RESUMO
Recently, progress in the study of lamins has been made in three areas: signals required for targetting newly synthesized lamins to the correct subnuclear compartment have been identified; information on lamina assembly has been obtained from in vitro studies using bacterially expressed proteins; and a mechanistic explanation for how the nuclear lamina is diassembled at the onset of mitosis is emerging.
Assuntos
Matriz Nuclear/fisiologia , Proteínas Nucleares/fisiologia , Animais , Ciclo Celular/fisiologia , Cromatina/fisiologia , Laminas , Mitose/fisiologia , FosforilaçãoRESUMO
Cyclin-dependent kinase (CDK) 7 was originally implicated in cell cycle control by virtue of its ability to phosphorylate and activate other CDKs. Subsequently, both CDK7 and its partner, cyclin H, were found to be associated with the general transcription factor TFIIH, suggesting additional roles for CDK7 in transcription and DNA repair. During the past year, a third subunit associated with CDK7 and cyclin H has been characterized, and the functional link between CDK7 and RNA polymerase II has been strengthened.
Assuntos
Ciclo Celular/genética , Quinases Ciclina-Dependentes , Reparo do DNA/fisiologia , DNA Fúngico/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transcrição Gênica/fisiologia , Animais , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Our current understanding of the eukaryotic cell cycle attributes a key regulatory role to cyclin-dependent protein kinases. It is important, therefore, to identify the physiological substrates of these kinases, and to understand how the phosphorylation of such proteins promotes cell cycle progression.
Assuntos
Ciclo Celular , Ciclinas , Proteínas Quinases , Animais , Replicação do DNA , Humanos , Complexos MultienzimáticosRESUMO
Present in organisms ranging from yeast to man, homologues of the Drosophila Polo kinase control multiple stages of cell division. At the onset of mitosis, Polo-like kinases (Plks) function in centrosome maturation and bipolar spindle formation, and they contribute to the activation of cyclin-dependent kinase (Cdk)1-cyclin B. Subsequently, they are required for the inactivation of Cdk1 and exit from mitosis. In the absence of Plk function, mitotic cyclins fail to be destroyed, indicating that Plks are important regulators of the anaphase-promoting complex/cyclosome (APC/C), a key component of the ubiquitin-dependent proteolytic degradation pathway. Finally, recent evidence implicates Plks in the temporal and spatial coordination of cytokinesis.
Assuntos
Proteínas de Drosophila , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Humanos , Fuso Acromático/metabolismoRESUMO
Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.
Assuntos
Dano ao DNA , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Cinetina , Mitose , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Purinas/farmacologia , Células Tumorais Cultivadas , Fosfatases cdc25/metabolismo , Quinase 1 Polo-LikeRESUMO
Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. Here, we have established a centrosome duplication assay using mammalian somatic cells. We show that centrosome duplication requires the activation of E2F transcription factors and Cdk2-cyclin A activity.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Ciclo Celular/fisiologia , Centrossomo/fisiologia , Centrossomo/ultraestrutura , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células CHO , Proteínas de Ciclo Celular/metabolismo , Cricetinae , Quinase 2 Dependente de Ciclina , Fatores de Transcrição E2F , Mamíferos , Proteínas Recombinantes/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , TransfecçãoRESUMO
Cyclin-dependent kinase (cdks) are key components of the engine that drives the cell proliferation cycle in all eukaryotes. These kinases are related to p34(cdc2) and associate with regulatory subunits belonging to the cyclin family. To understand how cdks promote cell cycle progression, it will be important to identify their physiological substrates and to determine how phosphorylation influences the functions of these substrates. This article discusses recent progress as well as some of the problems related to the quest for cdk substrates.
RESUMO
Named after the polo gene of Drosophila, POLO-like kinases (PLKs) constitute a novel, evolutionarily conserved family of essential cell-cycle regulators. As emphasized in this review, recent studies identify important roles for vertebrate PLKs at the onset of mitosis: Plx1, a Xenopus PLK, has been implicated in the activation of Cdc25 phosphatase (and hence the activation of Cdc2), while human Plk1 is required for the proper maturation of the poles of mitotic spindles. These studies suggest a major role for Plk1/Plx1 in coordinating spindle assembly with the activation of Cdc2-cyclin complexes, and they establish a direct link between PLKs and the core cell-cycle-regulatory machinery. Genetic and biochemical studies in yeasts and Drosophila point to additional roles for PLKs at later stages of mitosis. Finally, mammals express multiple PLKs, suggesting that different family members might function at distinct cell-cycle transitions, reminiscent of cyclin-dependent kinases.
RESUMO
Cyclin proteins form complexes with members of the p34cdc2 kinase family and they are essential components of the cell cycle regulatory machinery. They are thought to determine the timing of activation, the subcellular distribution, and/or the substrate specificity of cdc2-related kinases, but their precise mode of action remains to be elucidated. Here we report the cloning and sequencing of avian cyclin B2. Based on the use of monospecific antibodies raised against bacterially expressed protein, we also describe the subcellular distribution of cyclin B2 in chick embryo fibroblasts and in DU249 hepatoma cells. By indirect immunofluorescence microscopy we show that cyclin B2 is cytoplasmic during interphase of the cell cycle, but undergoes an abrupt translocation to the cell nucleus at the onset of mitotic prophase. Finally, we have examined the phenotypic consequences of expressing wild-type and mutated versions of avian cyclin B2 in HeLa cells. We found that expression of cyclin B2 carrying a mutation at arginine 32 (to serine) caused HeLa cells to arrest in a pseudomitotic state. Many of the arrested cells displayed multiple mitotic spindles, suggesting that the centrosome cycle had continued in spite of the cell cycle arrest.
Assuntos
Ciclo Celular , Núcleo Celular/metabolismo , Ciclinas/metabolismo , Mitose , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Embrião de Galinha , Galinhas , Ciclinas/genética , Fibroblastos/metabolismo , Biblioteca Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Transfecção , XenopusRESUMO
Casein kinase II (CK II) has been implicated in regulating multiple processes related to cell growth, proliferation, and differentiation. To better understand the function(s) and regulation of this ubiquitous kinase, it is important to know its subcellular distribution. However, this issue has been the subject of contradictory reports. In this study, we have used indirect immunofluorescence microscopy and cell fractionation to study the subcellular distribution of all three subunits of chicken CK II, alpha, alpha', and beta. We examined primary chick embryo fibroblasts, virally transformed chicken hepatoma cells, as well as HeLa cells transiently transfected with cDNAs encoding chicken CK II subunits. We found that each of the three CK II subunits was located predominantly in the cell nucleus, irrespective of the cell type analyzed or the procedure used for cell fixation. No major differences were detected in the subcellular distributions of individual CK II subunits, and no evidence was obtained for subunit redistributions during interphase of the cell cycle. During mitosis, the bulk of the enzyme was dispersed throughout the cell, though a fraction of all three subunits was associated with the mitotic spindle. Biochemical studies based on mechanical enucleation of chicken cells confirmed the predominantly nuclear location of all three CK II subunits. Finally, immunoblotting experiments were carried out to study the expression of CK II subunits. A survey of different adult chicken tissues revealed substantial tissue-specific differences in the levels of CK II protein, but no evidence was obtained for pronounced tissue specificity in the expression of individual CK II subunits. These results strongly suggest that CK II functions primarily in regulating nuclear activities, and that the two catalytic subunits, alpha and alpha', may carry out overlapping functions.
Assuntos
Núcleo Celular/enzimologia , Proteínas Serina-Treonina Quinases/análise , Animais , Anticorpos , Caseína Quinase II , Núcleo Celular/ultraestrutura , Células Cultivadas , Embrião de Galinha , Galinhas , Feminino , Fibroblastos/enzimologia , Imunofluorescência , Células HeLa , Humanos , Imuno-Histoquímica , Substâncias Macromoleculares , Masculino , Especificidade de Órgãos , Plasmídeos , Proteínas Serina-Treonina Quinases/genética , TransfecçãoRESUMO
Recent evidence suggests that the conserved COOH-terminal CaaX motif of nuclear lamins may play a role in targeting newly synthesized proteins to the nuclear envelope. We have shown previously that in rabbit reticulocyte lysates the cysteine residue of the CaaX motif of chicken lamin B2 is necessary for incorporation of a derivative of mevalonic acid, the precursor of isoprenoids. Here we have analyzed the properties of normal and mutated forms of chicken lamin B2 stably expressed in mouse L cells. Mutation of the cysteine residue of the CaaX motif to alanine or introduction of a stop codon immediately after the cysteine residue was found to abolish both isoprenylation and carboxyl methylation of transfected lamin B2. Concomitantly, although nuclear import of the mutant lamin B2 proteins was preserved, their association with the inner nuclear membrane was severely impaired. From these results we conclude that the COOH-terminal CaaX motif is required for isoprenylation and carboxyl methylation of lamins in vivo, and that these modifications are important for association of B-type lamins with the nucleoplasmic surface of the inner nuclear membrane.
Assuntos
Lamina Tipo B , Ácido Mevalônico/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Compartimento Celular , Galinhas , Cisteína , Análise Mutacional de DNA , Imunofluorescência , Técnicas In Vitro , Laminas , Metilação , Dados de Sequência Molecular , Proteínas Nucleares/química , Fosfatos de Poli-Isoprenil/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.
Assuntos
Anticorpos/farmacologia , Quinases relacionadas a CDC2 e CDC28 , Centrossomo/enzimologia , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/enzimologia , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , DNA/biossíntese , Epitopos/fisiologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Células HeLa/citologia , Células HeLa/enzimologia , Humanos , Masculino , Microinjeções , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mitose/fisiologia , Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Fuso Acromático/enzimologia , Tubulina (Proteína)/metabolismo , Quinase 1 Polo-LikeRESUMO
The intracellular distribution of p39, a 39,000-dalton substrate for a number of tyrosine protein kinases, has been determined by indirect immunofluorescence microscopy. No binding of anti-p39 antibodies to intact cells was observed, indicating that this protein is not accessible to antibody on the cell surface. Following detergent permeabilization of formaldehyde-fixed cells, a reasonably uniform cytoplasmic labeling was observed. This fluorescence was most pronounced in membrane ruffles, especially in the leading lamellae of migrating cells, and in areas of cell-cell contact. Brief permeabilization of cells with detergent prior to formaldehyde fixation resulted in the appearance of a reticular lattice. An identical staining pattern was observed when fluorescently-labeled lectins were used as plasma membrane markers, but not when antibodies to a variety of cytoskeletal proteins were used. Taken together, these results indicate that p39 is, at least in part, located at the cytoplasmic surface of the plasma membrane. Immunolabeling of Rous sarcoma virus-transformed cells with anti-p39 antibodies resulted in fluorescent staining patterns indistinguishable from those observed in untransformed cells. It is conceivable that p39 plays some structural role within a protein network underlying the plasma membrane.
Assuntos
Membrana Celular/análise , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismo , Animais , Especificidade de Anticorpos , Vírus do Sarcoma Aviário , Transformação Celular Viral , Imunofluorescência , Proteínas de Membrana/imunologia , Peso Molecular , Proteínas Tirosina Quinases , RatosRESUMO
In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.
Assuntos
Embrião de Galinha/análise , Retículo Endoplasmático/análise , Mitose , Membrana Nuclear/análise , Proteínas Nucleares/análise , Animais , Fracionamento Celular , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fibroblastos/análise , Secções Congeladas , Imunoensaio , Imuno-Histoquímica , Lamina Tipo A , Lamina Tipo B , Laminas , Microscopia EletrônicaRESUMO
The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.
Assuntos
Núcleo Celular/metabolismo , Lamina Tipo B , Proteínas Nucleares/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Células HeLa , Humanos , Interfase , Laminas , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Mapeamento de Peptídeos , Fosfopeptídeos/isolamento & purificação , Fosforilação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The nuclear lamina is an intermediate filament network that underlies the nuclear membrane in higher eukaryotic cells. During mitosis in higher eukaryotes, nuclear lamins are phosphorylated by a mitosis-specific kinase and this induces disassembly of the lamina structure. Recently, p34cdc2 protein kinase purified from starfish has been shown to induce phosphorylation of lamin proteins and disassembly of the nuclear lamina when incubated with isolated chick nuclei suggesting that p34cdc2 is likely to be the mitotic lamin kinase (Peter, M., J. Nakagawa, M. Dorée, J.C. Labbe, and E.A. Nigg. 1990b. Cell. 45:145-153). To confirm and extend these studies using genetic techniques, we have investigated the role of p34cdc2 in lamin phosphorylation in the fission yeast. As fission yeast lamins have not been identified, we have introduced a cDNA encoding the chicken lamin B2 protein into fission yeast. We report here that the chicken lamin B2 protein expressed in fission yeast is assembled into a structure that associates with the nucleus during interphase and becomes dispersed throughout the cytoplasm when cells enter mitosis. Mitotic reorganization correlates with phosphorylation of the chicken lamin B2 protein by a mitosis-specific yeast lamin kinase with similarities to the mitotic lamin kinase of higher eukaryotes. We show that a lamin kinase activity can be detected in cell-free yeast extracts and in p34cdc2 immunoprecipitates prepared from yeast cells arrested in mitosis. The fission yeast lamin kinase activity is temperature sensitive in extracts and immunoprecipitates prepared from strains bearing temperature-sensitive mutations in the cdc2 gene. These results in conjunction with the previously reported biochemical studies strongly suggest that disassembly of the nuclear lamina at mitosis in higher eukaryotic cells is a consequence of direct phosphorylation of nuclear lamins by p34cdc2.
Assuntos
Proteína Quinase CDC2/metabolismo , Lamina Tipo B , Mitose , Proteínas Nucleares/metabolismo , Schizosaccharomyces/enzimologia , Animais , Proteína Quinase CDC2/genética , Galinhas , Imunofluorescência , Expressão Gênica , Laminas , Mutação , Fosforilação , Testes de Precipitina , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Temperatura , TransfecçãoRESUMO
Using a mAb (R-7), we have characterized a 54-kD protein of the chicken nuclear envelope. Based on its biochemical properties and subnuclear distribution p54 is likely to be an integral membrane component specific to the inner nuclear membrane. Fractionation experiments indicate that p54 interacts, directly or indirectly, with the nuclear lamina, and analysis of p54 in cultured cells suggests that this interaction is controlled by cell cycle-dependent posttranslational modification, most likely phosphorylation. Modification of p54 results in a slightly reduced electrophoretic mobility, and it converts the protein from a detergent-resistant to a detergent-extractable form. Detergent solubilization of p54 can be induced in vivo by treating isolated nuclei or nuclear envelopes with highly purified cdc2 kinase, one of the most prominent kinases active in mitotic cells. These results suggest that mitotic phosphorylation of p54 might contribute to control nuclear envelope dynamics during mitosis in vivo.
Assuntos
Ciclo Celular , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Animais , Anticorpos Monoclonais , Proteína Quinase CDC2/metabolismo , Fracionamento Celular , Embrião de Galinha , Detergentes , Imunofluorescência , Immunoblotting , Laminas , Proteínas de Membrana/ultraestrutura , Microscopia Imunoeletrônica , Mitose , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/ultraestrutura , Fosforilação , Receptores de Superfície Celular/metabolismo , SolubilidadeRESUMO
The activity of cyclin-dependent kinases (cdks) depends on the phosphorylation of a residue corresponding to threonine 161 in human p34cdc2. One enzyme responsible for phosphorylating this critical residue has recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40MO15 as its catalytic subunit. In view of the cardinal role of cdks in cell cycle control, it is important to learn if and how CAK activity is regulated during the somatic cell cycle. Here, we report a molecular characterization of a human p40MO15 homologue and its associated CAK activity. We have cloned and sequenced a cDNA coding for human p40MO15, and raised specific polyclonal and monoclonal antibodies against the corresponding protein expressed in Escherichia coli. These tools were then used to demonstrate that p40MO15 protein expression and CAK activity are constant throughout the somatic cell cycle. Gel filtration suggests that active CAK is a multiprotein complex, and immunoprecipitation experiments identify two polypeptides of 34 and 32 kD as likely complex partners of p40MO15. The association of the three proteins is near stoichiometric and invariant throughout the cell cycle. Immunocytochemistry and biochemical enucleation experiments both demonstrate that p40MO15 is nuclear at all stages of the cell cycle (except for mitosis, when the protein redistributes throughout the cell), although the p34cdc2/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absence of obvious changes in CAK activity in exponentially growing cells constitutes a surprise. It suggests that the phosphorylation state of threonine 161 in p34cdc2 (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin substrates, and by phosphatase(s).
Assuntos
Ciclo Celular , Núcleo Celular/enzimologia , Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteína Quinase CDC2/metabolismo , Cromatografia em Gel , Clonagem Molecular , Ciclinas/metabolismo , DNA Complementar/química , DNA Complementar/genética , Fase G1 , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
By immunocytochemistry, quantitative immunoblotting, and two-dimensional gel electrophoresis, we have analyzed the distribution of nuclear lamin proteins during chicken embryonic development. Whereas no qualitative differences in the patterns of expression of lamins A, B1, and B2 were observed during gametogenesis in either the female or the male germ line, profound changes in the composition of the nuclear lamina occurred during the development of somatic tissues. Most unexpectedly, early chicken embryos were found to contain little if any lamin A, although they contained substantial amounts of lamins B1 and B2. During embryonic development, lamin A became increasingly prominent, whereas the amounts of lamin B1 decreased in many tissues. Interestingly, the extent and the developmental timing of these changes displayed pronounced tissue-specific variations. Lamin B2 was expressed in fairly constant amounts in all cell types investigated (except for pachytene-stage germ cells). These results have implications for the purported functional specializations of individual lamin proteins. In addition, they suggest that alterations in the composition of the nuclear lamina may be important for the establishment of cell- or tissue-specific differences in nuclear architecture.
Assuntos
Embrião de Galinha/metabolismo , Lamina Tipo B , Nucleoproteínas/biossíntese , Animais , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Feminino , Imunofluorescência , Regulação da Expressão Gênica , Imunoeletroforese , Lamina Tipo A , Laminas , Masculino , Meiose , Nucleoproteínas/genética , Oócitos/metabolismo , Espermatócitos/metabolismo , Xenopus laevis/metabolismoRESUMO
Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.