RESUMO
Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.
Assuntos
Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativadores de Enzimas/metabolismo , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteína Fosfatase 2/química , Subunidades ProteicasRESUMO
Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre-autophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Fagossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes.
Assuntos
Axônios/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/ultraestrutura , Núcleos da Rafe/citologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Neurônios/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-ets/genética , Ratos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Sinaptossomos/metabolismo , Triptofano Hidroxilase/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas de Ciclo Celular/metabolismo , Humanos , Metilação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.
Assuntos
Anticorpos Monoclonais , Epitopos , Proteínas Proto-Oncogênicas c-myc , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Camundongos , Células NIH 3T3 , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaRESUMO
The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies-including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit-are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B', an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.
Assuntos
Anticorpos/química , Multimerização Proteica , Proteína Fosfatase 2 , Animais , Linhagem Celular , Células HEK293 , Holoenzimas , Humanos , Camundongos , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , CoelhosRESUMO
Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.
Assuntos
Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Leucina/metabolismo , Fosfotirosina/metabolismo , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/efeitos dos fármacos , Reações Cruzadas/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Peptídeos/química , Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vanadatos/farmacologia , Quinases da Família src/metabolismoRESUMO
Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.
Assuntos
Anticorpos/metabolismo , Western Blotting/métodos , Corantes Fluorescentes/administração & dosagem , Proteínas/química , Animais , Biomarcadores , Corantes Fluorescentes/farmacologia , Imunização , Luminescência , Camundongos , Peso Molecular , Proteínas/metabolismo , Coloração e RotulagemRESUMO
The breast cancer-associated protein, BARD1, colocalizes with BRCA1 in nuclear foci in the S phase and after DNA damage, and the two proteins form a stable heterodimer implicated in DNA repair, protein ubiquitination, and control of mRNA processing. BARD1 has a BRCA1-independent proapoptotic activity; however, little is known about its regulation. Here, we show that BARD1 localization and apoptotic activity are regulated by nuclear-cytoplasmic shuttling. We identified a functional CRM1-dependent nuclear export sequence (NES) near the N-terminal RING domain of BARD1. The NES forms part of the BRCA1 dimerization domain, and coexpression of BRCA1 resulted in masking of the NES and nuclear retention of BARD1. In transient expression assays, BARD1 apoptotic activity was stimulated by nuclear export, and both apoptotic function and nuclear export were markedly reduced by BRCA1. Similar findings were obtained for endogenous BARD1. Silencing BRCA1 expression by siRNA, or disrupting the endogenous BARD1/BRCA1 interaction by peptide competition caused a reduction in BARD1 nuclear localization and foci formation, and increased the level of cytoplasmic BARD1 correlating with increased apoptosis. Our findings suggest that BRCA1/BARD1 heterodimer formation is important for optimal nuclear targeting of BARD1 and its role in DNA repair and cell survival.
Assuntos
Proteína BRCA1/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Animais , Apoptose , Sequência de Bases , Mama/citologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Primers do DNA , Dimerização , Células Epiteliais , Feminino , Citometria de Fluxo , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Transporte Proteico , Fase S , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Dedos de ZincoRESUMO
BACKGROUND: Disease-linked missense mutations can alter a protein's function with fatal consequences for the affected individual. How a single amino acid substitution in a protein affects its properties, is difficult to study in the context of the cellular proteome, because mutant proteins can often not be traced in cells due to the lack of mutation-specific detection tools. Antibodies, however, with their exquisite epitope specificity permit the detection of single amino acid substitutions but are not available for the vast majority of disease-causing mutant proteins. One of the most frequently missense-mutated human genes is the LMNA gene coding for A-type lamins. Mutations in LMNA cause phenotypically heterogenous, mostly autosomal-dominant inherited diseases, termed laminopathies. The molecular mechanisms underlying the phenotypic heterogeneity of laminopathies, however, are not well understood. Hence, the goal of this study was the development of monoclonal antibodies specific for disease-linked point-mutant A-type lamins. METHODOLOGY/PRINCIPAL FINDINGS: Using two different approaches of antigen presentation, namely KLH-coupled peptides and the display of a complete protein domain fused to the Hepatitis B virus capsid protein, we developed monoclonal antibodies against two disease-associated lamin A/C mutants. Both antibodies display exquisite specificity for the respective mutant proteins. We show that with the help of these novel antibodies it is now possible for the first time to study specifically the properties of the mutant proteins in primary patient cells in the background of wild-type protein. CONCLUSIONS: We report here the development of two point-mutant specific antibodies against A-type lamins. While synthetic peptides may be the prime choice of antigen, our results show that a given target sequence may have to be presented in alternative ways to ensure the induction of a mutant-specific immune response. Point-mutant specific antibodies will represent valuable tools for basic and clinical research on a number of hereditary as well as acquired diseases caused by dominant missense mutations.
Assuntos
Substituição de Aminoácidos/genética , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutação Puntual/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Epitopos/química , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lamina Tipo A/química , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Conformação Proteica , Transporte Proteico , Pele/patologiaRESUMO
Lamin A, a protein component of the nuclear lamina, is synthesized as a precursor named prelamin A, whose multi-step maturation process involves different protein intermediates. As demonstrated in laminopathies such as familial partial lipodystrophy, mandibuloacral dysplasia, Werner syndrome, Hutchinson-Gilford progeria syndrome and restrictive dermopathy, failure of prelamin A processing results in the accumulation of lamin A protein precursors inside the nucleus which dominantly produces aberrant chromatin structure. To understand if nuclear lamina components may be involved in prelamin A chromatin remodeling effects, we investigated barrier-to-autointegration factor (BAF) localization and expression in prelamin A accumulating cells. BAF is a DNA-binding protein that interacts directly with histones, lamins and LEM-domain proteins and has roles in chromatin structure, mitosis and gene regulation. In this study, we show that the BAF heterogeneous localization between nucleus and cytoplasm observed in HEK293 cycling cells changes in response to prelamin A accumulation. In particular, we observed that the accumulation of lamin A, non-farnesylated prelamin A and farnesylated carboxymethylated lamin A precursors induce BAF nuclear translocation. Moreover, we show that the treatment of human fibroblasts with prelamin A interfering drugs results in similar changes. Finally, we report that the accumulation of progerin, a truncated form of farnesylated and carboxymethylated prelamin A identified in Hutchinson-Gilford progeria syndrome cells, induces BAF recruitment in the nucleus. These findings are supported by coimmunoprecipitation of prelamin A or progerin with BAF in vivo and suggest that BAF could mediate prelamin A-induced chromatin effects.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Humanos , Lamina Tipo A , Progéria/metabolismo , Progéria/patologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , RatosRESUMO
The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint.