RESUMO
Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase.
Assuntos
Caseína Quinase II/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases/metabolismo , Quinases raf/metabolismo , Animais , Sítios de Ligação , Camundongos , Células NIH 3T3 , Estrutura Terciária de Proteína , XenopusRESUMO
Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [(3)H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [(3)H]P5 was not the end product of tasidotin metabolism. Large amounts of [(3)H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC(50) of tasidotin and a marked increase in intracellular [(3)H]tasidotin. BCPP also caused a 4-fold increase in the IC(50) of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC(50) obtained for P5 in the presence of BCPP.
Assuntos
Antineoplásicos/toxicidade , Depsipeptídeos/toxicidade , Oligopeptídeos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Depsipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Humanos , Concentração Inibidora 50 , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fatores de TempoRESUMO
Parafibromin, the product of the HRPT2 (hyperparathyroidism-jaw tumor syndrome 2) tumor suppressor gene, is the human homologue of yeast Cdc73, part of the yeast RNA polymerase II/Paf1 complex known to be important for histone modification and connections to posttranscriptional events. By purifying cellular parafibromin and characterizing its associated proteins, we have identified a human counterpart to the yeast Paf1 complex including homologs of Leo1, Paf1, and Ctr9. Like the yeast complex, the parafibromin complex associates with the nonphosphorylated and Ser2 and Ser5 phosphorylated forms of the RNA polymerase II large subunit. Immunofluorescence experiments show that parafibromin is a nuclear protein. In addition, cotransfection data suggest that parafibromin can interact with a histone methyltransferase complex that methylates histone H3 on lysine 4. Some mutant forms of parafibromin lack association with hPaf1 complex members and with the histone methyltransferase complex, suggesting that disruption of these complexes may correlate with the oncogenic process.
Assuntos
Complexos Multiproteicos , Proteínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Metiltransferases , Proteínas/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição , Proteínas Supressoras de TumorRESUMO
CCAAT/enhancer binding protein beta (C/EBPbeta) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPbeta is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPbeta in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPbeta activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPbeta, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPbeta as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPbeta on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , Transformação Celular Neoplásica , Genes ras , Transdução de Sinais , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Quinase 2 Dependente de Ciclina , Inibidores Enzimáticos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Alinhamento de Sequência , Serina/metabolismo , Treonina/metabolismo , Proteínas ras/genéticaRESUMO
The epigenetic programming of genomic DNA is accomplished, in part, by several DNA cytosine-5-methyltransferases that act by covalently modifying cytosines with the addition of a methyl group. This covalent modification is maintained by the DNA cytosine-5-methyltransferase-1 enzyme (DNMT1), which is capable of acting in concert with other similar enzymes to silence important tumor suppressor genes. IL-6 is a multifunctional mediator of inflammation, acting through several major signaling cascades, including the phosphatidylinositol-3-kinase pathway (PI-3-K), which activates protein kinase B (AKT/PKB) downstream. Here, we show that the subcellular localization of DNMT1 can be altered by the addition of IL-6, increasing the rate of nuclear translocation of the enzyme from the cytosolic compartment. The mechanism of nuclear translocation of DNMT1 is greatly enhanced by phosphorylation of the DNMT1 nuclear localization signal (NLS) by PKB/AKT kinase. Mutagenic alteration of the two AKT target amino acids within the NLS results in a major loss of DNMT1 nuclear translocation, while the creation of a "phospho-mimic" amino acid (mutation to acidic residues) restores this compartmentation ability. These observations suggest an interesting hypothesis regarding how mediators of chronic inflammation may disturb the delicate balance of cellular compartmentalization of important proteins, and reveals a potential mechanism for the induction or enhancement of tumor growth via alteration of the components involved in the epigenetic programming of a cell.
Assuntos
Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Interleucina-6/farmacologia , Sinais de Localização Nuclear/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sequência de Aminoácidos , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Marcação por Isótopo , Carioferinas/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Radioisótopos de Fósforo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de ProteínaRESUMO
Recent epidemiological studies suggest an association between higher blood levels of insulin-like growth factor I (IGF-I) and increased risk of prostate cancer. We evaluated the association between prediagnostic levels of IGF-I and insulin-like growth factor binding protein 3 (IGFBP-3) and prostate cancer risk in a nested case-control study within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Within the same cohort (using different cases and controls who had sequential serum samples available) we also examined changes in serum IGF-I and IGFBP-3 levels over time by case status. The risk association study included incident prostate cancer cases (n = 100) diagnosed at least 5 years after baseline blood draw (range, 5-12 years; median 9 years) and frequency-matched (4:1) controls. The sequential serum study included all of the prostate cancer cases (n = 21) with prediagnostic (2-3 years before diagnosis) and diagnostic serum available, and pair-matched controls (1:1). An ELISA was used to quantitate serum levels of IGF-I and IGFBP-3 for both studies. The association between IGF-I or IGFBP-3 and prostate cancer risk was assessed using conditional logistic regression, and paired t tests were used to evaluate case-control differences in change in serum analytes over time. We found no significant association between either IGF-I or IGFBP-3 and prostate cancer risk. In a multivariate analysis, we observed an odds ratio of 0.52 (95% confidence interval, 0.23-1.16) for the fourth versus the first quartile of serum IGF-I. Serum IGF-I, but not IGFBP-3, increased significantly over time in cases (18% increase) but not controls (4% decrease; P = 0.02). In contrast to previous reports, we found no evidence to support a causal association between serum IGF-I or IGFBP-3 and the risk of prostate cancer. It is possible that serum IGF-I may be serving as a tumor marker rather than an etiologic factor in prostate cancer.
Assuntos
Biomarcadores Tumorais/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias da Próstata/sangue , Idoso , Estudos de Casos e Controles , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
The HDM2 protein is a key regulator of the tumour suppressor, p53. Control of HDM2 function is critical for normal cell proliferation and stress responses, and it is becoming evident that multiple modifications of HDM2 can regulate its function within cells. In this study we show that HDM2 associated with the serine-threonine kinase, Akt, in response to growth factor stimulation of human primary cells. This association was concurrent with phosphorylation of Akt (at Ser 473), and resulted in elevated expression of HDM2 and enhanced nuclear localization. However, analysis of HDM2 proteins mutated at the consensus Akt recognition sites at serines 166 and 186 indicated that modification at these residues was not sufficient for the increased expression of the protein, which was blocked by the PI3 kinase inhibitor LY294002. Tryptic peptide and mutational analyses revealed evidence for an Akt phosphorylation site in HDM2 additional to the two consensus sites.
Assuntos
Proteínas Nucleares , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Sequência Consenso , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-mdm2 , TransfecçãoRESUMO
The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.
Assuntos
Endopeptidases/metabolismo , Potyvirus/enzimologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Endopeptidases/química , Dados de Sequência Molecular , Especificidade por Substrato , Proteínas Virais/químicaRESUMO
The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPbeta activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g<-->e' salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta in Ras(V12)-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPbeta-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , DNA/metabolismo , Zíper de Leucina , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , DNA/genética , Substâncias de Crescimento/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Alinhamento de Sequência , Ativação Transcricional , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Proteínas de Transporte/genética , Linhagem Celular Transformada , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas MetiltransferasesRESUMO
Polo-like kinase functions are essential for the establishment of a normal bipolar mitotic spindle, although precisely how Plk1 regulates the spindle is uncertain. In this study, we report that the small GTP/GDP-binding protein Ran is associated with Plk1. Plk1 is capable of phosphorylating co-immunoprecipitated Ran in vitro on serine-135 and Ran is phosphorylated in vivo at the same site during mitosis when Plk1 is normally activated. Cell cultures over-expressing a Ran S135D mutant have significantly higher numbers of abnormal mitotic cells than those over-expressing either wild-type or S135A Ran. The abnormalities in S135D mutant cells are similar to cells over-expressing Plk1. Our data suggests that Ran is a physiological substrate of Plk1 and that Plk1 regulates the spindle organization partially through its phosphorylation on Ran.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cães , Guanosina Trifosfato/química , Humanos , Mitose , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/química , Proteína ran de Ligação ao GTP/química , Quinase 1 Polo-LikeRESUMO
Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)theta. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCtheta in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCtheta to mediate NF-kappaB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCtheta's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCtheta with Akt/PKBalpha in NF-kappaB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCtheta in activated T cells. The PKCthetaT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCtheta at Thr-219 plays an important role in the correct targeting and cellular function of PKCtheta upon antigen receptor ligation.
Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Treonina/metabolismo , Membrana Celular/enzimologia , Ativação Enzimática , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Isoenzimas/genética , Células Jurkat , Fosforilação , Regiões Promotoras Genéticas , Proteína Quinase C/genética , Proteína Quinase C-theta , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Ativação TranscricionalRESUMO
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Linfócitos T CD4-Positivos/enzimologia , Ativação Enzimática , Humanos , Immunoblotting , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Mapeamento de Peptídeos , Fosforilação , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Proteína-Tirosina Quinase ZAP-70 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.
Assuntos
Regulação para Baixo , Retroalimentação Fisiológica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Isoenzimas/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase de Interação com NIMA , Peptídeos/genética , Peptídeos/metabolismo , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Xenopus laevis , Proteínas ras/metabolismoRESUMO
Cdc25C-associated kinase 1 (C-TAK1) has been implicated in cell cycle regulation and Ras signaling through its interactions with two putative substrates, the Cdc25C phosphatase and the MAPK scaffold KSR1. Here, we identify sequence motifs required for stable C-TAK1 association and substrate phosphorylation. Using a mutational approach to disrupt binding of C-TAK1 to KSR1 and Cdc25C, we demonstrate that C-TAK1 contributes to the regulation of these proteins in vivo through the generation of 14-3-3-binding sites. KSR1 proteins defective in C-TAK1 binding had severely reduced phosphorylation at the 14-3-3-binding site in vivo, were constitutively localized to the plasma membrane and had increased biological activity. Disruption of the Cdc25C-C-TAK1 interaction resulted in reduced 14-3-3-binding site phosphorylation and nuclear accumulation of Cdc25C in interphase cells. Finally, utilizing the acquired C-TAK1 binding and substrate phosphorylation data, we identify plakophilin 2 (PKP2) as a novel C-TAK1 substrate. Phosphorylation of PKP2 by C-TAK1 also generates a 14-3-3-binding site that influences PKP2 localization. These findings underscore the importance of C-TAK1 as a regulator of 14-3-3 binding and protein localization.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas 14-3-3 , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Humanos , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Fosforilação , Placofilinas , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Tirosina 3-Mono-Oxigenase/metabolismo , Xenopus , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismoRESUMO
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein flanked by Gag sequences (r-preNC) was expressed in Escherichia coli and purified. HIV-1 proteinase cleaved r-preNC to the "mature" NCp7 form, which is comprised of 55 residues. Further incubation resulted in cleavages of NCp7 itself between Phe16 and Asn17 of the proximal zinc finger domain and between Cys49 and Thr50 in the C-terminal part. Kinetic parameters determined for the cleavage of oligopeptides corresponding to the cleavage sites in r-preNC correlated well with the sequential processing of r-preNC. Mutations of Asn17 were introduced to alter the susceptibility of NC protein to HIV-1 proteinase. While mutating Asn17 to Ala resulted in a protein which was processed in a manner similar to that of the wild type, mutating it to Phe or Leu resulted in proteins which were processed at a substantially higher rate at this site than the wild type. Mutation of Asn17 to Lys or Gly resulted in proteins which were very poorly cleaved at this site. Oligopeptides containing the same amino acid substitutions at the cleavage site of the proximal zinc finger domain were also tested as substrates of the proteinase, and the kinetic parameters agreed well with the semiquantitative results obtained with the protein substrates.
Assuntos
Substituição de Aminoácidos/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Protease de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas Virais , Sequência de Aminoácidos , Análise Mutacional de DNA , HIV-1/enzimologia , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
Assuntos
Endopeptidases/metabolismo , Proteínas de Escherichia coli , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cinética , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The CCAAT/enhancer-binding proteins (C/EBPs) are basic leucine zipper transcription factors that play important roles in regulating cell growth and differentiation. C/EBP proteins form leucine zipper-mediated homodimers but are also capable of heterodimerizing with other C/EBPs in vitro. Here we show that C/EBPbeta occurs predominantly as a heterodimer that displays rapid mobility in gel shift assays. Biochemical fractionation and antibody supershift assays demonstrate that the C/EBPbeta heterodimeric partner is C/EBPgamma (Ig/EBP), a C/EBP protein that has been implicated as an inhibitor of other family members. Although most cell types express C/EBPbeta.C/EBPgamma heterodimers, macrophages contain a C/EBPbeta partner that is serologically distinct from C/EBPgamma. We found that C/EBPgamma blocked the ability of C/EBPbeta and C/EBPgamma to activate a reporter gene in L cell fibroblasts but did not inhibit a chimeric C/EBPbeta protein containing the GCN4 leucine zipper. Repression by C/EBPgamma occurs at the level of transactivation and requires heterodimerization with the C/EBP partner. C/EBPgamma was an ineffective repressor in HepG2 hepatoma cells despite forming C/EBP heterodimers, and C/EBPalpha was not effectively inhibited in either L or HepG2 cells. Our findings demonstrate that C/EBPgamma modulates C/EBP activity in a cell- and isoform-specific manner.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/química , Fatores de Transcrição , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína delta de Ligação ao Facilitador CCAAT , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Linhagem Celular , DNA/metabolismo , Dimerização , Humanos , Camundongos , Proteínas Repressoras/fisiologiaRESUMO
Caspases are important mediators of apoptotic cell death. Several cellular protein substrates of caspases contain potential phosphorylation site(s) at the cleavage-site region, and some of these sites have been verified to be phosphorylated. Since phosphorylation may affect substantially the substrate susceptibility towards proteolysis, phosphorylated, non-phosphorylated and substituted oligopeptides representing such cleavage sites were studied as substrates of apoptotic caspases 3, 7 and 8. Peptides containing phosphorylated serine residues at P4 and P1' positions were found to be substantially less susceptible towards proteolysis as compared with the serine-containing analogues, while phosphoserine at P3 did not have a substantial effect. P1 serine as well as P1-phosphorylated, serine-containing analogues of an oligopeptide representing the poly(ADP-ribose) polymerase cleavage site of caspase-3 were not hydrolysed by any of these enzymes, whereas the P1 aspartate-containing peptides were efficiently hydrolysed. These findings were interpreted with the aid of molecular modelling. Our results suggest that cleavage-site phosphorylation in certain positions could be disadvantageous or detrimental with respect to cleavability by caspases. Cleavage-site phosphorylation may therefore provide a regulatory mechanism to protect substrates from caspase-mediated degradation.
Assuntos
Caspases/metabolismo , Ácido Aspártico/metabolismo , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Humanos , Cinética , Modelos Moleculares , Fosforilação , Serina/metabolismo , Especificidade por SubstratoRESUMO
Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4 degrees C. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease.