Plays a key role in the control of the eukaryotic cell cycle by modulating the centrosome cycle as well as mitotic onset; promotes G2-M transition, and regulates G1 progress and G1-S transition via association with multiple interphase cyclins. Required in higher cells for entry into S-phase and mitosis. Phosphorylates PARVA/actopaxin, APC, AMPH, APC, BARD1, Bcl-xL/BCL2L1, BRCA2, CALD1, CASP8, CDC7, CDC20, CDC25A, CDC25C, CC2D1A, CSNK2 proteins/CKII, FZR1/CDH1, CDK7, CEBPB, CHAMP1, DMD/dystrophin, EEF1 proteins/EF-1, EZH2, KIF11/EG5, EGFR, FANCG, FOS, GFAP, GOLGA2/GM130, GRASP1, UBE2A/hHR6A, HIST1H1 proteins/histone H1, HMGA1, HIVEP3/KRC, LMNA, LMNB, LMNC, LBR, LATS1, MAP1B, MAP4, MARCKS, MCM2, MCM4, MKLP1, MYB, NEFH, NFIC, NPC/nuclear pore complex, PITPNM1/NIR2, NPM1, NCL, NUCKS1, NPM1/numatrin, ORC1, PRKAR2A, EEF1E1/p18, EIF3F/p47, p53/TP53, NONO/p54NRB, PAPOLA, PLEC/plectin, RB1, UL40/R2, RAB4A, RAP1GAP, RCC1, RPS6KB1/S6K1, KHDRBS1/SAM68, ESPL1, SKI, BIRC5/survivin, STIP1, TEX14, beta-tubulins, MAPT/TAU, NEDD1, VIM/vimentin, TK1, FOXO1, RUNX1/AML1 and RUNX2. CDK1/CDC2-cyclin-B controls pronuclear union in interphase fertilized eggs. Essential for early stages of embryonic development. During G2 and early mitosis, CDC25A/B/C-mediated dephosphorylation activates CDK1/cyclin complexes which phosphorylate several substrates that trigger at least centrosome separation, Golgi dynamics, nuclear envelope breakdown and chromosome condensation. Once chromosomes are condensed and aligned at the metaphase plate, CDK1 activity is switched off by WEE1- and PKMYT1-mediated phosphorylation to allow sister chromatid separation, chromosome decondensation, reformation of the nuclear envelope and cytokinesis. Inactivated by PKR/EIF2AK2- and WEE1-mediated phosphorylation upon DNA damage to stop cell cycle and genome replication at the G2 checkpoint thus facilitating DNA repair. Reactivated after successful DNA repair through WIP1-dependent signaling leading to CDC25A/B/C-mediated dephosphorylation and restoring cell cycle progression. In proliferating cells, CDK1-mediated FOXO1 phosphorylation at the G2-M phase represses FOXO1 interaction with 14-3-3 proteins and thereby promotes FOXO1 nuclear accumulation and transcription factor activity, leading to cell death of postmitotic neurons. The phosphorylation of beta-tubulins regulates microtubule dynamics during mitosis. NEDD1 phosphorylation promotes PLK1-mediated NEDD1 phosphorylation and subsequent targeting of the gamma-tubulin ring complex (gTuRC) to the centrosome, an important step for spindle formation. In addition, CC2D1A phosphorylation regulates CC2D1A spindle pole localization and association with SCC1/RAD21 and centriole cohesion during mitosis. The phosphorylation of Bcl-xL/BCL2L1 after prolongated G2 arrest upon DNA damage triggers apoptosis. In contrast, CASP8 phosphorylation during mitosis prevents its activation by proteolysis and subsequent apoptosis. This phosphorylation occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. EZH2 phosphorylation promotes H3K27me3 maintenance and epigenetic gene silencing. CALD1 phosphorylation promotes Schwann cell migration during peripheral nerve regeneration.
Nedd1 is a new member of the gamma-tubulin ring complex (gammaTuRC) and targets the gammaTuRC to the centrosomes for microtubule nucleation and spindle assembly in mitosis. Although its role is known, its functional regulation mechanism remains unclear. Here we report that the function of Nedd1 is regulated by Cdk1 and Plk1. During mitosis, Nedd1 is firstly phosphorylated at T550 by Cdk1, which creates a binding site for the polo-box domain of Plk1. Then, Nedd1 is further phosphorylated by Plk1 at four sites: T382, S397, S637 and S426. The sequential phosphorylation of Nedd1 by Cdk1 and Plk1 promotes its interaction with gamma-tubulin for targeting the gammaTuRC to the centrosome and is important for spindle formation. Knockdown of Plk1 by RNAi decreases Nedd1 phosphorylation and attenuates Nedd1 accumulation at the spindle pole and subsequent gamma-tubulin recruitment at the spindle pole for microtubule nucleation. Taken together, we propose that the sequential phosphorylation of Nedd1 by Cdk1 and Plk1 plays a pivotal role in targeting gammaTuRC to the centrosome by promoting the interaction of Nedd1 with the gammaTuRC component gamma-tubulin, during mitosis.
The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.
Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.
Transforming Growth Factor-beta (TGFbeta) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFbeta has any effect on G2 checkpoint kinases. We have found that TGFbeta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24h for cyclin B1 and cdc2 and by 48h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFbeta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48h after initiating TGFbeta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFbeta. In addition, TGFbeta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3h before a dramatic decrease of both molecules by 48h. Taken together, our data suggest that TGFbeta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFbeta-induced G1 arrest and growth inhibition.
The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.
Activation of cyclin-dependent kinase 1 (Cdk1) has been linked to cell death of postmitotic neurons in brain development and disease. We found that Cdk1 phosphorylated the transcription factor FOXO1 at Ser249 in vitro and in vivo. The phosphorylation of FOXO1 at Ser249 disrupted FOXO1 binding with 14-3-3 proteins and thereby promoted the nuclear accumulation of FOXO1 and stimulated FOXO1-dependent transcription, leading to cell death in neurons. In proliferating cells, Cdk1 induced FOXO1 Ser249 phosphorylation at the G2/M phase of the cell cycle, resulting in FOXO1-dependent expression of the mitotic regulator Polo-like kinase (Plk). These findings define a conserved signaling link between Cdk1 and FOXO1 that may have a key role in diverse biological processes, including the degeneration of postmitotic neurons.
Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr(161) by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr(14) and Tyr(15) phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, Cdc25A, -B, and -C. We find that expression of Cdc25A leads to an accelerated G(2)/M phase transition. In Cdc25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr(15). In addition, Tyr(15)-phosphorylated Cdk1 binds more cyclin B in Cdc25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, Cdc25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of Cdc25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G(2) and mitosis. Importantly, we find that Cdc25A overexpression leads to an activation of Cdk7 and increase in Thr(161) phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G(2)/M is tightly coupled and regulated by Cdc25 phosphatases.
Akt kinase-interacting protein 1 (Aki1)/Freud-1/CC2D1A is localized in the cytosol, nucleus, and centrosome. Aki1 plays distinct roles depending on its localization. In the cytosol, it acts as a scaffold protein in the phosphoinositide 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1)/Akt pathway. In the nucleus, it is a transcriptional repressor of the serotonin-1A (5-HT1A) receptor. In the centrosome, it regulates spindle pole localization of the cohesin subunit Scc1, thereby mediating centriole cohesion during mitosis. Although the function of Aki1 has been well clarified, the regulatory machinery of Aki1 is poorly understood. We previously found that Aki1 in mitotic cells displayed reduced mobility on immunoblot analysis, but the reason for this was unclear. Here we show that the electrophoretic mobility shift of Aki1 is derived from mitotic phosphorylation. The cyclin B1-cyclin-dependent kinase 1 (Cdk1) complex was found to be one of the kinases responsible for Aki1 phosphorylation during mitosis. We identified the Ser(208) residue of Aki1 as a cyclin B1-Cdk1 phosphorylation site. Furthermore, cyclin B1-Cdk1 inhibitor treatment was shown to attenuate the level of Aki1 in complex with Scc1, suggesting that Aki1 phosphorylation by cyclin B1-Cdk1 contributes to Aki1-Scc1 complex formation. Our results indicate that cyclin B1-Cdk1 is a kinase of Aki1 during mitosis and that its phosphorylation of Aki1 may regulate mitotic function.
Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-x(L) and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-x(L)/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-x(L) and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-x(L) mutant but not by a phosphomimetic Bcl-x(L) mutant, confirming Bcl-x(L) as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x(L)/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.
RUNX2 is a member of the runt family of DNA-binding transcription factors. RUNX2 mediates endothelial cell migration and invasion during tumor angiogenesis and is expressed in metastatic breast and prostate tumors. Our published studies showed that RUNX2 DNA-binding activity is low during growth arrest, but elevated in proliferating endothelial cells. To investigate its role in cell proliferation and cell cycle regulation, RUNX2 was depleted in human bone marrow endothelial cells using RNA interference. Specific RUNX2 depletion inhibited DNA-binding activity as measured by electrophoretic mobility shift assay resulting in inhibition of cell proliferation. Cells were synchronized at the G(1)/S boundary with excess thymidine or in mitosis (M phase) with nocodazole. Endogenous or ectopic RUNX2 activity was maximal at late G(2) and during M phase. Inhibition of RUNX2 expression by RNA interference delayed entry into and exit out of the G(2)/M phases of the cell cycle. RUNX2 was coimmunoprecipitated with cyclin B1 in mitotic cells, which further supported a role for RUNX2 in cell cycle progression. Moreover, in vitro kinase assays using recombinant cdc2 kinase showed that RUNX2 was phosphorylated at Ser(451). The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNA-binding activity during mitosis and the RUNX2 mutant S451A exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G(2) and M phases, thus promoting endothelial cell proliferation required for tumor angiogenesis.
In Xenopus embryos, the cell cycle is driven by an autonomous biochemical oscillator that controls the periodic activation and inactivation of cyclin B1-CDK1. The oscillator circuit includes a system of three interlinked positive and double-negative feedback loops (CDK1 -> Cdc25 -> CDK1; CDK1 -/ Wee1 -/ CDK1; and CDK1 -/ Myt1 -/ CDK1) that collectively function as a bistable trigger. Previous work established that this bistable trigger is essential for CDK1 oscillations in the early embryonic cell cycle. Here, we assess the importance of the trigger in the somatic cell cycle, where checkpoints and additional regulatory mechanisms could render it dispensable. Our approach was to express the phosphorylation site mutant CDK1AF, which short-circuits the feedback loops, in HeLa cells, and to monitor cell cycle progression by live cell fluorescence microscopy. We found that CDK1AF-expressing cells carry out a relatively normal first mitosis, but then undergo rapid cycles of cyclin B1 accumulation and destruction at intervals of 3-6 h. During these cycles, the cells enter and exit M phase-like states without carrying out cytokinesis or karyokinesis. Phenotypically similar rapid cycles were seen in Wee1 knockdown cells. These findings show that the interplay between CDK1, Wee1/Myt1, and Cdc25 is required for the establishment of G1 phase, for the normal approximately 20-h cell cycle period, and for the switch-like oscillations in cyclin B1 abundance characteristic of the somatic cell cycle. We propose that the HeLa cell cycle is built upon an unreliable negative feedback oscillator and that the normal high reliability, slow pace and switch-like character of the cycle is imposed by a bistable CDK1/Wee1/Myt1/Cdc25 system.
Cell division cycle 2 (Cdc2) protein is an essential subunit of M-phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double-stranded RNA-activated protein kinase (PKR) was involved in the process. The PKR-mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho-mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR-mediated Tyr 4-phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.
Caspase activation is a hallmark of apoptosis. However, the molecular mechanisms underlying the regulation of caspase-8 activation within the extrinsic death pathway are not well understood. In this study, we demonstrate that procaspase-8 is phosphorylated in mitotic cells by Cdk1/cyclin B1 on Ser-387, which is located at the N terminus of the catalytic subunit p10. This phosphorylation of procaspase-8 on Ser-387 occurs in cancer cell lines, as well as in primary breast tissues and lymphocytes. Furthermore, RNA interference-mediated silencing of cyclin B1 or treatment with the Cdk1 inhibitor RO-3306 enhances the Fas-mediated activation and processing of procaspase-8 in mitotic cells. A nonphosphorylatable procaspase-8 (S387A) facilitates Fas-induced apoptosis during mitosis. Our findings suggest that Cdk1/cyclin B1 activity shields human cells against extrinsic death stimuli and unravel the molecular details of the cross talk between cell cycle and extrinsic apoptotic pathways. Finally, this new mechanism may also contribute to tumorigenesis.
Interacting selectively and non-covalently with cyclins, proteins whose levels in a cell varies markedly during the cell cycle, rising steadily until mitosis, then falling abruptly to zero. As cyclins reach a threshold level, they are thought to drive cells into G2 phase and thus to mitosis.
Catalysis of the reaction: ATP + a protein = ADP + a phosphoprotein. This reaction requires the binding of a regulatory cyclin subunit and full activity requires stimulatory phosphorylation by a CDK-activating kinase (CAK).
Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.
The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34(cdc2) on the mitotic apparatus, and is phosphorylated on Thr(34) by p34(cdc2)-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr(34) resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34(cdc2)-cyclin B1 and suggest that survivin phosphorylation on Thr(34) may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.
Catalysis of the transfer of a phosphate group to a histone. Histones are any of a group of water-soluble proteins found in association with the DNA of plant and animal chromosomes.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
The Myt1 protein kinase functions to negatively regulate Cdc2-cyclin B complexes by phosphorylating Cdc2 on threonine 14 and tyrosine 15. Throughout interphase, human Myt1 localizes to the endoplasmic reticulum and Golgi complex, whereas Cdc2-cyclin B1 complexes shuttle between the nucleus and the cytoplasm. Here we report that overproduction of either kinase-active or kinase-inactive forms of Myt1 blocked the nuclear-cytoplasmic shuttling of cyclin B1 and caused cells to delay in the G2 phase of the cell cycle. The COOH-terminal 63 amino acids of Myt1 were identified as a Cdc2-cyclin B1 interaction domain. Myt1 mutants lacking this domain no longer bound cyclin B1 and did not efficiently phosphorylate Cdc2-cyclin B1 complexes in vitro. In addition, cells overproducing mutant forms of Myt1 lacking the interaction domain exhibited normal trafficking of cyclin B1 and unperturbed cell cycle progression. These results suggest that the docking of Cdc2-cyclin B1 complexes to the COOH terminus of Myt1 facilitates the phosphorylation of Cdc2 by Myt1 and that overproduction of Myt1 perturbs cell cycle progression by sequestering Cdc2-cyclin B1 complexes in the cytoplasm.
Evidence
2:
Inferred from Physical InteractionUniProtKB
Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1-Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein beta-TrCP. As with other substrates of SCF(beta-TrCP), the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF(beta-TrCP). Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis.
Evidence
3:
Inferred from Physical InteractionBHF-UCL
Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.
Evidence
4:
Inferred from Physical InteractionUniProtKB
The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34(cdc2) on the mitotic apparatus, and is phosphorylated on Thr(34) by p34(cdc2)-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr(34) resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34(cdc2)-cyclin B1 and suggest that survivin phosphorylation on Thr(34) may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.
Evidence
5:
Inferred from Physical InteractionIntAct
The process of cellular morphogenesis is highly conserved in eukaryotes and is dependent upon the function of proteins that are centrally involved in specification of the cell cycle. The human enhancer of invasion clone 10 (HEI10) protein was identified from a HeLa cell library based on its ability to promote yeast agar invasion and filamentation. Through two-hybrid screening, the mitotic cyclin B1 and an E2 ubiquitin-conjugating enzyme were isolated as HEI10-interacting proteins. Mutation of the HEI10 divergent RING finger motif (characteristic of E3 ubiquitin ligases) and Cdc2/cyclin binding and phosphorylation sites alter HEI10-dependent yeast phenotypes, including delay in G(2)/M transition. In vertebrates, the addition of HEI10 inhibits nuclear envelope breakdown and mitotic entry in Xenopus egg extracts. Mechanistically, HEI10 expression reduces cyclin B levels in cycling Xenopus eggs and reduces levels of the cyclin B ortholog Clb2p in yeast. HEI10 is itself a specific in vitro substrate of purified cyclin B/cdc2, with a TPVR motif as primary phosphorylation site. Finally, HEI10 is itself ubiquitinated in egg extracts and is also autoubiquitinated in vitro. These and other points lead to a model in which HEI10 defines a divergent class of E3 ubiquitin ligase, functioning in progression through G(2)/M.
Evidence
6:
Inferred from Physical InteractionIntAct
We have previously used mosaic flies to screen for tumour suppressors or negative regulators of cell proliferation. The cellular composition of these flies resembles that of cancer patients who are chimaeric individuals carrying a small number of mutated somatic cells. One of the genes we identified is the large tumour suppressor gene, lats (also known as wts), which encodes a putative serine/threonine kinase. Somatic cells mutant for lats undergo extensive proliferation and form large tumours in many tissues in mosaic adults. Homozygous mutants for various lats alleles display a range of developmental defects including embryonic lethality. Although many tumour suppressors have been identified in Drosophila melanogaster, it is not clear whether these fly genes are directly relevant to tumorigenesis in mammals. Here, we have isolated mammalian homologues of Drosophila lats. Human LATS1 suppresses tumour growth and rescues all developmental defects, including embryonic lethality in flies. In mammalian cells, LATS1 is phosphorylated in a cell-cycle-dependent manner and complexes with CDC2 in early mitosis. LATS1-associated CDC2 has no mitotic cyclin partner and no kinase activity for histone H1. Furthermore, lats mutant cells in Drosophila abnormally accumulate cyclin A. These biochemical observations indicate that LATS is a novel negative regulator of CDC2/cyclin A, a finding supported by genetic data in Drosophila demonstrating that lats specifically interacts with cdc2 and cyclin A.
Evidence
7:
Inferred from Physical InteractionIntAct
Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.
Evidence
8:
Inferred from Physical InteractionUniProtKB
Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.
Evidence
9:
Inferred from Physical InteractionIntAct
DYRKs are kinases that self-activate in vitro by autophosphorylation of a YTY motif in the kinase domain, but their regulation in vivo is not well understood. In C. elegans zygotes, MBK-2/DYRK phosphorylates oocyte proteins at the end of the meiotic divisions to promote the oocyte-to-embryo transition. Here we demonstrate that MBK-2 is under both positive and negative regulation during the transition. MBK-2 is activated during oocyte maturation by CDK-1-dependent phosphorylation of serine 68, a residue outside of the kinase domain required for full activity in vivo. The pseudotyrosine phosphatases EGG-4 and EGG-5 sequester activated MBK-2 until the meiotic divisions by binding to the YTY motif and inhibiting MBK-2's kinase activity directly, using a mixed-inhibition mechanism that does not involve tyrosine dephosphorylation. Our findings link cell-cycle progression to MBK-2/DYRK activation and the oocyte-to-embryo transition.
Evidence
10:
Inferred from Physical InteractionBHF-UCL
Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34CDC2-cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34CDC2-mediated phosphorylation and resulted in loss of p34CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34CDC2.
Evidence
11:
Inferred from Physical InteractionUniProtKB
In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.
Evidence
12:
Inferred from Physical InteractionIntAct
The FEZ1/LZTS1 (LZTS1) protein is frequently downregulated in human cancers of different histotypes. LZTS1 is expressed in normal tissues, and its introduction in cancer cells inhibits cell growth and suppresses tumorigenicity, owing to an accumulation of cells in G2/M. Here, we define its role in cell cycle regulation and tumor progression by generating Lzts1 knockout mice. In Lzts1(-/-) mouse embryo fibroblasts (MEFs), Cdc25C degradation was increased during M phase, resulting in decreased Cdk1 activity. As a consequence, Lzts1(-/-) MEFs showed accelerated mitotic progression, resistance to taxol- and nocodazole-induced M phase arrest, and improper chromosome segregation. Accordingly, Lzts1 deficiency was associated with an increased incidence of both spontaneous and carcinogen-induced cancers in mice.
Evidence
13:
Inferred from Physical InteractionIntAct
Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.
Evidence
14:
Inferred from Physical InteractionIntAct
The forkhead box O (FOXO) transcription factor FOXO1 functions as a tumor suppressor by regulating expression of genes involved in apoptosis, cell cycle arrest and oxidative detoxification. Here, we demonstrate that cyclin-dependent kinase 1 (CDK1) specifically phosphorylates FOXO1 at serine 249 (S249) in vitro and in vivo. Coimmunoprecipitation assays demonstrate that both endogenous CDK1 and ectopically expressed CDK1 form a protein complex with FOXO1 in prostate cancer (PCa) cells. In vitro protein binding assays reveal that CDK1 interacts directly with FOXO1. Accordingly, overexpression of CDK1 inhibits the transcriptional activity of FOXO1 in PCa cells through S249 phosphorylation on FOXO1. Consistent with the roles of FOXO3a and FOXO4 (two other members of the FOXO family) in cell cycle regulation, forced expression of FOXO1 causes a delay in the transition from G2 to M phase. This effect is blocked completely by overexpression of CDK1 and cyclin B1. Ectopic expression of constitutively active CDK1 also inhibits FOXO1-induced apoptosis in PCa cells. Moreover, we demonstrate that the inhibitory effect of FOXO1 on Ras oncogene-induced colony formation in fibroblasts is diminished by overexpression of CDK1. Given that CDK1 and cyclin B1 are often overexpressed in human cancers including PCa, our findings suggest that aberrant activation of CDK1 may contribute to tumorigenesis by promoting cell proliferation and survival via phosphorylation and inhibition of FOXO1.
Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.
Catalysis of the reaction: ATP + (DNA-directed RNA polymerase II) = ADP + phospho-(DNA-directed RNA polymerase II); phosphorylation occurs on residues in the carboxy-terminal domain (CTD) repeats.
The transcription and processing of pre-mRNA in eukaryotic cells are regulated in part by reversible phosphorylation of the C-terminal domain of the largest RNA polymerase (RNAP) II subunit. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. This study describes a family of small CTD phosphatases (SCPs) that preferentially catalyze the dephosphorylation of Ser5 within the consensus repeat. The preferred substrate for SCP1 is RNAP II phosphorylated by TFIIH. Like FCP1, the activity of SCP1 is enhanced by the RAP74 subunit of TFIIF. Expression of SCP1 inhibits activated transcription from a number of promoters, whereas a phosphatase-inactive mutant of SCP1 enhances transcription. Accordingly, SCP1 may play a role in the regulation of gene expression, possibly by controlling the transition from initiation/capping to processive transcript elongation.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
An aging process that has as participant a cell after a cell has stopped dividing. Cell aging may occur when a cell has temporarily stopped dividing through cell cycle arrest (GO:0007050) or when a cell has permanently stopped dividing, in which case it is undergoing cellular senescence (GO:0090398). May precede cell death (GO:0008219) and succeed cell maturation (GO:0048469).
Schwann cell migration facilitates peripheral nerve regeneration after injury. We have recently found increased activation of Cdc2 kinase in regenerating sciatic nerves. Here we show that Cdc2 phosphorylation of caldesmon regulates Schwann cell migration and nerve regeneration. A robust but transient increase in Cdc2 expression was found in cultured Schwann cells prepared from the sciatic nerve in rats that had undergone crush injury for 7 days. These ;injury-preconditioned' Schwann cells exhibited enhanced migration compared with non-preconditioned control cells and treatment with the cdk inhibitor roscovitine prevented cell migration. After transduction with recombinant Cdc2 DNA adenoviral vectors, Schwann cells were implanted into sciatic nerves; those expressing wild-type Cdc2 migrated further in the distal direction than those expressing dominant-negative Cdc2. We identified caldesmon as a downstream substrate of Cdc2 in Schwann cells and its phosphorylation by Cdc2 changed its subcellular localization. Overexpression of dominant-negative caldesmon significantly counteracted the migration effect caused by Cdc2. Finally, neurite outgrowth of cultured DRG sensory neurons, facilitated by co-culture with injury-preconditioned Schwann cells, was suppressed by roscovitine treatment. The results indicate that activation of the Cdc2-caldesmon pathway is necessary for Schwann cell migration and suggest a role for this pathway in peripheral axonal growth.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a hydrogen peroxide (H2O2) stimulus.
The cell cycle process in which centrosome duplication and separation takes place. The centrosome cycle can operate with a considerable degree of independence from other processes of the cell cycle.
The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B-Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B-Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B-Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B-Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B-Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.
The progressive compaction of dispersed interphase chromatin into threadlike chromosomes prior to mitotic or meiotic nuclear division, or during apoptosis, in eukaryotic cells.
The process of restoring DNA after damage. Genomes are subject to damage by chemical and physical agents in the environment (e.g. UV and ionizing radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway.
Dysregulation of the cell cycle is the underlying mechanism of neoplasia. Healthy cells prevent propagation of DNA mutations to progeny by activation of cellular checkpoints, which allows time for DNA repair. On the other hand, activation of the DNA damage response is also the general principle of many current cancer treatments. Thus, recent advances in understanding how checkpoints in the cell cycle work at the molecular level open the door to new approaches to antitumor therapy.
The cellular metabolic process in which a cell duplicates one or more molecules of DNA. DNA replication begins when specific sequences, known as origins of replication, are recognized and bound by initiation proteins, and ends when the original DNA molecule has been completely duplicated and the copies topologically separated. The unit of replication usually corresponds to the genome of the cell, an organelle, or a virus. The template for replication can either be an existing DNA molecule or RNA.
Dysregulation of the cell cycle is the underlying mechanism of neoplasia. Healthy cells prevent propagation of DNA mutations to progeny by activation of cellular checkpoints, which allows time for DNA repair. On the other hand, activation of the DNA damage response is also the general principle of many current cancer treatments. Thus, recent advances in understanding how checkpoints in the cell cycle work at the molecular level open the door to new approaches to antitumor therapy.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of cytoskeletal structures comprising microtubules and their associated proteins.
Dysregulation of the cell cycle is the underlying mechanism of neoplasia. Healthy cells prevent propagation of DNA mutations to progeny by activation of cellular checkpoints, which allows time for DNA repair. On the other hand, activation of the DNA damage response is also the general principle of many current cancer treatments. Thus, recent advances in understanding how checkpoints in the cell cycle work at the molecular level open the door to new approaches to antitumor therapy.
A cell cycle process comprising the steps by which the nucleus of a eukaryotic cell divides; the process involves condensation of chromosomal DNA into a highly compacted form. Canonically, mitosis produces two daughter nuclei whose chromosome complement is identical to that of the mother cell.
The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34(cdc2) on the mitotic apparatus, and is phosphorylated on Thr(34) by p34(cdc2)-cyclin B1, in vitro and in vivo. Loss of phosphorylation on Thr(34) resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34(cdc2)-cyclin B1 and suggest that survivin phosphorylation on Thr(34) may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.
Any process that increases the frequency, rate or extent of gene expression. Gene expression is the process in which a gene's coding sequence is converted into a mature gene product or products (proteins or RNA). This includes the production of an RNA transcript as well as any processing to produce a mature RNA product or an mRNA (for protein-coding genes) and the translation of that mRNA into protein. Some protein processing events may be included when they are required to form an active form of a product from an inactive precursor form.
Cell cycle progression is controlled by both extracellular and intracellular signalling molecules. It has been generally believed that cdc2/CDK1 only control G(2)-M transition in mammalian and many other higher eukaryotic cells. Accumulating evidence shows that cdc2 not only promotes G(2)-M transition but is also capable of regulating G(1) progress and G(1)-S transition via association with multiple interphase cyclins; cdc2 activity can be inhibited by p21 and p27, two traditional G(1) CDK inhibitors. In addition, cdc2-cyclin B controls pronuclear union in interphase fertilized eggs. These data suggest that cdc2 may be a pluripotent CDK. Although mechanisms responsible for the multiple functions of cdc2 remain to be further investigated, interactions of cdc2 with pRb and with several important transcription factors may provide a clue to the pluripotent role of cdc2.
Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cyclin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of microtubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.
Cell cycle progression is controlled by both extracellular and intracellular signalling molecules. It has been generally believed that cdc2/CDK1 only control G(2)-M transition in mammalian and many other higher eukaryotic cells. Accumulating evidence shows that cdc2 not only promotes G(2)-M transition but is also capable of regulating G(1) progress and G(1)-S transition via association with multiple interphase cyclins; cdc2 activity can be inhibited by p21 and p27, two traditional G(1) CDK inhibitors. In addition, cdc2-cyclin B controls pronuclear union in interphase fertilized eggs. These data suggest that cdc2 may be a pluripotent CDK. Although mechanisms responsible for the multiple functions of cdc2 remain to be further investigated, interactions of cdc2 with pRb and with several important transcription factors may provide a clue to the pluripotent role of cdc2.
Schwann cell migration facilitates peripheral nerve regeneration after injury. We have recently found increased activation of Cdc2 kinase in regenerating sciatic nerves. Here we show that Cdc2 phosphorylation of caldesmon regulates Schwann cell migration and nerve regeneration. A robust but transient increase in Cdc2 expression was found in cultured Schwann cells prepared from the sciatic nerve in rats that had undergone crush injury for 7 days. These ;injury-preconditioned' Schwann cells exhibited enhanced migration compared with non-preconditioned control cells and treatment with the cdk inhibitor roscovitine prevented cell migration. After transduction with recombinant Cdc2 DNA adenoviral vectors, Schwann cells were implanted into sciatic nerves; those expressing wild-type Cdc2 migrated further in the distal direction than those expressing dominant-negative Cdc2. We identified caldesmon as a downstream substrate of Cdc2 in Schwann cells and its phosphorylation by Cdc2 changed its subcellular localization. Overexpression of dominant-negative caldesmon significantly counteracted the migration effect caused by Cdc2. Finally, neurite outgrowth of cultured DRG sensory neurons, facilitated by co-culture with injury-preconditioned Schwann cells, was suppressed by roscovitine treatment. The results indicate that activation of the Cdc2-caldesmon pathway is necessary for Schwann cell migration and suggest a role for this pathway in peripheral axonal growth.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an activity stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an amine stimulus. An amine is a compound formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an axon injury stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a cadmium (Cd) ion stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a copper ion stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a drug stimulus. A drug is a substance used in the diagnosis, treatment or prevention of a disease.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an ethanol stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an organic cyclic compound stimulus.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a toxin stimulus.
