycle block in G1, inhibited the activity of CDK2 by 40-60%. By contrast, those which caused an accumulation of cells in G2/M were without effect. On the other hand, while quercetin, daidzein and luteolin did not alter the activity of CDK1, kaempferol, apigenin and genistein inhibited this kinase by 50-70%. We demonstrate that the up-regulation of the CDK inhibitors p27(KIP1) and p21(CIP1) is likely responsible for the inhibition of CDK2 while inhibition of CDK1 was rather due to the phosphorylation of the kinase on Tyr15 residue.

CDC2 Protein Kinase↗
PubMed2000

Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C.

Nuclear events of mitosis are initiated when the protein kinase cyclin-B1-Cdk1 is translocated into the nucleus during prophase. Recent work has unveiled many of the mechanisms that govern the localization of cyclin-B1-Cdk1 and its regulator Cdc25C. Phosphorylation-dependent changes in the rate of nuclear import and export of these proteins help to control the onset of mitosis both in normal cells and in cells delayed before mitosis by DNA damage.

Animals↗