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1.
Life Sci Alliance ; 4(3)2021 03.
Article in English | MEDLINE | ID: mdl-33402344

ABSTRACT

Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.


Subject(s)
Cell Cycle Proteins/metabolism , Cyclin A2/metabolism , Cytoplasm/metabolism , G2 Phase/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , S Phase/genetics , Signal Transduction/genetics , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cell Nucleus/metabolism , Chromatin/metabolism , Cyclin A2/genetics , Cyclin-Dependent Kinase 2/deficiency , Cyclin-Dependent Kinase 2/genetics , DNA Damage/genetics , Enzyme Activation/genetics , HeLa Cells , Humans , Mitosis/genetics , Phosphorylation/genetics , Protein Binding , Transfection , Polo-Like Kinase 1
2.
J Biol Chem ; 293(50): 19387-19399, 2018 12 14.
Article in English | MEDLINE | ID: mdl-30366983

ABSTRACT

Bone mass is maintained by a balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Although recent genetic studies have uncovered various mechanisms that regulate osteoblast differentiation, the molecular basis of osteoblast proliferation remains unclear. Here, using an osteoblast-specific loss-of-function mouse model, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates osteoblast proliferation and differentiation. Quantitative RT-PCR analyses revealed that Cdk1 is highly expressed in bone and is down-regulated upon osteoblast differentiation. We also noted that Cdk1 is dispensable for the bone-anabolic effects of parathyroid hormone (PTH). Cdk1 deletion in osteoblasts led to osteoporosis in adult mice due to low bone formation, but did not affect osteoclast formation in vivo Cdk1 overexpression in osteoblasts promoted proliferation, and conversely, Cdk1 knockdown inhibited osteoblast proliferation and promoted differentiation. Of note, we provide direct evidence that PTH's bone-anabolic effects occur without enhancing osteoblast proliferation in vivo Furthermore, we found that Cdk1 expression in osteoblasts is essential for bone fracture repair. These findings may help reduce the risk of nonunion after bone fracture and identify patients at higher risk for nonresponse to PTH treatment. Collectively, our results indicate that Cdk1 is essential for osteoblast proliferation and that it functions as a molecular switch that shifts osteoblast proliferation to maturation. We therefore conclude that Cdk1 plays an important role in bone formation.


Subject(s)
Bone and Bones/drug effects , Bone and Bones/physiology , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Gene Knockout Techniques , Osteogenesis/genetics , Parathyroid Hormone/pharmacology , 3T3 Cells , Animals , Bone and Bones/cytology , Bone and Bones/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Fractures, Bone/physiopathology , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Wound Healing/drug effects
3.
Cell Cycle ; 15(23): 3203-3209, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27831832

ABSTRACT

Small molecule inhibitors targeting CDK1/CDK2 have been clinically proven effective against a variety of tumors, albeit at the cost of profound off target toxicities. To separate potential therapeutic from toxic effects, we selectively knocked down CDK1 or CDK2 in p53 mutated HACAT cells by siRNA silencing. Using dynamic, cell cycle wide proteome arrays, we observed minor changes in overall abundance of proteins critically involved in cell cycle transition despite profound G2/M or G1/S arrest, respectively. Employing phospho site specific analyses, we identified uncoupled mitogenic, yet pro-apoptotic signaling from counter balancing anti-apoptotic activity in CDK2 disrupted cells. Moreover, a crucial role of CDK2 activity in early serum response was observed, extending well-established roles of CDKs outside their cell cycle regulating functions. In contrast, disruption of CDK1 only marginally affected phosphorylation events of crucial signaling nodes prior to G2/S transition. The data presented here suggest that the temporal separation of pro- and anti-apoptotic pathways by selective inhibition of CDK2 disrupts coherent signaling modules and may synergize with anti-proliferative drugs, averting toxic side effects from CDK1 inhibition.


Subject(s)
Apoptosis , CDC2 Protein Kinase/metabolism , Gene Silencing , Mitosis , Signal Transduction , Synthetic Lethal Mutations/genetics , Tumor Suppressor Protein p53/metabolism , CDC2 Protein Kinase/deficiency , Cell Line , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Kinetics , Mitosis/genetics , Phosphorylation , RNA, Small Interfering/metabolism
4.
Development ; 141(17): 3388-98, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25139855

ABSTRACT

Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIß (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Differentiation , Cell Nucleus/metabolism , Lens, Crystalline/cytology , Lens, Crystalline/enzymology , Mitosis , Animals , CDC2 Protein Kinase/deficiency , Cell Cycle Proteins , DNA/biosynthesis , Embryo, Mammalian/cytology , Embryo, Mammalian/enzymology , Endodeoxyribonucleases/metabolism , Endoplasmic Reticulum/metabolism , Endoreduplication , Epithelial Cells/cytology , Epithelial Cells/enzymology , Female , Integrases/metabolism , Lamins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/metabolism , Nuclear Proteins/metabolism , Phosphorylation
5.
PLoS One ; 7(7): e42129, 2012.
Article in English | MEDLINE | ID: mdl-22848730

ABSTRACT

Apoptosis of post-mitotic neurons plays a significant role in secondary tissue damage following traumatic spinal cord injury (SCI). Activation of E2F1-dependent transcription promotes expression of pro-apoptotic factors, including CDK1; this signal transduction pathway is believed to represent an important mechanism for the physiological or pathological neuronal cell death. However, a specific role for this pathway in neuronal apoptosis induced by SCI has not yet been reported. Here we demonstrate up-regulation of the E2F1/CDK1 pathway that is associated with neuronal apoptosis following impact SCI in rats. Expression of E2F1 and CDK1 were robustly up-regulated as early as 15 min after injury and sustained until 3 days post-injury. CDK1 activity and E2F1 downstream targets bim and c-Myb were significantly increased after SCI. Activation of E2F1/CDK1 signaling also was associated with death of neurons in vitro; this was attenuated by shRNA knockdown or pharmacological inhibition of the E2F1/CDK1 pathway. CR8, a novel and potent CDK1 inhibitor, blocked apoptosis of primary cortical neurons at low-micromolar concentrations. Moreover, SCI-induced up-regulation of E2F1/CDK1 and associated neuronal apoptosis was significantly attenuated by systemic injection of CR8 (1 mg/kg, i.p.) at 5 min after injury. CR8 significantly decreased posttraumatic elevation of biochemical markers of apoptosis, such as products of caspase-3 and α-fodrin cleavage, as well as neuronal cell death, as indicated by TUNEL staining. Importantly, CR8 treatment also increased the number of surviving neurons at 5 weeks after injury. Together, these findings indicate that activation of the E2F1/CDK1 pathway contributes to the pathophysiology of SCI and that selective inhibition of this signaling cascade may represent an attractive therapeutic strategy.


Subject(s)
Apoptosis/drug effects , CDC2 Protein Kinase/metabolism , E2F1 Transcription Factor/metabolism , Neurons/drug effects , Neurons/pathology , Signal Transduction/drug effects , Spinal Cord Injuries/pathology , Animals , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cell Line, Tumor , Down-Regulation/drug effects , Down-Regulation/genetics , E2F1 Transcription Factor/deficiency , E2F1 Transcription Factor/genetics , Gene Silencing , Humans , Male , Neurons/cytology , Neurons/metabolism , Purines/pharmacology , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Spinal Cord Injuries/genetics , Spinal Cord Injuries/metabolism
6.
Proc Natl Acad Sci U S A ; 106(9): 3184-9, 2009 Mar 03.
Article in English | MEDLINE | ID: mdl-19221029

ABSTRACT

Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR-Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2-Cdk1 in the regulation of late origin firing. Activity of cyclin A2-Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2-Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2-Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2-Cdk1 is a key regulator of S-phase programs.


Subject(s)
CDC2 Protein Kinase/metabolism , Cyclin A/metabolism , Animals , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cell Line , Cyclin A/genetics , Enzyme Activation , Humans , Kinetics , Mice , Mice, Knockout , Mutation/drug effects , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S Phase
7.
Nature ; 448(7155): 811-5, 2007 Aug 16.
Article in English | MEDLINE | ID: mdl-17700700

ABSTRACT

Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle , Embryo, Mammalian/cytology , Embryo, Mammalian/enzymology , Animals , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cells, Cultured , Cyclin-Dependent Kinases/deficiency , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Fibroblasts/cytology , Fibroblasts/drug effects , Genes, Essential/genetics , Interphase , Mice , Mitogens/pharmacology , Organogenesis
8.
Cancer Res ; 66(18): 9270-80, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16982772

ABSTRACT

Selective cyclin-dependent kinase (cdk) 2 inhibition is readily compensated. However, reduced cdk2 activity may have antiproliferative effects in concert with other family members. Here, inducible RNA interference was used to codeplete cdk2 and cdk1 from NCI-H1299 non-small cell lung cancer and U2OS osteosarcoma cells, and effects were compared with those mediated by depletion of either cdk alone. Depletion of cdk2 slowed G1 progression of NCI-H1299 cells and depletion of cdk1 slowed G2-M progression in both cell lines, with associated endoreduplication in U2OS cells. However, compared with the incomplete cell cycle blocks produced by individual depletion, combined depletion had substantial consequences, with G2-M arrest predominating in NCI-H1299 cells and apoptosis the primary outcome in U2OS cells. In U2OS cells, combined depletion affected RNA polymerase II expression and phosphorylation, causing decreased expression of the antiapoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis (XIAP), effects usually mediated by inhibition of the transcriptional cdk9. These events do not occur after individual depletion of cdk2 and cdk1, suggesting that reduction of cdk2, cdk1, and RNA polymerase II activities all contribute to apoptosis in U2OS cells. The limited cell death induced by combined depletion in NCI-H1299 cells was significantly increased by codepletion of cdk9 or XIAP or by simultaneous treatment with the cdk9 inhibitor flavopiridol. These results show the potency of concomitant compromise of cell cycle and transcriptional cdk activities and may guide the selection of clinical drug candidates.


Subject(s)
Apoptosis/physiology , CDC2 Protein Kinase/deficiency , Cyclin-Dependent Kinase 2/deficiency , Neoplasms/enzymology , Neoplasms/pathology , Apoptosis/genetics , Bone Neoplasms/enzymology , Bone Neoplasms/genetics , Bone Neoplasms/pathology , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/genetics , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Division/physiology , Cell Line, Tumor , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Flavonoids/pharmacology , G1 Phase/physiology , G2 Phase/physiology , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasms/therapy , Osteosarcoma/enzymology , Osteosarcoma/genetics , Osteosarcoma/pathology , Piperidines/pharmacology , RNA Polymerase II/antagonists & inhibitors , RNA Polymerase II/biosynthesis , RNA Polymerase II/genetics , RNA, Small Interfering/genetics
9.
J Cell Sci ; 116(Pt 1): 137-43, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12456723

ABSTRACT

The centriole, organizer of the centrosome, duplicates by assembling a unique daughter identical to itself in overall organization and length. The centriole is a cylindrical structure composed of nine sets of microtubules and is thus predicted to have nine-fold symmetry. During duplication, a daughter lacking discrete microtubular organization first appears off the wall of the mother centriole. It increases in length perpendicularly away from the mother and terminates growth when it matches the length of the mother. How a unique daughter of the correct length and overall organization is assembled is unknown. Here, we describe three types of unusual centriole configurations observed in wing imaginal discs of Drosophila following inactivation of Cdk1. First, we observed centriole triplets consisting of one mother and two daughters, which suggested that centrioles have more than one potential site for the assembly of daughters. Second, we observed centriole triplets comprising a grandmother, mother and daughter, which suggested that subsequent centriole duplication cycles do not require separation of mother and daughter centrioles. Finally, we observed centriole pairs in which the daughter is longer than its mother. These findings suggest that regulatory events rather than rigid structural constraints dictate features of the stereotyped duplication program of centrioles.


Subject(s)
CDC2 Protein Kinase/deficiency , Cell Division/genetics , Centrioles/pathology , Drosophila melanogaster/growth & development , Larva/growth & development , Wings, Animal/growth & development , Animals , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/genetics , Centrioles/genetics , Centrioles/ultrastructure , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental/genetics , Genes, cdc/physiology , Larva/cytology , Larva/genetics , Microscopy, Electron , Microtubules/genetics , Microtubules/metabolism , Microtubules/ultrastructure , Wings, Animal/cytology , Wings, Animal/metabolism
10.
Mol Microbiol ; 45(2): 321-32, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12123447

ABSTRACT

The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites. Expression of TPK2-HA-wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.


Subject(s)
Cyclin-Dependent Kinases/physiology , Protozoan Proteins/physiology , Toxoplasma/enzymology , Amino Acid Sequence , Animals , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , CDC28 Protein Kinase, S cerevisiae/deficiency , CDC28 Protein Kinase, S cerevisiae/genetics , COS Cells , Chlorocebus aethiops , Cloning, Molecular , Cyclin-Dependent Kinases/genetics , Cyclins/metabolism , Genetic Complementation Test , Molecular Sequence Data , Protozoan Proteins/genetics , Recombinant Fusion Proteins/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/physiology , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/physiology , Species Specificity , Toxoplasma/cytology
11.
J Cell Sci ; 114(Pt 24): 4557-65, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11792820

ABSTRACT

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both beta- and gamma-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.


Subject(s)
Gene Silencing , Genes/genetics , RNA, Untranslated/genetics , Animals , CDC2 Protein Kinase/deficiency , CDC2 Protein Kinase/genetics , Cell Line , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Gene Targeting/methods , HeLa Cells , Humans , Mice , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Mitosis/genetics , Nuclear Envelope/genetics , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , RNA, Small Interfering , Rats
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