RESUMO
Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona Desacetilases/fisiologia , Proteínas Repressoras/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Complexo Correpressor Histona Desacetilase e Sin3 , Ativação Transcricional , TransfecçãoRESUMO
Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.
Assuntos
Glutationa/análogos & derivados , Mercaptoetanol , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Compostos Nitrosos/metabolismo , Proteínas/metabolismo , S-Nitrosotióis , Animais , Biotinilação , Cerebelo/química , Genes ras , Glutationa/metabolismo , Hidrazinas/metabolismo , Immunoblotting , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxidos de Nitrogênio , Proteoma/genética , Proteoma/metabolismo , Ratos , S-NitrosoglutationaRESUMO
The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.
Assuntos
Neutrófilos/enzimologia , Oxirredutases/sangue , Oxirredutases/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/farmacologia , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação , Grupo dos Citocromos b/efeitos dos fármacos , Grupo dos Citocromos b/metabolismo , Membranas Artificiais , Oxirredução , Fosfoproteínas/química , Estrutura Terciária de Proteína , Superóxidos/metabolismo , SuínosRESUMO
Eighteen histone deacetylases (HDACs) are present in humans, categorized into two groups: zinc-dependent enzymes (HDAC1-11) and NAD(+)-dependent enzymes (sirtuins 1-7). Among zinc-dependent HDACs, HDAC6 is unique. It has a cytoplasmic localization, two catalytic sites, a ubiquitin-binding site, and it selectively deacetylases alpha-tubulin and Hsp90. Here, we report the discovery that the redox regulatory proteins, peroxiredoxin (Prx) I and Prx II are specific targets of HDAC6. Prx are antioxidants enzymes whose main function is H(2)O(2) reduction. Prx are elevated in many cancers and neurodegenerative diseases. The acetylated form of Prx accumulates in the absence of an active HDAC6. Acetylation of Prx increases its reducing activity, its resistance to superoxidation, and its resistance to transition to high-molecular-mass complexes. Thus, HDAC6 and Prx are targets for modulating intracellular redox status in therapeutic strategies for disorders as disparate as cancers and neurodegenerative diseases.
Assuntos
Histona Desacetilases/metabolismo , Peroxirredoxinas/metabolismo , Acetilação , Linhagem Celular Tumoral , Desacetilase 6 de Histona , Histona Desacetilases/análise , Humanos , Oxirredução , Estresse Oxidativo , Peróxidos/metabolismoRESUMO
During terminal differentiation, epithelia become columnar and develop specialized apical membrane structures (microvilli) and functions (regulated endocytosis and exocytosis). Using a clonal intercalated epithelial cell line, we found that high seeding density induced these characteristics, whereas low density seeding maintained a protoepithelial state. When cells were plated at low density, but on the extracellular matrix of high density cells, they converted to the more differentiated phenotype. The extracellular matrix (ECM) protein responsible for this activity was purified and found to be a large 230-kD protein, which we termed hensin. High density seeding caused hensin to be polymerized and deposited in the extracellular matrix, and only this form of hensin was able to induce terminal differentiation. Antibodies to hensin blocked the change in phenotype. However, its purification to homogeneity resulted in loss of activity, suggesting that an additional protein might be necessary for induction of terminal differentiation. Here, we found that a 29-kD protein specifically associates with hensin in the ECM. Addition of purified p29 restored the activity of homogenously purified hensin. Mass fingerprinting identified p29 as galectin 3. Purified recombinant galectin 3 was able to bind to hensin and to polymerize it in vitro. Seeding cells at high density induced secretion of galectin 3 into the ECM where it bundled hensin. Hence, the high density state causes a secretion of a protein that acts on another ECM protein to allow the new complex to signal the cell to change its phenotype. This is a new mechanism of inside-out signaling.
Assuntos
Aglutininas , Antígenos de Diferenciação/metabolismo , Células Epiteliais/citologia , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Membrana , Receptores Imunológicos/metabolismo , Receptores de Lipoproteínas , Animais , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Polaridade Celular , Colo/citologia , Proteínas de Ligação a DNA , Células Epiteliais/metabolismo , Galectina 3 , Ligação Proteica , Ratos , Receptores de Superfície Celular , Receptores Depuradores , Receptores Depuradores Classe B , Transdução de Sinais , Proteínas Supressoras de TumorRESUMO
We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.
