RESUMO
Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. The activity of some of them is deregulated in tumor cells and to find specific inhibitors is an important goal to be achieved. We report here the current methods to determine their in vitro activity in order to facilitate the identification of specific inhibitors. Mainly, the activity can be determined by using immunoprecipitates from cell samples with antibodies against specific Cdks as a source of the enzymes.
Assuntos
Ciclo Celular/genética , Quinases Ciclina-Dependentes/isolamento & purificação , Ciclinas/isolamento & purificação , Imunoprecipitação/métodos , Animais , Soluções Tampão , Corantes/química , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Cabras , Humanos , Camundongos , Biologia Molecular/métodos , Coelhos , Ratos , OvinosRESUMO
Cyclin-dependent kinases (Cdks) belong to a family of key regulators of cell division cycle and transcription. Their activity is mainly regulated by association with regulatory subunits named cyclins but their activities are also regulated by phosphorylation, acetylation, and the association with specific inhibitory proteins (CKIs). The activity of different Cdks is deregulated in many different type of tumors, and thus, Cdks are considered targets for antitumoral therapy. For large screenings of inhibitors the use of purified recombinant Cdks and cyclins is recommended. We report here the current methods to determine their in vitro activity for large screenings of inhibitors.
Assuntos
Quinases Ciclina-Dependentes/isolamento & purificação , Ciclinas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Bactérias/metabolismo , Proteína Quinase CDC2/química , Proteína Quinase CDC2/isolamento & purificação , Ciclo Celular , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinases Ciclina-Dependentes/química , Ciclinas/química , Humanos , Neoplasias/metabolismo , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/químicaRESUMO
Cyclin H (CycH), a member of the large cyclin family, participates in every process of cell division. Its biological functions and importance have received wide attention in mammalians, but not in higher plants. This work reports a protein purification protocol for obtaining Arabidopsis CycH;1 (AtCycH;1) from prokaryotic expression system, followed by characterization of its biophysical properties. The protein was constructed with a His-tag at its N-terminus. One-step nickel-affinity purification yielded high pure target protein, which behaved as a monomer in the testing condition. Circular Dichroism spectrum revealed that AtCycH;1 is a helical protein containing a significant amount of disordered structures. Further assays indicated that AtCycH;1 exhibits poor heat-resistance and can be easily degraded in room temperature, suggesting low stability for the protein. The flexible and unstable properties may be intrinsic to the protein in vivo as it has to bind with different partners during the cell cycle and be promptly degraded to meet the phase transition. The instability, however, can be improved by adding SO4(2-) ion in the protein buffer. The presence of a high concentration of SO4(2-) is capable of increasing the thermal stability and inhibiting the degradation. Irrespective of whether the association of SO4(2-) with AtCycH;1 drives the protein into more compact form or not, the current results may provide clues for a successful crystallization of AtCycH;1 and its subsequent structural analysis in the future.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/química , Ciclinas/química , Ciclinas/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Dicroísmo Circular , Ciclinas/metabolismo , Temperatura Alta , Dados de Sequência Molecular , Estabilidade Proteica , Alinhamento de SequênciaRESUMO
This work describes the purification and preliminary crystallographic analysis of the CBS-domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient domain protein 2; ACDP2), which consists of a pair of cystathionine ß-synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1-4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P2(1)2(1)2 and I222 (or I2(1)2(1)2(1)) and diffracted X-rays to 2.0 and 3.6â Å resolution, respectively, using synchrotron radiation.
Assuntos
Ciclinas/química , Animais , Proteínas de Transporte de Cátions , Cristalização , Cristalografia por Raios X , Ciclinas/isolamento & purificação , Humanos , CamundongosRESUMO
Cyclin-dependent kinases (CDKs) play essential roles in coordinate control of cell cycle progression. Activation of CDKs requires interaction with specific cyclin partners and phosphorylation of their T-loops by CDK-activating kinases (CAKs). The Arabidopsis thaliana genome encodes four potential CAKs. CAK2At (CDKD;3) and CAK4At (CDKD;2) are closely related to the vertebrate CAK, CDK7/p40MO15; they interact with cyclin H and phosphorylate CDKs, as well as the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. CAK1At (CDKF;1) shows cyclin H-independent CDK-kinase activity and can activate a heterologous CAK, Mcs6, in fission yeast. In Arabidopsis, CAK1At is a subunit of a protein complex of 130 kD, which phosphorylates the T-loop of CAK2At and CAK4At and activates the CTD-kinase activity of CAK4At in vitro and in root protoplasts. These results suggest that CAK1At is a novel CAK-activating kinase that modulates the activity of CAK2At and CAK4At, thereby controlling CDK activities and basal transcription in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Células Cultivadas , Ciclina H , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Ciclinas/isolamento & purificação , Ciclinas/metabolismo , Ativação Enzimática/genética , Insetos/genética , Insetos/metabolismo , Substâncias Macromoleculares/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Protoplastos/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Dinoflagellates are protists with permanently condensed chromosomes that lack histones and whose nuclear membrane remains intact during mitosis. These unusual nuclear characters have suggested that the typical cell cycle regulators might be slightly different than those in more typical eukaryotes. To test this, a cyclin has been isolated from the dinoflagellate Gonyaulax polyedra by functional complementation in cln123 mutant yeast. This GpCyc1 sequence contains two cyclin domains in its C-terminal region and a degradation box typical of mitotic cyclins. Similar to other dinoflagellate genes, GpCyc1 has a high copy number, with approximately 5000 copies found in the Gonyaulax genome. An antibody raised against the N-terminal region of the GpCYC1 reacts with a 68kDa protein on Western blots that is more abundant in cell cultures enriched for G2-phase cells than in those containing primarily G1-phase cells, indicating its cellular level follows a pattern expected for a mitotic cyclin. This is the first report of a cell cycle regulator cloned and sequenced from a dinoflagellate, and our results suggest control of the dinoflagellate cell cycle will be very similar to that of other organisms.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Dinoflagellida/metabolismo , Mitose/fisiologia , Análise de Sequência de Proteína , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Células Cultivadas , Clonagem Molecular/métodos , Ciclinas/genética , Ciclinas/isolamento & purificação , Dinoflagellida/genética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
We have cloned and sequenced a DNA fragment (approximately 1 kb) containing a complete open reading frame from a cDNA library of Leishmania donovani promastigotes. The alignment of the derived polypeptide sequence and the modeling studies revealed that the protein is highly homologous to the mammalian cyclins having conserved cyclin box and substrate-docking motif. Northern blot analysis of the RNA isolated from synchronized L. donovani promastigotes showed periodic expression of the message with maximum abundance at S-phase suggesting its involvement in the events related to the regulation of DNA replication. The results confirm that we have isolated a cyclin molecule from L. donovani (LdCyc1) which may play an important role in the regulation of the parasite cell cycle.
Assuntos
Ciclinas/química , Ciclinas/isolamento & purificação , Leishmania donovani/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , Ciclinas/genética , Replicação do DNA/genética , Regulação da Expressão Gênica , Biblioteca Gênica , Leishmania donovani/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA Mensageiro/análise , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins.
Assuntos
Ciclinas/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Ciclinas/genética , Ciclinas/isolamento & purificação , DNA de Protozoário/genética , Eritrócitos/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Protozoários , Humanos , Técnicas In Vitro , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmodium falciparum/citologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
B-type cyclin-dependent kinases (CDKs) are unique to plants and are assumed to be involved in the control of the G2-to-M phase progression and mitotic events. However, little is known about their cyclin partners. In Arabidopsis, we isolated cDNA encoding the D-type cyclin CYCD4;1 by a yeast (Saccharomyces cerevisiae) two-hybrid screening using CDKB2;1 as bait. In vitro pull-down assay showed that CYCD4;1 bound to CDKB2;1 and CDKA;1. Protein complexes of CYCD4;1-CDKA;1 and CYCD4;1-CDKB2;1 in insect cells exhibited histone H1-kinase activity. Promoter analysis using the luciferase reporter gene showed that CDKB2;1 was expressed from early G2 to M phase, whereas CYCD4;1 was expressed throughout the cell cycle. In situ hybridization of plant tissues revealed that both CDKB2;1 and CYCD4;1 transcripts accumulated in the shoot apical meristem, leaf primordia, vasculature of leaves, and tapetal cells in anthers. Our results suggest that CDKB2;1 and CYCD4;1 may form an active kinase complex during G2/M phase and control the development of particular tissues.
Assuntos
Arabidopsis/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/genética , Ciclina D , Ciclinas/genética , Ciclinas/isolamento & purificação , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Brotos de Planta/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Previous studies have shown that the viral Jun (v-Jun) oncoprotein induces marked alterations in cell cycle control, which are associated with, and may be caused by, increased cdk2 kinase activity. Since p21 CIP1 is an important regulator of cdk2, we investigated whether aberrant expression of this cyclin-dependent kinase inhibitor might contribute to cell cycle deregulation by v-Jun. We find that the basal levels of p21 CIP1 mRNA and protein expression are greatly reduced in chick embryo fibroblasts (CEF) transformed by v-Jun, and that v-Jun blocks the increases in p21 CIP1 expression that normally accompany growth inhibition induced by serum deprivation or confluency in untransformed CEF. Importantly, ectopic expression of p21 CIP1 in v-Jun-transformed CEF inhibits both cdk2 kinase activity and cell cycle progression, indicating that these alterations in p21 CIP1 expression are likely to be functionally significant for growth deregulation. We also investigated the mechanism through which v-Jun disturbs p21 CIP1 expression and the possible involvement of a known p21 CIP1 regulator, p53, as an intermediate in this process. This analysis revealed that repression is mediated primarily at the level of p21 CIP1 gene transcription, however the mechanism is complex; both p53-dependent and -independent mechanisms contribute as judged by analysis of p21 CIP1 promoter mutants and other assays of p53 transcriptional activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Fase G1/fisiologia , Proteína Oncogênica p65(gag-jun)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/isolamento & purificação , Regulação para Baixo , Regulação da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Proteína Oncogênica p65(gag-jun)/genética , Fase S/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cyclin-dependent kinase inhibitors (CDKIs) are considered as novel anticancer agents because of their ability to induce growth arrest or apoptosis in tumour cells. It has not yet been fully determined, however, which CDKI is the best candidate for the treatment of malignant gliomas and whether normal brain tissues are affected by CDKI expression. Using recombinant adenoviral vectors that express CDKIs (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)), we compared the antitumour effect of CDKIs on malignant glioma cell lines (A172, GB-1, T98G, U87-MG, U251-MG and U373-MG). p27(KIP1) showed higher ability to suppress the growth of all tumour cells tested than other CDKIs. Interestingly, overexpression of p27(KIP1) induced autophagic cell death, but not apoptosis in tumour cells. On the other hand, p27(KIP1) overexpression did not inhibit the viability of cultured astrocytes (RNB) nor induced autophagy. Overall, our findings suggest that gene transfer of p27(KIP1) may be a promising approach for the therapy of malignant gliomas.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/isolamento & purificação , Inibidor p16 de Quinase Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/isolamento & purificação , Inibidores Enzimáticos/uso terapêutico , Glioma/patologia , Proteínas Supressoras de Tumor/isolamento & purificação , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p19 , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , HumanosRESUMO
Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.
Assuntos
Quinases Ciclina-Dependentes , Ciclinas/genética , Ciclinas/metabolismo , Epsilonretrovirus/genética , Epsilonretrovirus/metabolismo , RNA Polimerase II/metabolismo , Células 3T3 , Animais , Quinase 8 Dependente de Ciclina , Ciclinas/química , Ciclinas/isolamento & purificação , Genes Virais , Células HeLa , Holoenzimas/metabolismo , Humanos , Camundongos , Mutação , Fosforilação , Testes de Precipitina , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , RNA Polimerase II/isolamento & purificação , Splicing de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição , TransfecçãoRESUMO
The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Ciclinas/metabolismo , Ligases/metabolismo , Proteínas Quinases/metabolismo , Complexos Ubiquitina-Proteína Ligase , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Bivalves , Bovinos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Ciclinas/isolamento & purificação , Feminino , Células HeLa , Humanos , Ligases/isolamento & purificação , Mitose , Oócitos/enzimologia , Fosforilação , Proteínas Serina-Treonina Quinases , Subunidades Proteicas , Proteínas Proto-Oncogênicas , Quinase 1 Polo-LikeRESUMO
The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclinas/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Baculoviridae/genética , Linhagem Celular , Clonagem Molecular , Ciclina A/metabolismo , Ciclina H , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/isolamento & purificação , Ciclinas/metabolismo , Substâncias Macromoleculares , Espectrometria de Massas , Fosforilação , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Spodoptera , Treonina , Proteínas de Xenopus , Xenopus laevis , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A novel cyclin, CycJ18, was isolated by complementation of G1 cyclin-deficient budding yeast with an Arabidopsis cDNA library. CycJ18 shares only 20% identity in its conserved cyclin box domain with other cyclins, and is predominantly expressed in young seedlings. CycJ18 is a member of a potential new plant cyclin class.
Assuntos
Arabidopsis/metabolismo , Ciclinas/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Ciclinas/genética , Biblioteca Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Alinhamento de SequênciaRESUMO
Cyclin T1 was recently identified, together with cdk9 (previously named PITALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue.
Assuntos
Ciclinas/isolamento & purificação , Imuno-Histoquímica/métodos , Adulto , Especificidade de Anticorpos , Sistema Cardiovascular/química , Sistema Cardiovascular/citologia , Ciclina T , Ciclinas/imunologia , Células Epiteliais/química , Células Epiteliais/citologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/citologia , Humanos , Queratinócitos/química , Queratinócitos/citologia , Sistema Nervoso/química , Sistema Nervoso/citologia , Distribuição TecidualRESUMO
CCAAT/enhancer-binding protein-alpha (C/EBP alpha) is a basic leucine zipper protein that controls transcription of genes important for liver function, white adipose tissue development, and granulocyte differentiation. In addition to its function in controlling gene expression in differentiated tissues, C/EBP alpha is also associated with an antimitotic activity. We have previously demonstrated that C/EBP alpha interacts with p21, a cyclin-dependent kinase (CDK) inhibitor, and that C/EBP alpha inhibits proliferation when expressed in several different cell types (Timchenko, N. A., Harris, T. E., Wilde, M., Bilyeu, T. A., Burgess-Beusse, B. L., Finegold, M. J., and Darlington, G. J. (1997) Mol. Cell. Biol. 17, 7353--7361). Here we define the regions of C/EBP alpha required for interaction with p21 and demonstrate that CDK2 also interacts with C/EBP alpha. We show that C/EBP alpha can cooperate with p21 to inhibit CDK2 activity in vitro. The effect of C/EBP alpha on CDK2 activity requires the p21 and CDK2 interaction sites within C/EBP alpha. C/EBP alpha mutants incapable of inhibiting CDK2 activity in vitro do not inhibit proliferation in cultured cells. However, C/EBP alpha mutants defective in DNA binding inhibit proliferation as effectively as the wild-type protein. These findings show that C/EBP alpha-mediated growth arrest occurs through protein interactions and is independent of its transcriptional activity.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/fisiologia , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Substituição de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/isolamento & purificação , Divisão Celular , Linhagem Celular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Fosforilação , Mutação Puntual , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Spodoptera , TransfecçãoRESUMO
Transgenic mice expressing specific oncogenes usually develop tumors in a stochastic fashion suggesting that tumor progression is a multi-step process. To gain further understanding of the interactions between oncogenes and tumor suppressor genes during tumorigenesis, we have crossed a transgenic strain (TG.NK) carrying an activated c-neu oncogene driven by the MMTV enhancer/promoter with p53-deficient mice. c-neu transgenic mice have stochastic breast tumor formation and normal appearing salivary glands. However, c-neu mice heterozygous for a p53 deletion develop parotid gland tumors and loose their wild type p53 allele. c-neu mice with a homozygous p53 deletion have increased rates of parotid tumor onset suggesting that inactivation of p53 is required and sufficient for parotid gland transformation in the presence of activated c-neu. In contrast to the dramatic effect of p53 in parotid gland transformation, p53 loss has little effect on the rate or stochastic appearance of mammary tumors. In addition, p53 loss was accompanied by the down regulation of p21 in parotid gland tumors but not breast tumors. The parotid gland tumors were aneuploid and demonstrated increased levels of Cyclin D1 expression. These observations suggest that in c-neu transgenic mice, p53 alterations have differential tissue effects and may be influenced by the tissue specific expression of genes influencing p53 activity.
Assuntos
Genes erbB-2 , Genes p53 , Neoplasias das Glândulas Salivares/genética , Aneuploidia , Animais , Transformação Celular Neoplásica/genética , Ciclina D1/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/isolamento & purificação , Feminino , Regulação Neoplásica da Expressão Gênica , Perda de Heterozigosidade , Masculino , Neoplasias Mamárias Animais/etiologia , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Neoplasias Parotídeas/genética , Neoplasias das Glândulas Salivares/etiologia , Glândulas Salivares/metabolismo , Processos Estocásticos , Distribuição Tecidual , Proteína Supressora de Tumor p53/isolamento & purificaçãoRESUMO
Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.
Assuntos
Ciclinas/isolamento & purificação , Proteínas Quinases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Proteína Quinase CDC2 , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/isolamento & purificação , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Citoplasma/enzimologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Histonas/metabolismo , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Cinetina , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Piperidinas/farmacologia , Testes de Precipitina , Proteínas Quinases/isolamento & purificação , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/metabolismoRESUMO
The functions of some CLC Cl(-) channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC-2 Cl(-) channel is less clear. Several important functions have been attributed to ClC-2, but contrary to these expectations ClC-2-deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC-2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC-2 disruption entails the death of two cell types which depend on supporting cells that form the blood-testes and blood-retina barriers. We propose that ClC-2 is crucial for controlling the ionic environment of these cells.