RESUMO
Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.
Assuntos
Proteína Quinase CDC2/farmacologia , Ciclinas/farmacologia , Endocitose/efeitos dos fármacos , Organelas/metabolismo , Linhagem Celular , Citosol/metabolismo , Células HeLa , Humanos , Organelas/efeitos dos fármacosRESUMO
The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.
Assuntos
Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes , Ciclinas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Quinase 2 Dependente de Ciclina , Ciclinas/química , Ciclinas/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Mutação , Oócitos/química , Mutação Puntual , Testes de Precipitina , Protamina Quinase/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , Proteínas de Xenopus , Xenopus laevis/metabolismoRESUMO
E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.
Assuntos
Ciclinas/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fase G2 , Humanos , Proteínas E7 de Papillomavirus , Testes de Precipitina , Protamina Quinase/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase SRESUMO
In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes , Ciclinas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Animais , Proteína Quinase CDC2/metabolismo , Células Cultivadas , Chlorocebus aethiops , Quinase 2 Dependente de Ciclina , Técnicas In Vitro , Testes de Precipitina , Protamina Quinase/metabolismo , Ligação ProteicaRESUMO
The retinoblastoma gene (Rb) product is a negative regulator of cellular proliferation, an effect that could be mediated in part at the transcriptional level through its ability to complex with the sequence-specific transcription factor DRTF1. This interaction is modulated by adenovirus E1a, which sequesters the Rb protein and several other cellular proteins, including cyclin A, a molecule that undergoes cyclical accumulation and destruction during each cell cycle and which is required for cell cycle progression. Cyclin A, which also complexes with DRTF1, facilitates the efficient assembly of the Rb protein into the complex. This suggests a role for cyclin A in regulating transcription and defines a transcription factor through which molecules that regulate the cell cycle in a negative fashion, such as Rb, and in a positive fashion, such as cyclin A, interact. Mutant loss-of-function Rb alleles, which occur in a variety of tumour cells, also fail to complex with E1a and large T antigen. Here we report on a naturally occurring loss-of-function Rb allele encoding a protein that fails to complex with DRTF1. This might explain how mutation in the Rb gene prevents negative growth control.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Proteínas de Ligação a DNA , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Animais , Bovinos , Linhagem Celular , Ciclinas/isolamento & purificação , Fatores de Transcrição E2F , Genes do Retinoblastoma , Imunoensaio , Substâncias Macromoleculares , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína do Retinoblastoma/isolamento & purificação , Proteína 1 de Ligação ao Retinoblastoma , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.
Assuntos
Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Quinase 2 Dependente de Ciclina , Ciclinas/fisiologia , Ativação Enzimática , Escherichia coli , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fosforilação , Testes de Precipitina , Protamina Quinase/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Xenopus , Proteínas de Xenopus , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes , Ciclinas/fisiologia , Proteínas de Ligação a DNA , Proteínas Quinases , Proteínas Serina-Treonina Quinases , Fatores de Transcrição/metabolismo , Sequência de Bases , Ciclo Celular , Quinase 2 Dependente de Ciclina , Fatores de Transcrição E2F , Dados de Sequência Molecular , Proteína do Retinoblastoma , Proteína 1 de Ligação ao Retinoblastoma , Transcrição GênicaRESUMO
MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.
Assuntos
Quinases Ciclina-Dependentes , Ciclinas/química , Proteínas de Neoplasias/química , Proteínas Serina-Treonina Quinases/química , Fatores de Transcrição TFII , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Ciclo Celular , Linhagem Celular , Ciclina H , Ciclinas/genética , Ciclinas/efeitos da radiação , Dano ao DNA , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/efeitos da radiação , Reparo do DNA , Substâncias Macromoleculares , Dados de Sequência Molecular , Estrutura Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/efeitos da radiação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/efeitos da radiação , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/efeitos da radiação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/efeitos da radiação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/efeitos da radiação , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição/efeitos da radiação , Raios Ultravioleta , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
A protein kinase activity that phosphorylates the C-terminal domain (CTD) of RNA polymerase II and is associated with the basal transcription-repair factor TFIIH (also called BTF2) resides with MO15, a cyclin-dependent protein kinase that was first found to be involved in cell cycle regulation. Using in vivo and in vitro repair assays, we show that MO15 is important for nucleotide excision repair, most likely through its association with TFIIH, thus providing an unexpected link among three important cellular mechanisms.