Assuntos
Proteínas de Transporte/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/metabolismo , Animais , Sequência de Bases , Transporte Biológico/fisiologia , Células CHO/química , Células CHO/fisiologia , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Membrana Celular/química , Membrana Celular/enzimologia , Clonagem Molecular , Cricetinae , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/genética , Complexo de Golgi/ultraestrutura , Leucemia Basofílica Aguda , Mamíferos , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratos , Proteínas SNARE , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Células Tumorais CultivadasRESUMO
We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Ligação Competitiva/fisiologia , Transporte Biológico/fisiologia , Células CHO/química , Células CHO/metabolismo , Células CHO/ultraestrutura , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Fracionamento Celular , Vesículas Revestidas/química , Cricetinae , Citoplasma/química , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/química , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE , Proteínas de Ligação a Fator Solúvel Sensível a N-EtilmaleimidaRESUMO
Progress through the cell cycle is governed by the cyclin-dependent kinases (CDKs), the activation of which requires phosphorylation by the CDK-activating kinase (CAK). In vertebrates, CAK is a trimeric enzyme containing CDK7, cyclin H, and MAT1. CAK from the budding yeast Saccharomyces cerevisiae was identified as an unusual 44-kilodalton protein kinase, Cak1, that is only distantly related to CDKs. Cak1 accounted for most CAK activity in yeast cell lysates, and its activity was constant throughout the cell cycle. The CAK1 gene was essential for cell viability. Thus, the major CAK in S. cerevisiae is distinct from the vertebrate enzyme, suggesting that budding yeast and vertebrates may have evolved different mechanisms of CDK activation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ativação Enzimática , Deleção de Genes , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The Rel-associated protein pp40 is functionally related to I kappa B, an inhibitor of the transcription factor NF-kappa B. Purified pp40 inhibits the DNA binding activity of the NF-kappa B protein complex (p50:p65 heterodimers), p50:c-Rel heteromers, and c-Rel homodimers. The sequence of the complementary DNA encoding pp40 revealed similarity to the gene encoding MAD-3, a protein with mammalian I kappa B-like activity. Protein sequencing of I kappa B purified from rabbit lung confirmed that MAD-3 encodes a protein similar to I kappa B. The sequence similarity between MAD-3 and pp40 includes a casein kinase II and consensus tyrosine phosphorylation site, as well as five repeats of a sequence found in the human erythrocyte protein ankyrin. These results suggest that rel-related transcription factors, which are capable of cytosolic to nuclear translocation, may be held in the cytosol by interaction with related cytoplasmic anchor molecules.
Assuntos
NF-kappa B/antagonistas & inibidores , Fosfoproteínas/genética , Proteínas Oncogênicas de Retroviridae/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , Sondas de DNA , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Oncogênicas v-rel , Fases de Leitura Aberta , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Telomeres are multifunctional elements that shield chromosome ends from degradation and end-to-end fusions, prevent activation of DNA damage checkpoints, and modulate the maintenance of telomeric DNA by telomerase. A major protein component of human telomeres has been identified and cloned. This factor, TRF, contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. Immunofluorescent labeling shows that TRF specifically colocalizes with telomeric DNA in human interphase cells and is located at chromosome ends during metaphase. The presence of TRF along the telomeric TTAGGG repeat array demonstrates that human telomeres form a specialized nucleoprotein complex.
Assuntos
Proteínas de Ligação a DNA/química , Telômero/química , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/química , Clonagem Molecular , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Células HeLa , Humanos , Interfase , Metáfase , Dados de Sequência Molecular , Peso Molecular , Sequências Repetitivas de Ácido Nucleico , Alinhamento de Sequência , TransfecçãoRESUMO
Acetylation of core histone tails plays a fundamental role in transcription regulation. In addition to acetylation, other posttranslational modifications, such as phosphorylation and methylation, occur in core histone tails. Here, we report the purification, molecular identification, and functional characterization of a histone H4-specific methyltransferase PRMT1, a protein arginine methyltransferase. PRMT1 specifically methylates arginine 3 (Arg 3) of H4 in vitro and in vivo. Methylation of Arg 3 by PRMT1 facilitates subsequent acetylation of H4 tails by p300. However, acetylation of H4 inhibits its methylation by PRMT1. Most important, a mutation in the S-adenosyl-l-methionine-binding site of PRMT1 substantially crippled its nuclear receptor coactivator activity. Our finding reveals Arg 3 of H4 as a novel methylation site by PRMT1 and indicates that Arg 3 methylation plays an important role in transcriptional regulation.
Assuntos
Arginina/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Receptores Androgênicos/metabolismo , Ativação Transcricional , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Células HeLa , Histonas/química , Humanos , Ácidos Hidroxâmicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Dados de Sequência Molecular , Mutação , Oócitos , Proteína-Arginina N-Metiltransferases , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , XenopusRESUMO
Platelet-derived growth factor (PDGF) has been previously shown to be homologous to the transforming gene of simian sarcoma virus (v-sis), and inappropriate expression of the cellular counterpart of the v-sis gene (c-sis) has been implicated in the generation of mesenchymal tumors. The U-2 OS human osteosarcoma line was shown to contain multiple c-sis transcripts. Immunoprecipitation experiments with antiserum to PDGF identified a variety of polypeptides ranging in size from 18,000 to 165,000 daltons that were immunoprecipitated specifically from U-2 OS cell extracts. The osteosarcoma also was shown to secrete a 29,000-dalton protein having the serological and structural characteristics of PDGF.
Assuntos
Proteínas de Neoplasias/genética , Oncogenes , Osteossarcoma/genética , Fator de Crescimento Derivado de Plaquetas , Transcrição Gênica , Linhagem Celular , Replicação do DNA , Humanos , Peso Molecular , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA MensageiroRESUMO
Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/enzimologia , Clonagem Molecular , DNA Complementar , Drosophila , Proteínas de Drosophila , Ativação Enzimática , Humanos , Lipossomos/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Recombinantes/metabolismo , OvinosRESUMO
Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas de Homeodomínio , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/isolamento & purificação , Dano ao DNA , DNA Fúngico/biossíntese , DNA Fúngico/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Antígenos de Histocompatibilidade Menor , Peso Molecular , Proteína de Replicação C , Saccharomyces cerevisiae/genéticaRESUMO
Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)) is a well-known messenger molecule that releases calcium from intracellular stores. Homologues with up to six phosphates have been characterized and recently, homologues with seven or eight phosphate groups, including pyrophosphates, have been identified. These homologues are diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)) and bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP(4)/InsP(8)) [1], the rapid turnover of which [2] is regulated by calcium [2] and adrenergic receptor activity [3]. It has been proposed that the high-energy pyrophosphates might participate in protein phosphorylation [4]. We have purified InsP(6) kinase [5] and PP-InsP(5) kinase [6], both of which display ATP synthase activity, transferring phosphate to ADP. Here, we report the cloning of two mammalian InsP(6) kinases and a yeast InsP(6) kinase. Furthermore, we show that the yeast protein, ArgRIII, is an inositol-polyphosphate kinase that can convert InsP(3) to InsP(4), InsP(5) and InsP(6). We have identified a new family of highly conserved inositol-polyphosphate kinases that contain a newly identified, unique consensus sequence.
Assuntos
Proteínas Fúngicas/metabolismo , Fosfatos de Inositol/biossíntese , Família Multigênica , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Sequência Consenso , Feminino , Proteínas Fúngicas/química , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Fosfotransferases (Aceptor do Grupo Fosfato)/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas Sanguíneas/genética , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/metabolismo , Galinhas , Ativação Enzimática , Ácidos Graxos/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Fosfoproteínas/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Suínos , Vesículas Transportadoras/metabolismo , Tirosina/metabolismoRESUMO
Development of the embryonic kidney results from reciprocal signaling between the ureteric bud and the metanephric mesenchyme. To identify the signaling molecules, we developed an assay in which metanephric mesenchymes are rescued from apoptosis by factors secreted from ureteric bud cells (UB cells). Purification and sequencing of one such factor identified the tissue inhibitor of metalloproteinase-2 (TIMP-2) as a metanephric mesenchymal growth factor. Growth activity was unlikely due to TIMP-2 inhibition of matrix metalloproteinases because ilomastat, a synthetic inhibitor of these enzymes, had no mesenchymal growth action. TIMP-2 was also involved in morphogenesis of the ureteric bud, inhibiting its branching and changing the deposition of its basement membrane; these effects were due to TIMP-2 inhibition of matrix metalloproteinases, as they were reproduced by ilomastat. Thus, TIMP-2 regulates kidney development by at least 2 distinct mechanisms. In addition, TIMP-2 was secreted from UB cells by mesenchymal factors that are essential for ureteric bud development. Hence, the mesenchyme synchronizes its own growth with ureteric morphogenesis by stimulating the secretion of TIMP-2 from the ureteric bud.
Assuntos
Epitélio/crescimento & desenvolvimento , Mesoderma , Néfrons/embriologia , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Animais , Apoptose/fisiologia , Sequência de Bases , Linhagem Celular , Ácidos Hidroxâmicos , Indóis/farmacologia , Mesoderma/efeitos dos fármacos , Metaloendopeptidases/antagonistas & inibidores , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Morfogênese , Néfrons/metabolismo , Néfrons/ultraestrutura , Inibidores de Proteases/farmacologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ureter/embriologia , Ureter/metabolismoRESUMO
Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.
Assuntos
Divisão Celular/fisiologia , Proteínas de Drosophila , Inibidores do Crescimento/metabolismo , Histona Desacetilases/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular , Linhagem Celular , DNA Helicases , DNA Complementar/genética , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Inibidores do Crescimento/genética , Células HeLa , Histona Desacetilase 2 , Histona Desacetilases/química , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Homologia de Sequência de Aminoácidos , Complexo Correpressor Histona Desacetilase e Sin3 , Fatores de Transcrição/química , Proteínas Supressoras de TumorRESUMO
We have identified a cellular protein, RBP-2N, a presumed recombinase, as a repressor of transcription. Inhibition of transcription by RBP-2N was dependent on its DNA recognition site and was demonstrated in vitro and in vivo. This repression appears to be general, as transcription mediated by SP1 and Gal4/VP16 was inhibited by RBP-2N. The protein was purified to near homogeneity from human cells on the basis of its binding to a site present in the promoter of the adenovirus pIX gene. The DNA recognition sequence is 5'-TGGGAAAGAA, which is markedly different from the recombination signal sequence originally identified as the target site for this protein. The sequence of the purified protein is 97% identical with that published for the mouse RBP-2N protein. The reported homolog in Drosophila is Suppressor of Hairless. RBP-2N binding sites are present in a number of cellular and viral promoters, so RBP-2N may have a general role in transcriptional repression.