RESUMO
Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short ß-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.
RESUMO
Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, translocation of phospholipase-Cγ1 (PLC-γ1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-γ1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-γ1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-γ1 activation are regulated both independently and additively by recruitment of PLC-γ1 to phosphorylated LAT, by formation of the LAT-Gads-SLP-76-PLC-γ1 tetramer, and by tyrosine phosphorylation of PLC-γ1. The recently solved structure of PLC-γ1 indicates that, in the resting state, several PLC-γ1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-γ1, enabling contact with its membrane substrate, disinhibition of PLC-γ1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.
Assuntos
Fosfolipase C gama , Transdução de Sinais , Linfócitos T , Ativação Enzimática , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Linfócitos T/metabolismoRESUMO
This year marks the 40th anniversary of the initial identification of p53 as a transformation-related Ag, which was the result of our effort to identify an antigenically distinct tumor Ag of a chemically induced mouse tumor and develop a cancer vaccine. Many researchers at the time viewed this effort as folly. Since then, its characterization has progressed from being an attractive cancer vaccine candidate to recognition as a key player in regulating critical pathways controlling the cell cycle and oncogenesis. Advances in molecular immunology and oncology have enhanced the role of p53 in both fields. It is now apparent that p53 plays a critical role in controlling immune recognition and responses in normal tissues as well as the tumor microenvironment. Together with the advances in clinical implementation of p53-based cancer immunotherapy, they highlight the importance of p53 in many areas of basic and translational cancer research.
Assuntos
Imunoterapia/métodos , Proteína Supressora de Tumor p53/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Carcinogênese/imunologia , Ciclo Celular/imunologia , Humanos , Neoplasias/imunologia , Neoplasias/terapia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologiaRESUMO
Because of microbicide noncompliance and lack of a durable, highly effective vaccine, a combined approach might improve HIV prophylaxis. We tested whether a vaccine-microbicide combination would enhance protection against SIV infection in rhesus macaques. Four macaque groups included vaccine only, vaccine-microbicide, microbicide only, and controls. Vaccine groups were primed twice mucosally with replicating adenovirus type 5 host range mutant SIV env/rev, gag, and nef recombinants and boosted twice i.m. with SIV gp120 proteins in alum. Controls and the microbicide-only group received adenovirus type 5 host range mutant empty vector and alum. The microbicide was SAMT-247, a 2-mercaptobenzamide thioester that targets the viral nucleocapsid protein NCp7, causing zinc ejection and preventing RNA encapsidation. Following vaccination, macaques were challenged intravaginally with repeated weekly low doses of SIVmac251 administered 3 h after application of 0.8% SAMT-247 gel (vaccine-microbicide and microbicide groups) or placebo gel (vaccine-only and control groups). The microbicide-only group exhibited potent protection; 10 of 12 macaques remained uninfected following 15 SIV challenges. The vaccine-only group developed strong mucosal and systemic humoral and cellular immunity but did not exhibit delayed acquisition compared with adjuvant controls. However, the vaccine-microbicide group exhibited significant acquisition delay compared with both control and vaccine-only groups, indicating further exploration of the combination strategy is warranted. Impaired protection in the vaccine-microbicide group compared with the microbicide-only group was not attributed to a vaccine-induced increase in SIV target cells. Possible Ab-dependent enhancement will be further investigated. The potent protection provided by SAMT-247 encourages its movement into human clinical trials.
Assuntos
Anti-Infecciosos/farmacologia , Benzamidas/farmacologia , Macaca mulatta/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Adenoviridae/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/imunologia , Células Cultivadas , Feminino , Produtos do Gene gag/imunologia , Vetores Genéticos/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade Humoral/imunologia , Macaca mulatta/virologia , Glicoproteínas de Membrana/imunologia , Projetos Piloto , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.
Assuntos
Fatores de Transcrição NFATC/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Calcineurina , Comunicação Celular , Humanos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-fos , Receptores de Antígenos de Linfócitos T/fisiologia , Especificidade por Substrato , Linfócitos T , Fatores de TranscriçãoRESUMO
The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.
Assuntos
Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Recombinantes , Termodinâmica , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.
Assuntos
Genes Codificadores dos Receptores de Linfócitos T/fisiologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica , Humanos , Células Jurkat , Fosforilação , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
Assuntos
Ativadores de Enzimas/química , Fosfopeptídeos/química , Proteína Fosfatase 2C/química , Bibliotecas de Moléculas Pequenas/química , Ativadores de Enzimas/isolamento & purificação , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Proteína Fosfatase 2C/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Proteína Supressora de Tumor p53/químicaRESUMO
Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.
Assuntos
Metais/metabolismo , Fosfopeptídeos/metabolismo , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Proteína Fosfatase 2C/genética , Homologia de Sequência , Especificidade por SubstratoRESUMO
PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.
Assuntos
Medição da Troca de Deutério , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/metabolismo , Sítios de Ligação , Humanos , Espectrometria de Massas , Conformação ProteicaRESUMO
Protein interactions are fundamental to the proper functioning of cells, and aberrant formation or regulation of protein interactions is at the heart of many diseases, including cancer. The advancement of methods to study the identity, function, and regulation of protein complexes makes possible the understanding of how those complexes malfunction in human diseases. New methodologies in mass spectrometry, microscopy, and protein structural analysis are rapidly advancing the amount and quality of the data, as well as the level of detail that can be obtained from experiments. With this progress, the questions that can be addressed and the biological landscape are changing. This series of minireviews highlights methodological advances and how they have been applied in novel ways to explore the function and regulation of pathways and dynamic networks in cells.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Animais , Humanos , Conformação Proteica , Relação Estrutura-AtividadeAssuntos
Interleucina-8/isolamento & purificação , Monócitos/imunologia , Neutrófilos/imunologia , Peptídeos/genética , Sequência de Aminoácidos , Células Cultivadas , Humanos , Doenças do Sistema Imunitário , Imunidade , Imunidade Inata , Interleucina-1/metabolismo , Interleucina-8/genética , Transtornos Leucocíticos , Lipopolissacarídeos/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The Cdc25A phosphatase positively regulates cell-cycle transitions, is degraded by the proteosome throughout interphase and in response to stress, and is overproduced in human cancers. The kinases targeting Cdc25A for proteolysis during early cell-cycle phases have not been identified, and mechanistic insight into the cause of Cdc25A overproduction in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates Cdc25A to promote its proteolysis in early cell-cycle phases. Phosphorylation by GSK-3beta requires priming of Cdc25A, and this can be catalyzed by polo-like kinase 3 (Plk-3). Importantly, a strong correlation between Cdc25A overproduction and GSK-3beta inactivation was observed in human tumor tissues, indicating that GSK-3beta inactivation may account for Cdc25A overproduction in a subset of human tumors.
Assuntos
Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Neoplasias/enzimologia , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Fosfatases cdc25/metabolismo , Animais , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Ativação Enzimática/efeitos da radiação , Estabilidade Enzimática/efeitos da radiação , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos da radiação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Ligação Proteica/efeitos da radiação , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos da radiação , Radiação Ionizante , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
The p53 tumor suppressor is a critical mediator of the cellular response to stress. The N-terminal transactivation domain of p53 makes protein interactions that promote its function as a transcription factor. Among those cofactors is the histone acetyltransferase p300, which both stabilizes p53 and promotes local chromatin unwinding. Here, we report the nuclear magnetic resonance solution structure of the Taz2 domain of p300 bound to the second transactivation subdomain of p53. In the complex, p53 forms an α-helix between residues 47 and 55 that interacts with the α1-α2-α3 face of Taz2. Mutational analysis indicated several residues in both p53 and Taz2 that are critical for stabilizing the interaction. Finally, further characterization of the complex by isothermal titration calorimetry revealed that complex formation is pH-dependent and releases a bound chloride ion. This study highlights differences in the structures of complexes formed by the two transactivation subdomains of p53 that may be broadly observed and play critical roles in p53 transcriptional activity.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Histona Acetiltransferases/metabolismo , Modelos Moleculares , Proteína Supressora de Tumor p53/metabolismo , Substituição de Aminoácidos , Varredura Diferencial de Calorimetria , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/genética , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias/tratamento farmacológico , Fosfoproteínas Fosfatases/metabolismo , Proteína Supressora de Tumor p53/deficiência , Animais , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Primers do DNA/genética , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Neoplasias/metabolismo , Plasmídeos/genética , Proteína Fosfatase 2C , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/- nor Chk2-/- mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/-Chk2-/- and Chk1+/-Chk2+/- mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.
Assuntos
Ciclo Celular/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Senescência Celular , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Aberrações Cromossômicas , Dano ao DNA , Reparo do DNA , Feminino , Fibroblastos/citologia , Fibroblastos/fisiologia , Deleção de Genes , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genéticaRESUMO
T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Humanos , Células Jurkat , Ativação Linfocitária , Proteínas Oncogênicas/genética , Fosfoproteínas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-vav/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Domínios de Homologia de srcRESUMO
A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.
Assuntos
Antineoplásicos Hormonais/farmacologia , Benzamidas/farmacologia , Neoplasias da Mama/tratamento farmacológico , DNA/metabolismo , Receptor alfa de Estrogênio/fisiologia , Tamoxifeno/farmacologia , Animais , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Camundongos , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Elementos de RespostaRESUMO
The PPM phosphatases require millimolar concentrations of Mg²âº or Mn²âº to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg²âº binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg²âº, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg²âº was bound to the low-affinity site, whether Mg²âº or Mn²âº ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg²âº ion to the low-affinity site.
Assuntos
Domínio Catalítico , Magnésio/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Alanina/química , Sequência de Aminoácidos , Ácido Aspártico/química , Catálise , Células HEK293 , Humanos , Manganês/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Alinhamento de SequênciaRESUMO
Modulation of tumor suppressor activities may provide new opportunities for cancer therapy. Here we show that disruption of the gene Ppm1d encoding Wip1 phosphatase activated the p53 and p16 (also called Ink4a)-p19 (also called ARF) pathways through p38 MAPK signaling and suppressed in vitro transformation of mouse embryo fibroblasts (MEFs) by oncogenes. Disruption of the gene Cdkn2a (encoding p16 and p19), but not of Trp53 (encoding p53), reconstituted cell transformation in Ppm1d-null MEFs. In vivo, deletion of Ppm1d in mice bearing mouse mammary tumor virus (MMTV) promoter-driven oncogenes Erbb2 (also called c-neu) or Hras1 impaired mammary carcinogenesis, whereas reduced expression of p16 and p19 by methylation-induced silencing or inactivation of p38 MAPK correlated with tumor appearance. We conclude that inactivation or depletion of the Wip1 phosphatase with resultant p38 MAPK activation suppresses tumor appearance by modulating the Cdkn2a tumor-suppressor locus.