RESUMO
We have recently reported that protein kinase CK2 phosphorylates both in vivo and in vitro residue serine-46 of the cell cycle regulating protein Cdc28 of budding yeast Saccharomyces cerevisiae, confirming a previous observation that the same site is phosphorylated in Cdc2/Cdk1, the human homolog of Cdc28. In addition, S. cerevisiae in which serine-46 of Cdc28 has been mutated to alanine show a decrease of 33% in both cell volume and protein content, providing the genetic evidence that CK2 is involved in the regulation of budding yeast cell division cycle, and suggesting that this regulation may be brought about in G1 phase of the mammalian cell cycle. Here, we extended this observation reporting that the mutation of serine-46 of Cdc28 to glutamic acid doubles, at least in vitro, the H1-kinase activity of the Cdc28/cyclin A complex. Since this mutation has only little effects on the cell size of the cells, we hypothesize multiple roles of yeast CK2 in regulating the G1 transition in budding yeast.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/química , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alanina/química , Sequência de Aminoácidos , Sítios de Ligação , Caseína Quinase II , Domínio Catalítico , Ciclo Celular , Ciclina A/metabolismo , Fase G1 , Genótipo , Histonas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina/químicaRESUMO
Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.
Assuntos
Transplante de Células , Feto/fisiologia , Sobrevivência de Enxerto/fisiologia , Mesoderma/citologia , Células-Tronco/citologia , Transplante Heterólogo/fisiologia , Adipócitos/citologia , Adulto , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Condrócitos/citologia , Feminino , Feto/citologia , Idade Gestacional , Humanos , Músculo Esquelético/citologia , Miocárdio/citologia , Reação em Cadeia da Polimerase , Gravidez , OvinosRESUMO
The nuclear receptor and transcription factor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), regulates the activity of other transcription factors in the adipogenic differentiation and inflammatory response pathways. We examined the possible function of the PPAR-gamma pathway in osteoclast (Ocl) formation from CD34(+) hematopoietic stem cells (CD34(+) HSCs), using a co-culture system comprised of human mesenchymal stem cells (hMSCs) and CD34(+) HSCs, both derived from bone marrow. Ocl formation in this co-culture system is enhanced by the addition of exogenous osteoprotegerin ligand (OPGL), an essential Ocl differentiation factor, and macrophage-colony stimulating factor (M-CSF). The data indicate that soluble OPGL (sOPGL) and M-CSF stimulate Ocl formation in the co-cultures up to 4-fold compared with CD34(+) HSCs alone treated with sOPGL and M-CSF. CD34(+) HSCs, but not hMSCs, express PPAR-gamma, and 15-deoxy-Delta(12, 14)-prostaglandin-J2 (15d-PG-J2), a PPAR-gamma agonist, completely blocked the effects of sOPGL and M-CSF on Ocl formation and activity. The inhibitory effect of 15d-PG-J2 is specific to the Ocl lineage in both human and mouse models of osteoclastogenesis. Accordingly, parallel experiments demonstrate that sOPGL activates the NF-kappaB pathway within mouse Ocl progenitors, and this effect was abolished by 15d-PG-J2. These data establish a link between PPAR-gamma and OPGL signaling within Ocl progenitors, and support a role for PPAR-gamma pathway in the modulation of osteoclastogenesis.
Assuntos
Diferenciação Celular , Osteoclastos/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Animais , Antígenos CD34/imunologia , Sequência de Bases , Proteínas de Transporte/metabolismo , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Osteoclastos/citologia , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de SinaisRESUMO
Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
Assuntos
Adipócitos/citologia , Linhagem da Célula , Condrócitos/citologia , Mesoderma/citologia , Osteócitos/citologia , Células-Tronco/citologia , Adulto , Apoptose , Células da Medula Óssea/citologia , Diferenciação Celular , Divisão Celular , Separação Celular , Células Cultivadas , Fibroblastos/citologia , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , FenótipoRESUMO
Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits. Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII. We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro. L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE). However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times. Additionally, L41 protein enhances the autophosphorylation of CKIIalpha. The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates. The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Isoenzimas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias , Sequência de Bases , Sítios de Ligação , Caseína Quinase II , Proteínas de Ligação a DNA , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Proteínas Ribossômicas/genética , Saccharomyces cerevisiaeRESUMO
Butyrate, a dietary fiber derivative, is a well-known differentiating agent in cultured cell lines. In addition, its antineoplastic activity toward colon-rectum cancers has been documented both in vivo and in vitro. Despite the large amount of information on the potential clinical efficacy of butyrate, its mechanism of action at the molecular level has only been partially investigated. Here, we show that serine/threonine protein kinase CKII is a target of butyrate activity. In the human adenocarcinoma cell line, HT29, treated with 2 mM sodium butyrate, CKII activity decreases 50% at 24 and 48 hours after drug addition. The enzyme down-regulation is not due to changes in protein amount since the levels of the different CKII subunits remain constant during butyrate treatment. The data reported provide the first evidence that CKII down-regulation is involved in the signal transduction pathway started by butyrate.
Assuntos
Butiratos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Adenocarcinoma/enzimologia , Sequência de Aminoácidos , Ácido Butírico , Caseína Quinase II , Diferenciação Celular/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Oligopeptídeos/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Human mesenchymal stem cells can be isolated from bone marrow aspirates, purified and cultured for many passages without losing their unique properties. One of the hallmarks of stem cells is pluripotency, and human mesenchymal stem cells can be induced to assume phenotypes of mesenchymal tissues including, but not limited to, those of osteocytes, chondrocytes and adipocytes. Due to their ability to form cartilage, bone, fat and other connective tissue, human mesenchymal stem cells have great potential in regenerating diseased or injured tissues. Successful growth of human mesenchymal stem cells is essential to this process, and we have examined the response of human mesenchymal stem cells towards FGF1 and FGF2, two potent growth factors for human tissues. We provide evidence that: 1) human mesenchymal stem cells produce mRNA for receptors for FGF1 and FGF2; 2) these receptors can be detected on the surface of human mesenchymal stem cells; 3) FGF1 and FGF2 increase the rate at which human mesenchymal stem cells proliferate.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Células-Tronco/citologia , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
The transforming growth factor beta (TGF-beta) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-beta isoforms (TGF-beta 1, -beta 2 and -beta 3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-beta 3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-beta 3 was the major TGF-beta isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-beta 3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-beta 3 specific antibody showed TGF-beta 3 localization in perivascular smooth muscle.
Assuntos
Proteínas Recombinantes/isolamento & purificação , Fator de Crescimento Transformador beta/isolamento & purificação , Cordão Umbilical/química , Sequência de Aminoácidos , Aminoácidos/análise , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Divisão Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida/genética , Proteínas Recombinantes/farmacologia , Análise de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Crescimento Transformador beta/classificação , Fator de Crescimento Transformador beta/metabolismoRESUMO
S100 beta, a calcium-binding protein synthesized by CNS astrocytes, has trophic effects in vitro (neurite extension and glial proliferation). In Alzheimer's disease and Down's syndrome, severely afflicted brain regions exhibit up to 20-fold higher levels of S100 beta protein, and astrocytes surrounding neuritic plaques exhibit highly elevated levels of S100 beta immunostaining. A major constituent of plaques, beta-amyloid, has been reported to have neurotoxic and neurotrophic effects in vitro. In our study we examined the responses of CNS glia to beta-amyloid. C6 glioma cells and primary rat astrocyte cultures were treated with beta A(1-40) peptide at doses up to 1 microM. Weak mitogenic activity, measured by [3H]thymidine incorporation, was observed. Northern blot analysis revealed increases of S100 beta mRNA within 24 h in a dose-dependent manner. Nuclear run-off transcription assays showed that beta A(1-40) specifically induced new synthesis of S100 beta mRNA in cells maintained in serum, but under serum-free conditions, there was a general elevation of several mRNA species. Corresponding increases of S100 beta protein synthesis were observed by immunoprecipitation of 35S-labeled cellular proteins. To evaluate whether this effect of beta-amyloid was mediated via neurokinin receptors or by calcium fluxes, various agonists and antagonists were tested and found to be ineffective at stimulating S100 beta synthesis. In sum, these in vitro data suggest that in neuropathological conditions, beta-amyloid itself is an agent which may provoke chronic gliosis and the production of trophic substances by astrocytes.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica/fisiologia , Glioma/metabolismo , Fatores de Crescimento Neural/genética , Neuroglia/metabolismo , Proteínas S100 , Doença de Alzheimer/metabolismo , Células Cultivadas , Síndrome de Down/metabolismo , Glioma/patologia , Humanos , RNA Mensageiro/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Células Tumorais CultivadasRESUMO
A 64-kDa protein was purified from an octyl glucoside/cholate extract of spinach thylakoids. N-Terminal analysis yielded 23 residues of sequence, of which the first 15 were identical to a sequence reported [Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-II). We report the complete sequence of this 64-kDa protein, deduced from cDNA clones. The transit peptide has a chloroplast import signal at the N-terminus and a C-terminal hydrophobic span bounded by basic amino acids that predicts localization of the protein to the thylakoid lumen. The mature protein sequence is about 50% identical to several polyphenol oxidases (PPOs). Canonical protein kinase motifs are absent, as are sequences characteristic of ATP-binding sites. The mature protein resembles arthropodan hemocyanin (Hc), possessing three major domains. The N-terminal domain is rich in cysteine residues and predicted alpha-helices. The central domain has a conserved motif, N-terminal to a presumptive Cu-A site, that is not found in tyrosinases or Hc and is proposed as the provider of a third imidazole ligand to Cu-A. An unusual 13-residue, glutamine-rich link begins a C-terminal domain containing 7 predicted beta-strands which, by analogy with Hc, may form an antiparallel beta-barrel. We conclude that this 64-kDa polypeptide is a lumenal PPO and the precursor of a 42.5-kDa PPO form described previously [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Catecol Oxidase/genética , Clonagem Molecular , Proteínas Quinases/química , Spinacia oleracea/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Catecol Oxidase/química , Cloroplastos/enzimologia , Sequência Conservada , Cobre/química , Cisteína/química , DNA Complementar/química , Hemocianinas/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de SequênciaRESUMO
Protein kinase CKII (formerly casein kinase II) can be isolated as a heterotetramer, containing two catalytic (alpha or alpha') and two regulatory (beta) subunits. We have characterized the forms of CKII in HeLa cells using antibodies specific for the alpha or alpha' subunits. Following metabolic labeling with [35S]methionine, whole cell soluble extracts were analyzed by immunoprecipitation and gel electrophoresis. Both alpha and alpha' coprecipitate with beta and with each other. However, when extracts are depleted of alpha, a pool of CKII containing only alpha' and beta is identified. Similarly, depletion of alpha' revealed a pool exclusively of alpha and beta. Therefore, we propose that there are three distinct isoforms of CKII within HeLa cells with different catalytic subunit stoichiometries (alpha 2 beta 2, alpha alpha' beta 2, and alpha' 2 beta 2). With our immunodepletion procedure we have characterized the isoforms by activity analysis, turnover of pulse-labeled subunits, and by localization in subcellular fractions obtained from labeled cells. We have also analyzed complex formation between the catalytic and regulatory subunits by examining the differences in the rate of signal incorporation into subunits in immunoprecipitates obtained from continuously labeled and pulse-labeled cells. We have found that the alpha 2 beta 2 and alpha alpha' beta 2 isoforms assemble relatively slowly (12-16 h), whereas complex formation of the alpha' 2 beta 2 isoform occurs more rapidly (2-4 h). Analysis of isoform complex formation in subcellular fractions from pulse-labeled cells revealed that the majority of nuclear CKII is assembled in the nucleus from free catalytic and regulatory subunit polypeptides.
Assuntos
Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Anticorpos , Caseína Quinase II , Células HeLa , Humanos , Immunoblotting , Isoenzimas/química , Cinética , Substâncias Macromoleculares , Metionina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/química , Especificidade por SubstratoRESUMO
Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.
Assuntos
Substâncias de Crescimento/isolamento & purificação , Substâncias de Crescimento/fisiologia , Tumores Neuroectodérmicos Primitivos Periféricos/química , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Sequência de Aminoácidos , Anticorpos/farmacologia , Especificidade de Anticorpos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Fenômenos Químicos , Físico-Química , Meios de Cultura , Substâncias de Crescimento/química , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Homologia de Sequência de Aminoácidos , Fator de Crescimento Transformador beta/análise , Fator de Crescimento Transformador beta/imunologia , Células Tumorais CultivadasRESUMO
Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.
Assuntos
Proteína Quinase CDC2/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Células HeLa , Humanos , MAP Quinase Quinase 1 , MAP Quinase Quinase 2 , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptídeos/química , Fosfotreonina/metabolismo , Proteínas RecombinantesRESUMO
Accumulation of the amyloid A beta peptide, which is derived from a larger precursor protein (APP), and the formation of plaques, are major events believed to be involved in the etiology of Alzheimer's disease. Abnormal regulation of the metabolism of APP may contribute to the deposition of plaques. APP is an integral membrane protein containing several putative phosphorylation sites within its cytoplasmic domain. We report here that APP is phosphorylated at Thr668 by p34cdc2 protein kinase (cdc2 kinase) in vitro, and in a cell cycle-dependent manner in vivo. At the G2/M phase of the cell cycle, when APP phosphorylation is maximal, the levels of mature APP (mAPP) and immature APP (imAPP) do not change significantly. However, imAPP is altered qualitatively. Furthermore, the level of the secreted extracellular N-terminal domain (APPS) is decreased and that of the truncated intracellular C-terminal fragment (APPCOOH) is increased. These findings suggest the possibility that phosphorylation-dependent events occurring during the cell cycle affect the metabolism of APP. Alterations in these events might play a role in the pathogenesis of Alzheimer's disease.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ciclo Celular/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/biossíntese , Animais , Eletroforese em Gel Bidimensional , Fase G2 , Células HeLa , Humanos , Cinética , Metionina/metabolismo , Mitose , Dados de Sequência Molecular , Células PC12 , Mapeamento de Peptídeos , Peptídeos/síntese química , Fosfatos/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosforilação , TreoninaRESUMO
Protein kinase CKII is a prevalent serine/threonine protein kinase whose structure is highly conserved among eukaryotic organisms. Its involvement in the eukaryotic cell division cycle has been implicated by genetic experiments in yeast, antisense DNA, and inhibitory antibody experiments in mammalian cells, changes in activity during growth stimulation experiments, and protection of cells from radiation damage to replicating DNA. In addition, the cdc2 protein kinase, which is central to cell division cycle control, serves as a substrate for CKII specifically during the G1 phase of human cells. In this report, extracts of HeLa cells were prepared using neutral, aqueous buffers at low ionic strength. The cells were enriched for specific stages of the cell division cycle by treatment with drugs or by centrifugal elutriation. The results indicate that CKII activity in these extracts is highest during the G1 phase, and there appears to be a reduction in soluble CKII activity during the S phase. These data are consistent with the hypothesis that high CKII is necessary for a normal G1 phase but that progression through the S phase requires inhibition of CKII.
Assuntos
Ciclo Celular , Regulação Enzimológica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteína Quinase CDC2/metabolismo , Caseína Quinase II , Divisão Celular , Separação Celular , Fase G1 , Células HeLa/efeitos dos fármacos , Células HeLa/enzimologia , Humanos , Hidroxiureia/farmacologia , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Protein kinase CK2 is very abundant in rat brain when compared with other rat tissues. The enzyme is an oligomeric protein with the structures alpha 2 beta 2, alpha alpha'beta 2 and alpha'2 beta 2. The alpha and alpha' subunits are catalytic and have a high degree of homology, whereas the beta subunit seems regulatory. Using specific antibodies to synthetic peptide antigens that discriminate among the alpha, alpha', and beta subunits of CK2, we have observed a significant increase in the amount of alpha' subunit during the late postnatal neocortical maturation period. The increased alpha' expression occurs at a parallel time to synaptogenesis. As for its distribution, the alpha' subunit of CK2 is much more abundant in neurons (particularly in large size neurons) than it is in glia. These results are consistent with a hypothetical role for CK2 isoforms containing alpha' subunits in the regulation of specific functions in fully differentiated neurons.
Assuntos
Córtex Cerebral/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Sequência de Aminoácidos , Animais , Caseína Quinase II , Células Cultivadas , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Indução Enzimática , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neuroglia/enzimologia , Neurônios/enzimologia , Peptídeos/síntese química , Peptídeos/metabolismo , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , RatosRESUMO
p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20 degrees C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.
Assuntos
Mutação/genética , Proteína Supressora de Tumor p53/química , Sequência de Bases , Sítios de Ligação/genética , Humanos , Dados de Sequência Molecular , Mutação/fisiologia , Oligonucleotídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Termolisina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
An Epstein-Barr virus-encoded protein, ZEBRA, mediates the switch from latency to the viral lytic life cycle. ZEBRA's domain structure and DNA binding specificity resemble that of cellular transcriptional activators such as c-Fos/c-Jun. We show that ZEBRA, like c-Jun, is phosphorylated by casein kinase II (CKII). The principal site of phosphorylation is serine-173 (S173), five amino acids upstream of the basic DNA recognition domain. CKII phosphorylation abrogated ZEBRA's capacity to bind its target DNA sequences. S173 is a functional component of ZEBRA's DNA binding domain, since mutation of S173 to alanine (S173A) reduced DNA binding in vitro to 10% of wild-type levels. Transcriptional activation of a native viral promoter in vivo by mutant S173A was also reduced markedly. Reversible phosphorylation of S173 is likely to be an important means of regulating ZEBRA's activity in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina , Transativadores/metabolismo , Proteínas Virais/metabolismo , Alanina , Sequência de Aminoácidos , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação , Caseína Quinase II , Clonagem Molecular , Primers do DNA , Escherichia coli , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Mutação Puntual , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Phosphorylation of simian virus 40 (SV40) T antigen on threonine 124 activates viral DNA replication in vivo and in vitro. We have manipulated the modification of T-antigen residue 124 both genetically and biochemically and have investigated individual replication functions of T antigen under conditions suitable for in vitro DNA replication. We find that the hexamer assembly, helicase, DNA polymerase alpha-binding, and transcriptional-autoregulation functions are independent of phosphorylation of threonine 124. In contrast, neither T antigen with an alanine mutation of threonine 124 made in human cells nor unphosphorylated T antigen made in Escherichia coli binds the SV40 replication origin as stably as phosphorylated wild-type T antigen does. Furthermore, modification of threonine 124 is essential for complete unwinding of the SV40 replication origin. We conclude that phosphorylation of threonine 124 enhances specific interactions of T antigen with SV40 origin DNA. Our findings do not exclude the possibility that phosphorylation of threonine 124 may affect additional undefined steps in DNA replication. We also show that DNase footprinting and KMnO4 modification assays are not as stringent as immunoprecipitation and origin-dependent strand displacement assays for detecting defects in the origin-binding and -unwinding functions of T antigen. Differences in the assays may explain discrepancies in previous reports on the role of T-antigen phosphorylation in DNA binding.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteína Quinase CDC2/metabolismo , DNA Helicases/metabolismo , Vírus 40 dos Símios/fisiologia , Treonina , Replicação Viral , Sequência de Aminoácidos , Antígenos Transformantes de Poliomavirus/isolamento & purificação , DNA Polimerase II/metabolismo , DNA Super-Helicoidal/isolamento & purificação , DNA Super-Helicoidal/metabolismo , Desoxirribonuclease I , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Células HeLa , Homeostase , Humanos , Cinética , Modelos Estruturais , Fosforilação , Plasmídeos , Ligação Proteica , Conformação Proteica , Vírus 40 dos Símios/metabolismo , Transcrição GênicaRESUMO
Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcriptional factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here we show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34cdc2 (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation.