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1.
Oncogene ; 29(44): 5911-22, 2010 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-20802526

RESUMO

Fine tuning of Ras activity is widely known as a mechanism to induce different cellular responses. Recently, we have shown that calmodulin (CaM) binds to K-Ras and that K-Ras phosphorylation inhibits its interaction with CaM. In this study we report that CaM inhibits K-Ras phosphorylation at Ser181 by protein kinase C (PKC) in vivo, and this is a mechanism to modulate K-Ras activity and signaling. Although CaM inhibition increased the activation of endogenous K-Ras, PKC inhibition decreased its activation status. We demonstrate that K-Ras phosphorylation decreased susceptibility to p120GAP activity. Accordingly, we also observed that non-phosphorylable K-Ras mutant exhibits a less sustained activation profile and do not efficiently activate AKT at low growth factor doses compared with wild-type K-Ras. It is interesting that the physiological responses induced by K-Ras are affected by this phosphorylation; when K-Ras cannot be phosphorylated it exhibits a remarkably decreased ability to stimulate proliferation in non-saturated serum conditions. Finally, we demonstrate that phosphorylation also regulates oncogenic K-Ras functions, as focus formation capacity, mobility and apoptosis resistance upon adriamycin treatment of cells expressing oncogenic K-Ras that cannot be phosphorylated are highly compromised. Moreover, at low serum concentration proliferation and survival is practically inhibited when cells cannot phosphorylate oncogenic K-Ras. In this condition, K-Ras phosphorylation is essential to ensure a proper activation of mitogen-activated protein kinase and PI3K/AKT pathways. In summary, our findings suggest that the interplay between CaM interaction and PKC phosphorylation is essential to regulate non-oncogenic and oncogenic K-Ras activity and functionality.


Assuntos
Calmodulina/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Acetato de Tetradecanoilforbol/farmacologia
2.
Oncogene ; 28(29): 2654-66, 2009 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-19483727

RESUMO

Cyclin A accumulates at the onset of S phase, remains high during G(2) and early mitosis and is degraded at prometaphase. Here, we report that the acetyltransferase P/CAF directly interacts with cyclin A that as a consequence becomes acetylated at lysines 54, 68, 95 and 112. Maximal acetylation occurs simultaneously to ubiquitylation at mitosis, indicating importance of acetylation on cyclin A stability. This was further confirmed by the observation that the pseudoacetylated cyclin A mutant can be ubiquitylated whereas the nonacetylatable mutant cannot. The nonacetylatable mutant is more stable than cyclin A WT (cycA WT) and arrests cell cycle at mitosis. Moreover, in cells treated with histone deacetylase inhibitors cyclin A acetylation increases and its stability decreases, thus supporting the function of acetylation on cyclin A degradation. Although the nonacetylatable mutant cannot be ubiquitylated, it interacts with the proteins needed for its degradation (cdks, Cks, Cdc20, Cdh1 and APC/C). In fact, its association with cdks is increased and its complexes with these kinases display higher activity than control cycA WT-cdk complexes. All these results indicate that cyclin A acetylation at specific lysines is crucial for cyclin A stability and also has a function in the regulation of cycA-cdk activity.


Assuntos
Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Células COS , Chlorocebus aethiops , Ciclina A/genética , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Mutação
3.
Analyst ; 133(1): 105-11, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18087621

RESUMO

The qualitative evaluation of chromatographic data in the framework of external quality assurance schemes is considered in this paper. The homogeneity in the evaluation of chromatographic data among human experts in samples with analytes close to the limit of detection of analytical methods was examined and also a Support Vector Machine (SVM) was developed as an alternative to experts for a more homogeneous and automatic evaluation. A set of 105 ion chromatograms obtained by anti-doping control laboratories was used in this study. The quality of the ion chromatograms was evaluated qualitatively by nine independent experts (associating a score from 0 to 4) and also more objectively taking into account chromatographic parameters (peak width, asymmetry, resolution and S/N ratio). Results obtained showed a high degree of variability among experts when judging ion chromatograms. Experts applying extremely outlying evaluation criteria were identified and excluded from the data used to develop the SVM. This machine was built providing the system with qualitative information (scores assigned by experts) and with objective data (parameters) of the ion chromatograms. A seven-fold cross-validation approach was used to train and to evaluate the predictive ability of the machine. According to the results obtained, the SVM developed was found to be close to the reasoning process followed by the homogeneous human expert group. This machine also could provide a scoring system to sort laboratories according to the quality of their results. The qualitative evaluation of analytical records using a scoring system allowed the identification of the main factors affecting the quality of chromatographic analytical data, such as the specific analytical technique applied and the adherence to guidelines for reporting positive results.


Assuntos
Cromatografia por Troca Iônica/normas , Interpretação Estatística de Dados , Estudos de Avaliação como Assunto , Controle de Qualidade , Dopagem Esportivo , Humanos , Laboratórios/normas , Sensibilidade e Especificidade , Detecção do Abuso de Substâncias/métodos
4.
Oncogene ; 25(29): 4033-42, 2006 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-16474839

RESUMO

We report here that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts in vitro and in vivo with the protein SET. This interaction is performed through the acidic domain of SET located at the carboxy terminal region. On analysing the functional relevance of SET-GAPDH interaction, we observed that GAPDH reverses in a dose-dependent manner, the inhibition of cyclin B-cdk1 activity produced by SET. Similarly to SET, GAPDH associates with cyclin B, suggesting that the regulation of cyclin B-cdk1 activity might be mediated not only by the interaction of GAPDH with SET but also with cyclin B. To analyse the putative role of GAPDH on cell cycle progression, HCT116 cells were transfected with a GAPDH expression vector. Results indicate that overexpression of GAPDH does not affect the timing of DNA replication but induces an increase in the number of mitosis, an advancement of the peak of cyclin B-cdk1 activity and an acceleration of cell cycle progression. All these results suggest that GAPDH might be involved in cell cycle regulation by modulating cyclin B-cdk1 activity.


Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Ciclina B/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , DNA de Neoplasias/biossíntese , Proteínas de Ligação a DNA , Ativação Enzimática , Vetores Genéticos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Chaperonas de Histonas , Humanos , Ligação Proteica , Estrutura Terciária de Proteína/genética , Transfecção
5.
Oncogene ; 25(2): 260-70, 2006 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-16170352

RESUMO

The oncoprotein SET participates in a diversity of cellular functions including cell proliferation. Its role on cell cycle progression is likely mediated by inhibiting cyclin B-cdk1 and the protein phosphatase 2A (PP2A). On identifying new SET cellular partners, we found that SET interacts in vitro and in vivo with the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2); a protein involved in various aspects of mRNA biogenesis. The SET-binding region of hnRNPA2 is the RNP1 sequence that belongs to the RNA-binding domain (RBD) of this protein. We also found that hnRNPA2 has much higher affinity for single-standed DNA than for SET. On analysing the effect of hnRNPA2 on PP2A inhibition by SET, we observed that hnRNPA2 cooperates with SET on PP2A inhibition. This is because we found that hnRNPA2 is also a PP2A inhibitor. HnRNPA2 interacts with PP2A by the RNP1 sequence; however, to inhibit PP2A activity, the complete RBD is needed. We also observed that overexpression of hnRNPA2 inhibits PP2A activity and stimulates cell proliferation. Interestingly, the overexpression of the complete RBD is sufficient to stimulate proliferation. As hnRNPA2 is overexpressed in a variety of human tumors, our results suggest that hnRNPA2 might participate in oncogenesis by stimulating cell proliferation.


Assuntos
Proliferação de Células , Proteínas Cromossômicas não Histona/metabolismo , DNA de Cadeia Simples/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células COS , Ciclo Celular , Células Cultivadas , Chlorocebus aethiops , Cromatografia de Afinidade , Proteínas Cromossômicas não Histona/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA , Eletroforese em Gel Bidimensional , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Chaperonas de Histonas , Humanos , Imunoprecipitação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Plasmídeos , Ligação Proteica , Proteína Fosfatase 2 , RNA/metabolismo , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Transcrição/genética
6.
Mol Pharmacol ; 60(6): 1235-42, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11723230

RESUMO

KARs, new semisynthetic antitumor bis-indole derivatives, were found to be inhibitors of tubulin polymerization with lower toxicity than vinblastine or vincristine, used in chemotherapy. Here, we compare the effect of KARs with those of vinblastine and vincristine on cell viability, cell proliferation, and cell cycle in neuroblastoma cell line (SH-SY5Y). At concentrations of the different compounds equivalent in causing 50% of inhibition of cell growth, KARs induced a complete arrest in the G2/M phase, whereas vinblastine and vincristine induced a partial arrest in both G0/G1 and G2/M. Moreover, a combination of KAR-2 and W13 (an anticalmodulin drug) qualitatively caused a similar arrest in both G0/G1 and G2/M than vinblastine. Levels of cyclin A and B1 were higher in KARs-treated cells than in vinblastine- or vincristine-treated cells. Cdc2 activity was much higher in KAR-2 than in vinblastine-treated cells, indicating a stronger mitotic arrest. The effect of KAR2 and vinblastine on microtubules network was analyzed by immunostaining with anti-tubulin antibody. Results indicated that KAR-2-induces the formation of aberrant mitotic spindles, with not apparent effect on interphase microtubules, whereas vinblastine partially destroyed interphase microtubules coexisting with normal and aberrant mitotic spindles.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fase G2/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neuroblastoma/patologia , Fuso Acromático/efeitos dos fármacos , Vimblastina/análogos & derivados , Apoptose , Proteína Quinase CDC2/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Divisão Celular/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Ciclina A/efeitos dos fármacos , Ciclina A/metabolismo , Ciclina B/efeitos dos fármacos , Ciclina B/metabolismo , Ciclina B1 , Humanos , Interfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Células Tumorais Cultivadas , Vimblastina/farmacologia
7.
Mol Cell Biol ; 21(21): 7345-54, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11585916

RESUMO

Activation of Ras induces a variety of cellular responses depending on the specific effector activated and the intensity and amplitude of this activation. We have previously shown that calmodulin is an essential molecule in the down-regulation of the Ras/Raf/MEK/extracellularly regulated kinase (ERK) pathway in cultured fibroblasts and that this is due at least in part to an inhibitory effect of calmodulin on Ras activation. Here we show that inhibition of calmodulin synergizes with diverse stimuli (epidermal growth factor, platelet-derived growth factor, bombesin, or fetal bovine serum) to induce ERK activation. Moreover, even in the absence of any added stimuli, activation of Ras by calmodulin inhibition was observed. To identify the calmodulin-binding protein involved in this process, calmodulin affinity chromatography was performed. We show that Ras and Raf from cellular lysates were able to bind to calmodulin. Furthermore, Ras binding to calmodulin was favored in lysates with large amounts of GTP-bound Ras, and it was Raf independent. Interestingly, only one of the Ras isoforms, K-RasB, was able to bind to calmodulin. Furthermore, calmodulin inhibition preferentially activated K-Ras. Interaction between calmodulin and K-RasB is direct and is inhibited by the calmodulin kinase II calmodulin-binding domain. Thus, GTP-bound K-RasB is a calmodulin-binding protein, and we suggest that this binding may be a key element in the modulation of Ras signaling.


Assuntos
Calmodulina/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Células 3T3 , Animais , Bombesina/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Regulação Enzimológica da Expressão Gênica , Genes ras/genética , Humanos , Immunoblotting , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas ras/química
8.
J Biol Chem ; 275(45): 35091-7, 2000 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-10952989

RESUMO

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway. The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.


Assuntos
Ciclina D1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osmose , Arsenitos/farmacologia , Western Blotting , Cloreto de Cálcio/farmacologia , Divisão Celular , Linhagem Celular , Cisteína Endopeptidases , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Glutationa Transferase/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases , Cloreto de Magnésio/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Estresse Oxidativo , Fosforilação , Mutação Puntual , Testes de Precipitina , Complexo de Endopeptidases do Proteassoma , Piridinas/farmacologia , Pirimidinas/farmacologia , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes de Fusão/metabolismo , Cloreto de Sódio/farmacologia , Treonina/química , Fatores de Tempo , Células Tumorais Cultivadas , Ubiquitinas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno
9.
J Hepatol ; 33(2): 266-74, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10952244

RESUMO

BACKGROUND/AIMS: The cell cycle inhibitors p21Cip1 and p27Kip1 regulate liver regeneration by modulating the activity of cyclin-dependent kinases (CDKs). However, the specific role of these inhibitors in the regulation of CDK2 activity during liver regeneration remains unknown. The aim of this study was to examine the association of p21Cip1 and p27Kip1 with cyclin E-CDK2 and cyclin A-CDK2 complexes during rat liver regeneration and to correlate the association of both inhibitors with CDK2 activity. METHODS: The association of p21Cip1 or p27Kip1 with cyclin E-CDK2 or cyclin A-CDK2 and the activities of these complexes were analyzed by immunoprecipitation of rat liver homogenates obtained at different times after a partial hepatectomy (PH), followed by Western blotting or kinase assays. RESULTS: High amounts of p27Kip1 bound to cyclin E-CDK2 were observed during the first 13 h after PH, when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound-p27Kip1 decreased strongly. The amount of p21Cip1 bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both inhibitors were detected and at 28 h, a significant increase in p21Cip1 and p27Kip1 associated with cyclin A-CDK2 was observed. CONCLUSIONS: p27Kip1 acts as a brake on cyclin E-CDK2 activity during the first 13 h after a PH. Both p21Cip1 and p27Kip1 down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclina E/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Ciclinas/fisiologia , Regeneração Hepática/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Supressoras de Tumor , Animais , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Genes Supressores de Tumor , Masculino , Ratos , Ratos Sprague-Dawley
10.
J Cell Physiol ; 184(3): 380-4, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10911370

RESUMO

Incubation of isolated rat soleus and EDL muscles in the presence of 10 mM leucine resulted in a decreased proteolytic rate as measured by the release of tyrosine into the incubation medium. The effect of this branched-chain amino acid (BCAA) is associated with a decreased activity of the lysosomal proteases and a decreased expression of the genes of the ATP-ubiquitin-dependent proteolysis (ubiquitin and C8). Incubation of muscles in the presence of actinomycin D revealed that the effects of the amino acid can be accounted for by an inhibition of the transcription rate. The presence of leucine did not influence the gene expression of other nonlysosomal (m-calpain) and lysosomal (cathepsin B) proteolytic systems. It is concluded that the well-known effect of BCAA on muscle proteolysis is mediated, in the short term, by the inhibition of lysosomal proteolysis. In a longer period, based on the inhibition of gene transcription observed, an involvement of the ATP-dependent proteolytic system is also likely to occur.


Assuntos
Aminoácidos de Cadeia Ramificada/farmacologia , Endopeptidases/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Endopeptidases/genética , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Masculino , Músculo Esquelético/metabolismo , Fosfocreatina/metabolismo , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ubiquitinas/genética , Ubiquitinas/metabolismo
11.
J Pept Res ; 55(2): 148-62, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10784031

RESUMO

Calmodulin is known to bind to various amphipathic helical peptide sequences, and the calmodulin-peptide binding surface has been shown to be remarkably tolerant sterically. D-Amino acid peptides, therefore, represent potential nonhydrolysable intracellular antagonists of calmodulin. In the present study, synthetic combinatorial libraries have been used to develop novel D-amino acid hexapeptide antagonists to calmodulin-regulated phosphodiesterase activity. Five hexapeptides were identified from a library containing over 52 million sequences. These peptides inhibited cell proliferation both in cell culture using normal rat kidney cells and by injection via the femoral vein following partial hepatectomy of rat liver cells. These hexapeptides showed no toxic effect on the cells. Despite their short length, the identified hexapeptides appear to adopt a partial helical conformation similar to other known calmodulin-binding peptides, as shown by CD spectroscopy in the presence of calmodulin and NMR spectroscopy in DMSO. The present peptides are the shortest peptide calmodulin antagonists reported to date showing potential in vivo activity.


Assuntos
Calmodulina/antagonistas & inibidores , Inibidores do Crescimento/farmacologia , Oligopeptídeos/farmacologia , Animais , Calmodulina/metabolismo , Células Cultivadas , Dicroísmo Circular , Técnicas de Química Combinatória , Inibidores do Crescimento/química , Inibidores do Crescimento/metabolismo , Masculino , Modelos Moleculares , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Biblioteca de Peptídeos , Inibidores de Fosfodiesterase/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Espectrometria de Fluorescência , Estereoisomerismo
12.
Oncogene ; 19(5): 690-9, 2000 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-10698514

RESUMO

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Núcleo Celular/química , Quinases Ciclina-Dependentes/metabolismo , Lisina/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteínas Proto-Oncogênicas , Células 3T3 , Animais , Divisão Celular , Meios de Cultura Livres de Soro , Ciclina D1/metabolismo , Ciclina G , Ciclina G1 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Ciclinas/metabolismo , Ativação Enzimática/genética , Regulação da Expressão Gênica , Camundongos , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína do Retinoblastoma/antagonistas & inibidores
13.
J Biol Chem ; 274(46): 33161-5, 1999 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-10551888

RESUMO

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Células Tumorais Cultivadas
14.
J Biol Chem ; 274(35): 24445-8, 1999 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-10455103

RESUMO

p21(Cip1), first described as an inhibitor of cyclin-dependent kinases, has recently been shown to have a function in the formation of cyclin D-Cdk4 complexes and in their nuclear translocation. The dual behavior of p21(Cip1) may be due to its association with other proteins. Different evidence presented here indicate an in vitro and in vivo interaction of p21(Cip1) with calmodulin: 1) purified p21(Cip1) is able to bind to calmodulin-Sepharose in a Ca(2+)-dependent manner, and this binding is inhibited by the calmodulin-binding domain of calmodulin-dependent kinase II; 2) both molecules coimmunoprecipitate when extracted from cellular lysates; and 3) colocalization of calmodulin and p21(Cip1) can be detected in vivo by electron microscopy immunogold analysis. The carboxyl-terminal domain of p21(Cip1) is responsible for the calmodulin interaction, since p21(145-164) peptide is also able to bind calmodulin and to compete with full-length p21(Cip1) for the calmodulin binding. Because treatment of cells with anti-calmodulin drugs decreases the nuclear accumulation of p21(Cip1), we hypothesize that calmodulin interaction with p21(Cip1) is important for p21(Cip1), and in consequence for cyclin D-Cdk4, translocation into the cell nucleus.


Assuntos
Calmodulina/metabolismo , Núcleo Celular/metabolismo , Ciclinas/metabolismo , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Linhagem Celular , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Ativação Enzimática , Microscopia Eletrônica , Dados de Sequência Molecular , Fosforilação , Testes de Precipitina , Ligação Proteica , Ratos , Sulfonamidas/farmacologia
15.
Neurochem Int ; 34(2): 137-40, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10213072

RESUMO

Histological features of neurogenic muscle involvement included type grouping, muscle fiber atrophy, and target fibers. In muscles with myofiber atrophy and target fibers, we found an increased expression of the genes encoding for the ubiquitin-ATP-dependent proteolytic system. Thus, in patients with target fibers, a 5.2- and a 3.9-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively, while in those with atrophic angulated hyperoxidative fibers, a 3.9- and a 4.4-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively. It is suggested that the activation of this proteolytic system may be responsible for the skeletal muscle alterations that often accompany human muscle neurogenic involvement.


Assuntos
Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Ubiquitinas/genética , Northern Blotting , Humanos , Técnicas Imunoenzimáticas , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/inervação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Hepatology ; 29(2): 385-95, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9918914

RESUMO

Partial hepatectomy (PH) triggers the entry of rat liver cells into the cell cycle. The signals leading to cell-cycle activation converge into a family of kinases named cyclin-dependent kinases (cdks). Specific cyclin-cdk complexes are sequentially activated during the cell cycle. Cyclin D-cdk4 and cyclin E-cdk2 are activated during the G1 phase, cyclin A-cdk2 is activated during the S phase, and cyclin B-cdk1 during mitosis. In the present study, we have examined the timing of the activation of cdk4 and cdk2, the intracellular location of G1/S cyclins and cdks, and the relationship between location and cdk4 and cdk2 activities during rat liver regeneration after a PH. Results showed that the activity of both kinases started at 13 hours and showed maximal levels at 24 hours after hepatectomy. In quiescent cells, cyclin D3 and cdk4 were cytoplasmatic, whereas cyclin D1 was nuclear. At 5 hours after hepatectomy, cyclin D3 and cdk4 began to move into the nucleus, and at 13 hours, they were mostly nuclear. During the first 13 hours after hepatectomy, significant amounts of cyclin D1-cdk4 and cyclin D3-cdk4 complexes were formed, but they were mostly inactive. At 24 hours, these complexes were maximally activated. This activation was associated with the accumulation of cyclin D1, cyclin D3, and cdk4 in a nuclear subfraction extractable with nucleases. At 28 hours, the activity of cdk4 in this nuclear subfraction decreased when cyclin D1 moved from this fraction to the nuclear matrix (NM) and the levels of cyclin D3 diminished. The maximal activation of cdk2 at 24 hours was also associated with the accumulation of cyclin E, cyclin A, and cdk2 in this nuclease-sensitive fraction. The inactivation of cdk2 at 28 hours was associated with a strong decrease in cdk2 in this nuclear subfraction. Thus, results reported here indicate that the activation of cdk4 and cdk2 observed in rat liver cells after a PH is associated with a specific intranuclear location of these cdks and their associated cyclins.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Núcleo Celular/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Regeneração Hepática , Fígado/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor , Animais , Ciclina D , Ciclina E/análise , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/análise , Ciclinas/análise , Inibidores Enzimáticos , Fase G1 , Hepatectomia , Imuno-Histoquímica , Cinética , Fígado/ultraestrutura , Masculino , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/análise , Ratos , Ratos Sprague-Dawley , Fase S
17.
J Biol Chem ; 273(50): 33279-86, 1998 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-9837900

RESUMO

Although it is known that calmodulin is involved in G1 progression, the calmodulin-dependent G1 events are not well understood. We have analyzed here the role of calmodulin in the activity, the expression, and the intracellular location of proteins involved in G1 progression. The addition of anti-calmodulin drugs to normal rat kidney cells in early G1 inhibited cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities, as well as retinoblastoma protein phosphorylation. Protein levels of cdk4, cyclin D1, cyclin D2, cyclin E, p21, and p27 were not affected after CaM inhibition, whereas decreases in the amount of cyclin A and Cdc2 were observed. The decrease of Cdk4 activity was due neither to changes in its association to cyclin D1 nor to changes in the amount of p21 or p27 bound to cyclin D1-Cdk4 complexes. Calmodulin inhibition also produced a translocation of nuclear cyclin D1 and Cdk4 to the cytoplasm. This translocation could be responsible for the decreased Cdk4 activity upon calmodulin inhibition. Immunoprecipitation, calmodulin affinity chromatography, and direct binding experiments indicated that calmodulin associates with Cdk4 and cyclin D1 through a calmodulin-binding protein. The facts that Hsp90 interacts with Cdk4 and that its inhibition induced Cdk4 and cyclin D1 translocation to the cytoplasm point to Hsp90 as a good candidate for being the calmodulin-binding protein involved in the nuclear accumulation of Cdk4 and cyclin D1.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Calmodulina/fisiologia , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fase G1 , Proteínas Proto-Oncogênicas , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Linhagem Celular , Núcleo Celular/enzimologia , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Ativação Enzimática , Imuno-Histoquímica , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Ratos
18.
Int J Mol Med ; 2(1): 69-73, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9854146

RESUMO

Human biopsies obtained from skeletal muscle of cachectic AIDS patients clearly showed an increased expression (in relation to that of healthy subjects) of the genes encoding for the ubiquitin-ATP-dependent proteolytic system. Increases of 120% and 42% were observed for the 2.4 and 1.2 kb ubiquitin transcripts, respectively. The expression of the C8 proteasome subunit was also increased by 60% in the cachectic AIDS patients in relation to the healthy control subjects. It is suggested that the activation of this proteolytic system (possibly via changes in circulating cytokines, such as TNF) may be responsible for the skeletal muscle waste that often accompanies AIDS.


Assuntos
Síndrome da Imunodeficiência Adquirida/genética , Cisteína Endopeptidases/genética , Síndrome de Emaciação por Infecção pelo HIV/genética , Complexos Multienzimáticos/genética , Músculo Esquelético/metabolismo , Ubiquitinas/genética , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/metabolismo , Regulação da Expressão Gênica , Humanos , Complexo de Endopeptidases do Proteassoma , Fator de Necrose Tumoral alfa/fisiologia
19.
Int J Mol Med ; 1(6): 971-7, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9852633

RESUMO

Induction of experimental insulin-deficiency by a single administration of streptozotocin to rats resulted in substantial changes in heart and skeletal muscle size and protein content. This was accompanied by a marked loss of total body (carcass) nitrogen and raised concentrations of circulating branched-chain amino acids. These changes were related to alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in both the fractional protein rates of synthesis (decreased by 37%) and degradation (increased by 141%). The increased protein degradation observed in the muscle of the diabetic animals was associated only with an increase in the expression of the genes controlling ubiquitin-dependent proteolysis. It may be suggested that the hormonal changes associated with the diabetic state play an important role in the regulation of the activity of the ubiquitin-dependent proteolytic system in skeletal muscle, highlighting the major role of this system in the diabetes-related cachexia.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Ácido 3-Hidroxibutírico/sangue , Acetoacetatos/sangue , Tecido Adiposo/crescimento & desenvolvimento , Animais , Peso Corporal , Feminino , Hidrólise , Hiperglicemia/sangue , Insulina/sangue , Isoleucina/metabolismo , Rim/crescimento & desenvolvimento , Leucina/metabolismo , Fígado/crescimento & desenvolvimento , Glicogênio Hepático/metabolismo , Metilistidinas/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Tamanho do Órgão , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ubiquitinas/genética , Ubiquitinas/fisiologia , Valina/metabolismo
20.
Mol Cell Endocrinol ; 142(1-2): 183-9, 1998 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-9783914

RESUMO

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the TNF-alpha receptor type I protein (Tnfr1 degree/Tnfr1 degree), resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the gene-knockout mice muscle wastage was not affected to the same extent. In both groups, tumour burden resulted in significant increases in circulating TNF-alpha, a cytokine which, as we have previously demonstrated, can induce protein breakdown in skeletal muscle. Muscle wastage in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result is a decreased rate of protein accumulation that accounts for the muscle weight loss observed as a result of tumour burden. In contrast, gene knockout mice did not have significantly lower rates of protein accumulation as a result of tumour implantation. The increase in protein degradation in the tumour-bearing wild mice was accompanied by an enhanced expression of both ubiquitin and proteasome subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. Tumour-bearing gene-deficient mice did not show any increase in gene expression. It is concluded that TNF-alpha (alone or in combination with other cytokines) is responsible for the activation of protein breakdown in skeletal muscle of tumour-bearing mice.


Assuntos
Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/complicações , Proteínas Musculares/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Caquexia/etiologia , Deleção de Genes , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinas/genética , Redução de Peso
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