RESUMO
Dystroglycan is frequently lost in adenocarcinoma, but the mechanisms and consequences are poorly understood. We report an analysis of ß-dystroglycan in prostate cancer in human tissue samples and in LNCaP cells in vitro. There is progressive loss of ß-dystroglycan immunoreactivity from basal and lateral surfaces of prostate epithelia which correlates significantly with increasing Gleason grade. In about half of matched bone metastases there is significant dystroglycan re-expression. In tumour tissue and in LNCaP cells there is also a tyrosine phosphorylation-dependent translocation of ß-dystroglycan to the nucleus. Analysis of gene expression data by microarray, reveals that nuclear targeting of ß-dystroglycan in LNCaP cells alters the transcription of relatively few genes, the most unregulated being the transcription factor ETV1. These data suggest that proteolysis, tyrosine phosphorylation and translocation of dystroglycan to the nucleus resulting in altered gene transcription could be important mechanisms in the progression of prostate cancer.
Assuntos
Androgênios/farmacologia , Núcleo Celular/metabolismo , Distroglicanas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Distroglicanas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Masculino , Ácido Mirístico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. METHODS: We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in ß-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of ß-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. CONCLUSIONS: This study suggests that changes in ß-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.
Assuntos
Distroglicanas/fisiologia , Invasividade Neoplásica/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Masculino , Neoplasias da Próstata/fisiopatologia , Células Tumorais CultivadasRESUMO
AIMS: Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. METHODS AND RESULTS: Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. CONCLUSIONS: There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.
Assuntos
Carcinoma/metabolismo , Distroglicanas/metabolismo , Linhagem Celular Tumoral , Epitélio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Modelos Biológicos , Análise Serial de TecidosRESUMO
Dystroglycan is a cell adhesion molecule that interacts with ezrin family proteins and also components of the extracellular signal-regulated kinase pathway. Ezrin and extracellular signal-regulated kinase are both involved in aspects of the cell division cycle. We therefore examined the role of dystroglycan during cytokinesis. Endogenous dystroglycan colocalised with ezrin at the cleavage furrow and midbody during cytokinesis in REF52 cells. Live cell imaging of green fluorescent protein-tagged dystroglycan in Swiss 3T3 and Hela cells revealed a similar localisation. Live cell imaging of a dystroglycan lacking its cytoplasmic domain revealed an even membrane localisation but no cleavage furrow or midbody localisation. Deletion of a previously identified ezrin-binding site in the dystroglycan cytoplasmic domain however only resulted in a slight reduction in cleavage furrow localisation but loss of midbody staining. There was no apparent cytokinetic defect in cells depleted for dystroglycan, however apoptosis levels were considerably higher in dystroglycan knockdown cells. Cell cycle analysis showed a delay in G2/M transition, possibly caused by a more than 50% reduction in extracellular signal-regulated kinase levels in the knockdown cells. Dystroglycan may therefore not only have a role in organising the contractile ring through direct or indirect associations with actin, but can also modulate the cell cycle by affecting extracellular signal-regulated kinase levels.
Assuntos
Citocinese , Distroglicanas/fisiologia , Citoesqueleto de Actina/química , Animais , Ciclo Celular , Distroglicanas/análise , Distroglicanas/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Camundongos , Estrutura Terciária de Proteína , Células Swiss 3T3RESUMO
BACKGROUND: The Polycomb Group protein EZH2 is implicated in prostate cancer progression. EZH2 promotes prostate cancer cell proliferation and invasiveness. We describe a link between EZH2 function and actin polymerization in prostate cancer cells. METHODS: Nuclear and cytoplasmic EZH2 expression in benign and malignant prostate tissue samples was assessed. An association between EZH2 function and actin polymerization in prostate cancer cells was investigated using siRNA-mediated knock-down of EZH2. Effects of EZH2 knock-down on actin polymerization dynamics were analyzed biochemically using immunoblot analysis of cell lysate fractions, and morphologically using immunocytochemistry. RESULTS: Cytoplasmic EZH2 is expressed at low levels in benign prostate epithelial cells and over-expressed in prostate cancer cells. Cytoplasmic EZH2 expression levels correlate with nuclear EZH2 expression in prostate cancer samples. Knock-down of EZH2 in PC3 prostate cancer cells increases the amount of F-actin polymerization, cell size, and formation of actin-rich filaments. CONCLUSIONS: Cytoplasmic EZH2 is over-expressed in prostate cancer cells. EZH2 function promotes a reduction in the pool of insoluble F-actin in invasive prostate cancer cells. EZH2 may regulate actin polymerization dynamics and thereby promote prostate cancer cell motility and invasiveness.
Assuntos
Actinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Tamanho Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Inativação Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Complexo Repressor Polycomb 2 , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Dystroglycan, a ubiquitous membrane-spanning cell adhesion molecule, is a crucial link between the actin cytoskeleton and the extracellular matrix. With a wide expression pattern and multiple interacting proteins, not only is dystroglycan now thought to be important as a structural molecule but also new research has suggested that it has a role in cell signalling, cytoskeleton reorganization and as a potential tumour suppressor.
Assuntos
Distroglicanas/metabolismo , Plaquinas/metabolismo , Transdução de Sinais/fisiologia , Actinas/metabolismo , Animais , Citoesqueleto/metabolismo , Distroglicanas/química , Matriz Extracelular/metabolismo , Humanos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Plaquinas/químicaRESUMO
Spectrin family proteins represent an important group of actin-bundling and membrane-anchoring proteins found in diverse structures from yeast to man. Arising from a common ancestral alpha-actinin gene through duplications and rearrangements, the family has increased to include the spectrins and dystrophin/utrophin. The spectrin family is characterized by the presence of spectrin repeats, actin binding domains, and EF hands. With increasing divergence, new domains and functions have been added such that spectrin and dystrophin also contain specialized protein-protein interaction motifs and regions for interaction with membranes and phospholipids. The acquisition of new domains also increased the functional complexity of the family such that the proteins perform a range of tasks way beyond the simple bundling of actin filaments by alpha-actinin in S. pombe. We discuss the evolutionary, structural, functional, and regulatory roles of the spectrin family of proteins and describe some of the disease traits associated with loss of spectrin family protein function.
Assuntos
Actinina/química , Distrofina/química , Espectrina/química , Actinina/fisiologia , Sequência de Aminoácidos , Animais , Distrofina/fisiologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Humanos , Ligação Proteica , Espectrina/fisiologiaRESUMO
Dystroglycan is part of an adhesion receptor complex linking the extracellular matrix to the actin cytoskeleton. Previous studies have implicated dystroglycan in basement membrane formation and as a crucial link between dystrophin and laminin in muscle. We report here a further novel function for dystroglycan which appears to be in addition to its role as an adhesion molecule. beta-dystroglycan has been localized to microvilli structures in a number of cell types where it associates with the cytoskeletal adaptor ezrin, through which it is able to modulate the actin cytoskeleton and induce peripheral filopodia and microvilli. Ezrin is able to interact with dystroglycan through a cluster of basic residues in the juxtamembrane region of dystroglycan, and mutation of these residues both prevents ezrin binding and the induction of actin-rich surface protrusions. These studies reveal novel functions and additional signalling roles for dystroglycan, raising the possibility of new avenues for therapeutic intervention in diseases such as Duchenne muscular dystrophy.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Distroglicanas/metabolismo , Fosfoproteínas/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Proteínas do Citoesqueleto , Distroglicanas/genética , Genes Reporter , Humanos , Distrofia Muscular de Duchenne/metabolismo , