RESUMO
The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inibidor de Quinase Dependente de Ciclina p21/imunologia , Memória Imunológica , Canal de Potássio Kv1.3/imunologia , Transdução de Sinais/imunologia , Proteína Smad3/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/imunologia , Divisão Celular/genética , Divisão Celular/imunologia , Células Cultivadas , Ciclina B1/genética , Ciclina B1/imunologia , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fase G2/genética , Fase G2/imunologia , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Fosforilação/genética , Fosforilação/imunologia , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
OBJECTIVE: The expression of cytokine-associated genes in dendritic cells (DCs) derived from umbilical cord blood (UCB) and adult peripheral blood (APB) was comprehensively compared in order to elucidate the difference in DC function between newborns and adults. STUDY DESIGN: Immature DCs were obtained from UCB and APB of healthy human donors. Several cytokines were added to generate mature DCs. Gene expression was compared using cDNA microarray containing 553 cytokine-associated genes. Eleven genes with differential expression were selected and determined their expression levels in DCs by quantitative real-time RT-PCR. RESULTS: The expression of the Th1 response-related genes (IL-12B and IL-18) and chemokine genes (CXCL9, CXCL13, CCL18 and CCL24) was significantly lower in UCB-DCs than in APB-DC in both maturation states. On the other hand, calgranulins A and B, which are speculated to induce immune tolerance, showed higher expression in UCB-DCs. The expression of cell cycle-related genes (CDC2 and cyclin B1) was significantly higher in UCB-DCs than in APB-DCs, and immature UCB-DCs proliferated more rapidly than immature APB-DCs. CONCLUSION: The expression of genes related to immune responses was significantly different between UCB- and APB-DCs, which may cause a decreased DC-mediated immunity and an increased susceptibility to infection in newborns.
Assuntos
Células Dendríticas , Sangue Fetal , Adulto , Apoptose/genética , Apoptose/imunologia , Proteína 3 com Repetições IAP de Baculovírus , Biomarcadores/sangue , Biomarcadores/metabolismo , Células Sanguíneas/citologia , Células Sanguíneas/imunologia , Células Sanguíneas/metabolismo , Proteína Quinase CDC2/sangue , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Técnicas de Cultura de Células , Ciclo Celular/genética , Ciclo Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Ciclina B/sangue , Ciclina B/genética , Ciclina B1 , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Sangue Fetal/citologia , Sangue Fetal/imunologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Humanos , Recém-Nascido , Inflamação/genética , Inflamação/imunologia , Proteínas Inibidoras de Apoptose/sangue , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/imunologia , Complexo Antígeno L1 Leucocitário/sangue , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Ubiquitina-Proteína LigasesRESUMO
The present study aimed to evaluate the inhibitory effects of an irinotecan derivative, ZBH1208, on brain tumors, and to explore the underlying molecular mechanisms. To determine the effects of ZBH1208, a brain tumor mouse model was established by transplanting B22 cells. Subsequently, the visceral indices of immune organs and white blood cell counts were determined, and the effects of ZBH1208 on the expression levels of cell cyclerelated proteins were assessed by western blotting. The tumor inhibition rates of 20 and 40 mg/kg ZBH1208 were 11.7 and 54.1%, respectively. Compared with the negative control group, ZBH1208 barely affected visceral indices or white blood cell count. Furthermore, the expression levels of p53, p21, cyclindependent kinase 7 (CDK7), Wee1, phosphorylated (p)cell division cycle 2 (CDC2) (Tyr15), pCDC2 (Thr161) and cyclin B1 proteins were upregulated, whereas the expression levels of cyclin E were downregulated, and those of CDC2, CDK2 and CDC25C were barely altered. In conclusion, the present study demonstrated that ZBH1208 suppressed the growth of B22 mouse brain tumor xenografts, but did not affect their visceral indices or white blood cell counts. It was suggested that ZBH1208 exerted its effects by regulating the expression of p53, p21, Wee1, pCDC2 (Tyr15) and cyclin E proteins.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Regulação Neoplásica da Expressão Gênica , Imunidade Inata/efeitos dos fármacos , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Camptotecina/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Linhagem Celular Tumoral , Ciclina B1/genética , Ciclina B1/imunologia , Ciclina E/genética , Ciclina E/imunologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/imunologia , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/imunologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Vida Livre de Germes , Irinotecano , Masculino , Camundongos , Camundongos Endogâmicos ICR , Transplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Quantitative measurement of enzyme activity is a valuable approach to study how cells function. We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other protein kinase assays.
Assuntos
Especificidade de Anticorpos , Western Blotting/métodos , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Ciclina B/imunologia , Ciclina B/metabolismo , Ensaios Enzimáticos/métodos , Proteína Quinase CDC2/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Genoma , Helicobacter pylori/imunologia , Macrófagos/metabolismo , Animais , Aurora Quinase B/genética , Aurora Quinase B/imunologia , Carga Bacteriana , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Pontos de Checagem do Ciclo Celular/imunologia , Proteínas de Ciclo Celular/imunologia , Linhagem Celular Transformada , Proliferação de Células , Replicação do DNA/genética , Replicação do DNA/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Helicobacter pylori/patogenicidade , Evasão da Resposta Imune , Macrófagos/microbiologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , FosforilaçãoRESUMO
Mice and rabbits were injected with various forms of a 16 amino acid synthetic peptide representing PSTAIR, the evolutionarily conserved region of the protein kinase p34cdc2, for polyclonal antisera and hybridoma-monoclonal antibody production. Antisera from mice injected with an unconjugated monomeric form of the peptide showed no reaction to the peptide. Of four animals injected with the monomeric form of the peptide conjugated to keyhole limpet hemocyanin via m-maleimidobenzoyl-N-hydroxysulfosuccinimide (MBS), antisera from only one mouse had a very low titer to the peptide, and all four animals produced antibody to the MBS bridge. Both mice injected with an octameric multiple antigen peptide (MAP) of PSTAIR produced antisera reactive to the octameric MAP form of the peptide in ELISA and also to the cdc2 protein expressed in bacteria in an immunoblotting assay. Splenocytes from one mouse injected with the octameric MAP form of the peptide were successfully used for hybridoma-monoclonal antibody production. A monoclonal antibody was produced that reacted with octamer, monomer and cdc2-expressed protein and specifically with the carboxyl terminus of the 16 amino acid peptide.
Assuntos
Anticorpos Monoclonais/imunologia , Proteína Quinase CDC2/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Coelhos , Proteínas Recombinantes/imunologiaRESUMO
Since malignant cell proliferation occurs at the expense of cell differentiation, we examined the possibility of differentiating malignant cells from mature cells by altering the regulation of the cell cycle. Using the human promyelocytic leukemia cell line HL-60, which expresses p34cdc2, the product of the cdc2 gene that controls the cell cycle, we showed that guided internalization of an anti-cdc2 monoclonal antibody could initiate differentiation not yet described for other cells of myeloid malignant origin. Experimental cellular manipulation may be employed as a method of inducing in vitro differentiation for transplantation purposes.
Assuntos
Leucemia Promielocítica Aguda/patologia , Anticorpos Monoclonais/farmacologia , Proteína Quinase CDC2/imunologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Células HL-60/enzimologia , Células HL-60/patologia , Humanos , Leucemia Promielocítica Aguda/enzimologiaRESUMO
p34cdc2 protein kinase is a component of M phase-promoting factor (MPF), which plays an important role in controlling the mitotic and meiotic cell cycle. p34cdc2 contains a unique 16 amino acid sequence (PSTAIR) that is conserved from fission yeast to human. Using polyclonal anti-PSTAIR antibody, we detected the p34cdc2 homologue in the central nervous system of adult mice by western blotting. By immunohistochemical technique, we found that the p34cdc2 homologue was located in the nucleoli of neurons and glia in the central and peripheral nervous systems. In the central nervous system, positive cells were widely distributed from the cerebral cortex to the spinal cord. Immunoreactive cells were also detected in retina and pituitary. The evidence that the p34cdc2 is present in neurons which have lost the ability of cell division predicts another function of p34cdc2 family proteins besides the one that has generally recognized.
Assuntos
Proteína Quinase CDC2/metabolismo , Nucléolo Celular/enzimologia , Glândulas Endócrinas/enzimologia , Sistema Nervoso/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Mesotelina , Camundongos , Dados de Sequência Molecular , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestruturaRESUMO
We here describe an alternative way to microinjection by which cellular transport of immunoglobulins through surface membranes can be achieved after binding to specific surface receptors either induced or constutively present, or via Fc receptors (Ig-mediated). In this report, the internalisation of two antibodies in two different cellular systems is analysed: the anti-p21ras monoclonal antibody (MoAb) after surface Ig binding on murine placental cells and anti-cdc2 MoAb that binds directly to its surface receptor expressed on the human promyelocytic leukemia cell line HL-60. In both cases, binding and internalisation is followed by Electron Microscopy (EM) and function is assessed by different assays. The first involves abrogation of class II antigen expression induced by Interferon-gamma (IFN-gamma) and 5-Azacytidine (5-AzaC) known to be mediated by activation of the ras pathway. The second involves growth cessation of HL-60 cells after antibody adsorption when a G1-S-specific culture supernatant containing anti-cdc2 activity is employed, whereas no growth hindrance is observed when a G2-M-specific anti-cdc2 MoAb is used. Thus, the antibodies do not follow the lysosyme pathway and do not lose their functional activity. This method may be applied in the future in order to achieve biological or clinical therapies.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Endocitose , Adsorção , Animais , Azacitidina/farmacologia , Proteína Quinase CDC2/imunologia , Feminino , Células HL-60 , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microinjeções , Proteínas Proto-Oncogênicas p21(ras)/imunologiaRESUMO
A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34cdc2, is encoded by cdc2 gene. Immunoblotting with a polyclonal antibody raised against PSTAIRE sequence (found in the N-terminal region of all cdc2 and cdc2 related proteins throughout the phylogenetic scale including higher plants), was used to study the presence of p34cdc2 in onion scale leaves and root tip cells. p34cdc2 homologues are beyond the detection level in scale leaves. PSTAIRE antibody was used to estimate p34cdc2 kinase protein levels during cell cycle in highly synchronous population of Allium cepa L. root meristem cells. p34cdc2 kinase protein showed gradual increase in their levels from S phase to G2 phase boundary. Immunoprecipitation followed by in vitro histone H1 kinase assays also depicted that its kinase activity increased parallel to the increase in p34cdc2 level.
Assuntos
Proteína Quinase CDC2/metabolismo , Cebolas/citologia , Cebolas/metabolismo , Sequência de Aminoácidos , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/imunologia , Ciclo Celular , Epitopos/genética , Células HeLa , Humanos , Meristema/citologia , Meristema/metabolismo , Dados de Sequência MolecularAssuntos
Apoptose/fisiologia , Serina Endopeptidases/fisiologia , Animais , Apoptose/imunologia , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/fisiologia , Ciclo Celular/imunologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/imunologia , Quinases Ciclina-Dependentes/fisiologia , Citotoxicidade Imunológica , Humanos , Células Matadoras Naturais/imunologia , Necrose , Serina Endopeptidases/imunologia , Linfócitos T Citotóxicos/imunologiaAssuntos
Anticorpos Monoclonais/imunologia , Proteína Quinase CDC2/imunologia , Proteínas de Ciclo Celular/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reações Cruzadas , Células HEK293 , Humanos , CamundongosRESUMO
CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7(+) cells. This could be demonstrated for human and murine T cells and was valid for both CD4(+) and CD8(+) T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7(-/-) T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular/imunologia , Proliferação de Células , Quimiocina CCL19/imunologia , Quimiocina CCL21/imunologia , Receptores CCR7/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL19/metabolismo , Quimiocina CCL19/farmacologia , Quimiocina CCL21/metabolismo , Quimiocina CCL21/farmacologia , Inibidor de Quinase Dependente de Ciclina p27/imunologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta Imunológica , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Interleucina-2/imunologia , Interleucina-2/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transplante de Órgãos , Receptores CCR7/genética , Receptores CCR7/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Transplante HomólogoRESUMO
The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/deficiência , Ureia/análogos & derivados , Anticorpos/farmacologia , Western Blotting , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Camptotecina/farmacologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quinase 1 do Ponto de Checagem , Dano ao DNA , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Células HeLa , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Ureia/química , Ureia/farmacologia , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
At the onset of mitosis, eukaryotic cells display an abrupt increase in a Ca2(+)- and cyclic nucleotide-independent histone H1 kinase activity, referred to as growth-associated or M phase-specific H1 kinase. The molecular basis for this activity is generally attributed to a kinase complex that consists of the p34cdc2 protein and cyclin, and exhibits maturation-promoting factor (MPF) activity. In the present study, we show that more than one kinase contributes to M phase-specific H1 kinase activity. When mature Xenopus oocyte extract prepared with ATP gamma S and NaF was fractionated by gel filtration, two prominent peaks of H1 kinase activity were detected, with apparent molecular masses of 600 and 150 kDa. The 150-kDa kinase copurified with the p34cdc2 protein and was immobilized by the suc 1 gene product p13 and anti-cyclin B2, which are specific for the cdc2 kinase complex. However, the 600-kDa kinase did not satisfy any of these criteria, thus identifying it as a novel M phase-specific H1 kinase. Only the 600-kDa kinase was recognized by the mitosis-specific monoclonal antibody, MPM-2, which inhibits Xenopus oocyte maturation and immunodepletes MPF activity. Furthermore, not only did the full activation of this kinase (MPM-2 kinase) coincide with the activation of MPF during the cell cycle, but also MPM-2 kinase-positive fractions obtained by gel filtration accelerated progesterone-induced oocyte maturation. It is, therefore, likely that MPM-2 kinase is a positive regulator in the M phase induction pathway.
Assuntos
Anticorpos Monoclonais/imunologia , Fator Promotor de Maturação/imunologia , Mitose , Animais , Especificidade de Anticorpos , Proteína Quinase CDC2/imunologia , Fator Promotor de Maturação/classificação , Fator Promotor de Maturação/metabolismo , Peso Molecular , Oócitos/metabolismo , Progesterona/farmacologia , Xenopus laevisRESUMO
Recent advances in the understanding of the molecular basis of cell cycle control, particularly that of mitotic control, are reviewed with respect to what is known about cell cycle control in early mouse embryos. The behaviour of the murine homologue of cdc2+, a component of Maturation Promotion Factor (MPF), is described at these early stages.
Assuntos
Blastocisto/fisiologia , Ciclo Celular/fisiologia , Fator Promotor de Maturação/metabolismo , Animais , Anticorpos Monoclonais , Proteína Quinase CDC2/imunologia , Proteína Quinase CDC2/metabolismo , Mesotelina , Camundongos , Peso Molecular , XenopusRESUMO
In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions. The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells. Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro. Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis. Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives. In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin. The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated. It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable. Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition. Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34. Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date. A species of protein with a mobility slightly less than true p34 was found, even in resting T cells. Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen. Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Proteína Quinase CDC2/metabolismo , Proteína do Retinoblastoma/metabolismo , Linfócitos T/fisiologia , Proteína Quinase CDC2/imunologia , Divisão Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Ativação Linfocitária , Peso Molecular , Fosforilação , Linfócitos T/citologia , Fatores de TempoRESUMO
Changes in protein kinase activities are thought to contribute to the alteration of gene expression after heat shock and related stresses. In an attempt to identify enzymes which might be involved in both chromatin structure modification and transcriptional switch in heat-shocked cells, we have studied protein kinase activities in heat-shocked cell lysates with two exogenous substrates: a tetramer of a heptapeptide (heptapeptide 4) corresponding to the RNA polymerase II C-terminal domain (CTD), and the histone H1. Heat-shock and arsenite stress were found to stimulate strongly CTD kinase activity. H1 kinase activity was also stimulated but more weakly. Stimulation of CTD and H1 kinases occurs mainly at the early phase of recovery and by a process which is independent of protein synthesis. The stress-induced H1 kinase is shown to contain a molecule related to the mitotic-promoting factor (MPF) Cdc2 component. On the other hand, though Cdc2-related protein has also been reported to be part of a CTD kinase complex, we show that the stress-induced CTD kinase activity corresponds to a distinct entity. It is proposed that stress activation of CTD kinase might be involved in changing the specificity of RNA polymerase II.
Assuntos
Temperatura Alta , Proteínas Quinases/metabolismo , RNA Polimerase II/metabolismo , Estresse Fisiológico/enzimologia , Sequência de Aminoácidos , Proteína Quinase CDC2/imunologia , Cicloeximida/farmacologia , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Células HeLa/enzimologia , Humanos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Protamina Quinase/metabolismo , Proteínas Quinases/imunologia , RNA Polimerase II/imunologiaRESUMO
We investigated plant cell division by testing for the presence and involvement in progress through the division cycle of the protein p34cdc2, a key participant in division control in other eukaryotes. A protein of the same m(r) 34,000 has structural similarity indicated by its reaction with three sorts of antibody raised against (1) cell division-specific regions within a 16-amino acid internal sequence that is perfectly conserved in p34cdc2 from all known sources, (2) the carboxy-terminal 127 amino acids of human p34cdc2 linked to beta-galactosidase, and (3) whole p34cdc2 of fission yeast. Participation of p34 in the division cycle of the green plant Chlamydomonas is indicated by phosphorylation of the protein only in proliferating cells. There is a consistent fivefold increase relative to other proteins when cells become committed to division and a maximum of phosphorylation at the time of nuclear division under conditions that alter by twofold the time of these events. A p34 protein is detectable in oats and Arabidopsis and in remote taxa, including red and brown algae. We conclude that the plant kingdom shares a division control involving p34cdc2 that was probably established in the common ancestral eukaryote prior to divergence of any of the major eukaryote taxa.
Assuntos
Proteína Quinase CDC2/química , Ciclo Celular/fisiologia , Chlamydomonas reinhardtii/química , Plantas/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Proteína Quinase CDC2/imunologia , Divisão Celular , Chlamydomonas reinhardtii/citologia , Imuno-Histoquímica , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosforilação , Homologia de Sequência do Ácido NucleicoRESUMO
Using probes obtained by PCR amplification, we have cloned Drosophila cDNAs encoding structural homologs of the p34cdc2 cell cycle kinase. Southern blot experiments and in situ hybridization to polytene chromosomes demonstrated that the isolated cDNAs, were derived from two distinct genes, Dm cdc2 (31E) and Dm cdc2c (92F). Northern blot and in situ hybridization experiments revealed that these two genes are coexpressed during embryogenesis and that expression is correlated with cell proliferation. However, despite the similarity in structure and expression, the two gene products differed in functional assays in yeasts. Expression of Dm cdc2 in Schizosaccharomyces pombe and Saccharomyces cerevisiae rescued cell cycle arrest caused by mutations in cdc2+ and CDC28, the genes encoding the p34cdc2 kinase homologs of these yeasts. In contrast, the Dm cdc2c gene product did not restore cell cycle progression. Thus, in addition to the identification of a functional homolog in Drosophila, our results indicate the presence of a closely related cognate of the p34cdc2 cell cycle kinase.