RESUMO
To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Segregação de Cromossomos , Ensaios Enzimáticos , Proteínas Associadas aos Microtúbulos/genética , Mitose , Mutação , Fosforilação/fisiologia , Multimerização Proteica/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Whole-genome duplications (WGDs) have played a central role in the evolution of genomes and constitute an important source of genome instability in cancer. Here, we show in Saccharomyces cerevisiae that abnormal accumulations of histones are sufficient to induce WGDs. Our results link these WGDs to a reduced incorporation of the histone variant H2A.Z to chromatin. Moreover, we show that high levels of histones promote Swe1WEE1 stabilisation thereby triggering the phosphorylation and inhibition of Cdc28CDK1 through a mechanism different of the canonical DNA damage response. Our results link high levels of histones to a specific type of genome instability that is quite frequently observed in cancer and uncovers a new mechanism that might be able to respond to high levels of histones.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Duplicação Cromossômica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , FosforilaçãoRESUMO
Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Carbono/metabolismo , Ciclo Celular , Proliferação de Células , Metabolismo Energético , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Ativação Enzimática , Pontos de Checagem da Fase G1 do Ciclo Celular , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Trealase/metabolismo , Trealose/metabolismoRESUMO
To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Anáfase , Nucléolo Celular/metabolismo , Ativação Enzimática , Metáfase , Peptídeos/química , FosforilaçãoRESUMO
CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons, a phenotype akin to animals lacking p27. We propose that the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Neurônios/metabolismo , Animais , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular , Diferenciação Celular , Células Cultivadas , Dano ao DNA , Ativação Enzimática , Camundongos , Camundongos Knockout , Neurônios/citologiaRESUMO
Pah1p, which functions as phosphatidate phosphatase (PAP) in the yeast Saccharomyces cerevisiae, plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The diacylglycerol produced by PAP is used for the synthesis of triacylglycerol as well as for the synthesis of phospholipids via the Kennedy pathway. Pah1p is a highly phosphorylated protein in vivo and has been previously shown to be phosphorylated by the protein kinases Pho85p-Pho80p and Cdc28p-cyclin B. In this work, we showed that Pah1p was a bona fide substrate for protein kinase A, and we identified by mass spectrometry and mutagenesis that Ser-10, Ser-677, Ser-773, Ser-774, and Ser-788 were the target sites of phosphorylation. Protein kinase A-mediated phosphorylation of Pah1p inhibited its PAP activity by decreasing catalytic efficiency, and the inhibitory effect was primarily conferred by phosphorylation at Ser-10. Analysis of the S10A and S10D mutations (mimicking dephosphorylation and phosphorylation, respectively), alone or in combination with the seven alanine (7A) mutations of the sites phosphorylated by Pho85p-Pho80p and Cdc28p-cyclin B, indicated that phosphorylation at Ser-10 stabilized Pah1p abundance and inhibited its association with membranes, PAP activity, and triacylglycerol synthesis. The S10A mutation enhanced the physiological effects imparted by the 7A mutations, whereas the S10D mutations attenuated the effects of the 7A mutations. These data indicated that the protein kinase A-mediated phosphorylation of Ser-10 functions in conjunction with the phosphorylations mediated by Pho85p-Pho80p and Cdc28p-cyclin B and that phospho-Ser-10 should be dephosphorylated for proper PAP function.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fosfatidato Fosfatase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismo , Cinética , Lipídeos/química , Mutação , Fosforilação , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Serina/química , Frações Subcelulares/metabolismo , Especificidade por Substrato , Triglicerídeos/químicaRESUMO
Genome stability depends on faithful chromosome segregation, which relies on maintenance of chromatid cohesion during S phase. In eukaryotes, Pds1/securin is the only known inhibitor that can prevent loss of cohesion. However, pds1Δ yeast cells and securin-null mice are viable. We sought to identify redundant mechanisms that promote cohesion within S phase in the absence of Pds1 and found that cells lacking the S-phase cyclins Clb5 and Clb6 have a cohesion defect under conditions of replication stress. Similar to the phenotype of pds1Δ cells, loss of cohesion in cells lacking Clb5 and Clb6 is dependent on Esp1. However, Pds1 phosphorylation by Cdk-cyclin is not required for cohesion. Moreover, cells lacking Clb5, Clb6, and Pds1 are inviable and lose cohesion during an unperturbed S phase, indicating that Pds1 and specific B-type cyclins promote cohesion independently of one another. Consistent with this, we find that Mcd1/Scc1 is less abundant on chromosomes in cells lacking Clb5 and Clb6 during replication stress. However, clb5Δ clb6Δ cells do accumulate Mcd1/Scc1 at centromeres upon mitotic arrest, suggesting that the cyclin-dependent mechanism is S phase specific. These data indicate that Clb5 and Clb6 promote cohesion which is then protected by Pds1 and that both mechanisms are required during replication stress.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Nucleares/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Ciclina B/genética , Quinases Ciclina-Dependentes/genética , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Hidroxiureia/farmacologia , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Securina , Separase , Fuso Acromático/metabolismoRESUMO
Entry into and precise progression through the cell cycle depends on the sequential expression and activation of cyclin dependent kinases (CDK). In accord, CDK dysregulation is a hallmark of many cancers. The function of Cdk2 is still an enigma as in vitro studies revealed that it is required for S phase-entry, whereas in vivo studies showed that Cdk2 is not an essential gene. Moreover, unlike other Cdks, or its cyclin E regulator, Cdk2-overexpressing tumors were reported only in one type of tumor. In this report we used budding yeast as a tool to explore Cdk2 function. We showed that hCdk2 promoted S phase in cells carrying a temperature-sensitive mutation in yCDK1, albeit, only when expressed at low or moderate levels. Overexpression of hCdk2 resulted in a defect in the G1 to S transition and a reduction in viability. The same phenotypes were observed in cells overexpressing its yeast functional homolog, Ime2, which is a meiosis-specific CDK-like kinase. A genetic interaction with the DNA damage checkpoint was demonstrated by showing an increased toxicity of hCdk2 and Ime2 in RAD53-deleted cells, and delayed Rad53 activation in response to MMS treatment in cells overexpressing hCdk2 or Ime2.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA , Fase G1 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Meiose , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Fase S , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF(Cdc4) ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC(Cdh1)). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF(Cdc4) target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclina B/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Ciclina B/genética , Cinesinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares/genética , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/genéticaRESUMO
The induction of middle meiotic promoters is a key regulatory event in the life cycle of Saccharomyces cerevisiae that controls exit from prophase, meiosis, and spore formation. The Sum1 repressor and Ndt80 activator proteins control middle promoters by binding to overlapping DNA elements. NDT80 is controlled by a tightly regulated middle meiotic promoter through a positive autoregulatory loop and is repressed in vegetative cells by Sum1. It has previously been shown that the meiosis-specific kinase Ime2 promotes the removal of Sum1 from DNA. Here, we show that Sum1 is also regulated by the cyclin-dependent kinase, Cdk1. While sum1 phosphosite mutants that are insensitive to Cdk1 or Ime2 complete meiosis and form spores, a mutant that is insensitive to both Ime2 and Cdk1 (sum1-ci) blocks meiotic development in prophase with an ndt80Delta-like phenotype. Ectopic expression of NDT80 or mutation of a Sum1-binding element in the NDT80 promoter bypasses the sum1-ci block. Hst1 is a NAD(+)-dependent histone deacetylase that is linked to Sum1 by the Rfm1 tethering factor. Deletion of HST1 or RFM1 also bypasses the sum1-ci block. These results demonstrate that Sum1 functions as a key meiotic brake through the NDT80 promoter and that Cdk1 and Ime2 trigger exit from meiotic prophase by inhibiting the Sum1 transcriptional repression complex.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Prófase Meiótica I , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Sítios de Ligação , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC28 de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Estradiol/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Prófase Meiótica I/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
START-dependent transcription in Saccharomyces cerevisiae is regulated by two transcription factors SBF and MBF, whose activity is controlled by the binding of the repressor Whi5. Phosphorylation and removal of Whi5 by the cyclin-dependent kinase (CDK) Cln3-Cdc28 alleviates the Whi5-dependent repression on SBF and MBF, initiating entry into a new cell cycle. This Whi5-SBF/MBF transcriptional circuit is analogous to the regulatory pathway in mammalian cells that features the E2F family of G1 transcription factors and the retinoblastoma tumor suppressor protein (Rb). Here we describe genetic and biochemical evidence for the involvement of another CDK, Pcl-Pho85, in regulating G1 transcription, via phosphorylation and inhibition of Whi5. We show that a strain deleted for both PHO85 and CLN3 has a slow growth phenotype, a G1 delay, and is severely compromised for SBF-dependent reporter gene expression, yet all of these defects are alleviated by deletion of WHI5. Our biochemical and genetic tests suggest Whi5 mediates repression in part through interaction with two histone deacetylases (HDACs), Hos3 and Rpd3. In a manner analogous to cyclin D/CDK4/6, which phosphorylates Rb in mammalian cells disrupting its association with HDACs, phosphorylation by the early G1 CDKs Cln3-Cdc28 and Pcl9-Pho85 inhibits association of Whi5 with the HDACs. Contributions from multiple CDKs may provide the precision and accuracy necessary to activate G1 transcription when both internal and external cues are optimal.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fase G1/fisiologia , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Fase G1/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild-type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable K(M)/V(max) values for mutant cdk2 and wild-type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Cinética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Especificidade por SubstratoRESUMO
Saccharomyces cerevisiae cells lacking ISC1 (inositol phosphosphingolipase C) exhibit sensitivity to genotoxic agents such as methyl methanesulfonate and hydroxyurea (HU). Cell cycle analysis by flow cytometry revealed a G(2)/M block in isc1Delta cells when treated with methyl methanesulfonate or HU. Further investigation revealed that the levels of Cdc28 phosphorylated on Tyr-19, which plays an essential role in the regulation of the G(2)/M checkpoint, were higher in synchronized and asynchronous cells lacking ISC1 in response to HU. Use of a Cdc28-Y19F mutant protected isc1Delta from the G(2)/M block. In wild type cells, HU induced a loss of the Swe1p kinase, the enzyme that phosphorylates Cdc28-Tyr-19, correlating with resumption of the cell cycle. In the isc1Delta cells, however, the levels of Swe1p remained at sustained high levels in response to HU. Significantly, deletion of SWE1 in an isc1Delta background overcame the G(2)/M block in response to HU. The double isc1Delta/swe1Delta mutant also overcame the growth defect on HU. Taken together, these findings implicate Isc1p as an upstream regulator of Swe1p levels and stability and Cdc28-Tyr-19 phosphorylation, in effect signaling recovery from the effects of genotoxic stress and allowing G(2)/M progression.
Assuntos
Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Hidroxiureia/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipases Tipo C/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Fase G2/fisiologia , Técnicas de Inativação de Genes , Mesilatos/farmacologia , Mutação , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/genéticaRESUMO
Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G(2), followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Desenvolvimento Embrionário/fisiologia , Proteínas Quinases/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Ciclina A/genética , Ciclina A/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclina B1 , Quinases Ciclina-Dependentes/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Knockout , Fases de Leitura Aberta , Fenótipo , Ploidias , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Interferência de RNARESUMO
The mammalian Cks family consists of 2 well-conserved small proteins, Cks1 and Cks2. Cks1 has been shown to promote cell-cycle progression by triggering degradation of p27(kip1). The function of Cks2 in somatic mammalian cells is not well understood although it is required for the first metaphase/anaphase transition during the meiosis. Emerging evidence shows that elevated expression of Cks1 and Cks2 is often found in a variety of tumors, and is correlated with poor survival rate of the patients. Here we demonstrated that expression of Cks1 and Cks2 were elevated in prostate tumors of human and animal models, as well as prostatic cancer cell lines. Forced expression of Cks1 and Cks2 in benign prostate tumor epithelial cells promoted cell population growth. Knockdown of Cks1 expression in malignant prostate tumor cells inhibited proliferation, anchorage-independent growth, and migration activities, whereas knockdown of Cks2 expression induced programmed cell death and inhibited the tumorigenicity. Collectively, the data suggest that elevated expression of Cks1 contributes to the tumorigenicity of prostate tumor cells by promoting cell growth and elevated expression of Cks2 protects the cells from apoptosis. Thus, the finding suggests a novel therapeutic strategy for prostatic cancer based on inhibiting Cks1 and Cks2 activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Quinases/metabolismo , Animais , Apoptose , Western Blotting , Quinases relacionadas a CDC2 e CDC28/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quinases Ciclina-Dependentes/genética , DNA Complementar/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Neoplasias da Próstata/patologia , Proteínas Quinases/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase,which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdh1 , Ciclo Celular , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Motores Moleculares , Fosforilação , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Quinase 1 Polo-LikeRESUMO
Ydj1 is a Saccharomyces cerevisiae Hsp40 molecular chaperone that functions with Hsp70 to promote polypeptide folding. We identified Ydj1 as being important for maintaining steady-state levels of protein kinases after screening several chaperones and cochaperones in gene deletion mutant strains. Pulse-chase analyses revealed that a portion of Tpk2 kinase was degraded shortly after synthesis in a ydj1Delta mutant, while the remainder was capable of maturing but with reduced kinetics compared to the wild type. Cdc28 maturation was also delayed in the ydj1Delta mutant strain. Ydj1 protects nascent kinases in different contexts, such as when Hsp90 is inhibited with geldanamycin or when CDC37 is mutated. The protective function of Ydj1 is due partly to its intrinsic chaperone function, but this is minor compared to the protective effect resulting from its interaction with Hsp70. SIS1, a type II Hsp40, was unable to suppress defects in kinase accumulation in the ydj1Delta mutant, suggesting some specificity in Ydj1 chaperone action. However, analysis of chimeric proteins that contained the chaperone modules of Ydj1 or Sis1 indicated that Ydj1 promotes kinase accumulation independently of its client-binding specificity. Our results suggest that Ydj1 can both protect nascent chains against degradation and control the rate of kinase maturation.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico , Estabilidade Enzimática , Deleção de Genes , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , SolubilidadeRESUMO
During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B , Ciclinas/genética , Ciclinas/metabolismo , Dineínas/genética , Dineínas/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutação , Proteínas Nucleares/genética , Fosforilação , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina , Fuso Acromático/fisiologiaRESUMO
Two-nanosecond molecular dynamics modeling of the crystalline lattice of an active complex of kinase pT160-CDK2/cyclin A/ATP-Mg2+ substrate has been performed. The results of modeling indicated that the structures of the nonmutant CDK2 complex and mutant CDK2 complex, which involves the G 16S-CD K2 substitution corresponding to that of yeast, markedly differ, the differences in structural conformations being particularly well pronounced in those regions that play a key role in the functioning of kinase. Based on the results of computations, structural elements are considered that may affect the kinase activity and regulatory phosphorylation, and the binding of protein kinase to cyclins and substrates.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/química , Quinase 2 Dependente de Ciclina/química , Modelos Moleculares , Mutação Puntual , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Magnésio/química , Ligação Proteica/genética , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
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.