The process whose specific outcome is the progression of a ventricular cardiac muscle cell over time, from its formation to the mature state. Cardiac muscle cells are striated muscle cells that are responsible for heart contraction. The ventricle is the part of the heart that pumps blood out of the organ.
IEAOrtholog Compara
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reactions
Phosphorylation at Thr-14 or Tyr-15 inactivates the enzyme, while phosphorylation at Thr-161 activates it. Activated through a multistep process; binding to cyclin-B is required for relocation of cyclin-kinase complexes to the nucleus, activated by CAK/CDK7-mediated phosphorylation on Thr-161, and CDC25-mediated dephosphorylation of inhibitory phosphorylation on Thr-14 and Tyr-15. Inhibited by flavopiridol and derivatives, pyrimidine derivatives, pyridine derivatives, purine derivatives, staurosporine, paullones, oxoindoles, indazole analogs, indolin-2-ones, pyrazolo[3,4-b]pyridines, imidazo[1,2-a]pyridine (AZ703), thiazolinone analogs(RO-3306), thiazol urea, macrocyclic quinoxalin-2-one, pyrrolo[2,3-a]carbazole, pyrazolo[1,5-a]-1,3,5-triazine, pyrazolo[1,5-a]pyrimidine (Dinaciclib, SCH 727965), 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine), olomoucine, AG-024322, AT-7519, P276-00, R547/Ro-4584820 and SNS-032/BMS-387032. Repressed by the CDK inhibitors CDKN1A/p21 and CDKN1B/p27 during the G1 phase and by CDKN1A/p21 at the G1-S checkpoint upon DNA damage. Transient activation by rapid and transient dephosphorylation at Tyr-15 triggered by TGFB1.
Eur. J. Biochem. 243, 527-536 (1997)[PubMed:9030781]
Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.
Transforming Growth Factor-beta (TGFbeta) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFbeta has any effect on G2 checkpoint kinases. We have found that TGFbeta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24h for cyclin B1 and cdc2 and by 48h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFbeta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48h after initiating TGFbeta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFbeta. In addition, TGFbeta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3h before a dramatic decrease of both molecules by 48h. Taken together, our data suggest that TGFbeta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFbeta-induced G1 arrest and growth inhibition.
Cyclin dependent kinases (CDKs) are a family of proteins involved in the regulation of cell cycle progression and attractive targets in oncology. The regulation of CDKs activities is achieved by their association with cyclin partners and kinases, phosphatases and specific inhibitors. Different CDKs complexes exert their functions at different phases. CDK1 is a master modulator in the initiation and transition process through mitosis of the cell cycle. Previous studies have shown that loss of CDK1 activity or the aberrant expression of CDK1 involved in G2 phase arrest and many tumor types, thereby validating CDK1 as a therapeutic target. Therefore, a surge of interest has been devoted to searching for potent CDK1 inhibitors as effective chemotherapeutic agents. Herein we focus, in this review, mainly on the studies about the structure, functions and different structure classes of potent CDK1 inhibitors.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in the complex series of events by which the cell duplicates its contents and divides into two. The eukaryotic cell cycle can be divided in four phases termed G1 (first gap period), S (synthesis, phase during which the DNA is replicated), G2 (second gap period) and M (mitosis). The prokaryotic cell cycle typically involves a period of growth followed by DNA replication, partition of chromosomes, formation of septum and division into two similar or identical daughter cells.
Protein involved in the separation of one cell into two daughter cells. In eukaryotic cells, cell division includes the nuclear division (mitosis) and the subsequent cytoplasmic division (cytokinesis).
Protein involved in mitosis, the nuclear division in eukaryotic cells involving the exact duplication and separation of the chromosome threads so that each daughter nucleus carries a chromosome complement identical to that of the parent nucleus. Mitosis is divided into four substages: prophase, metaphase, anaphase and telophase.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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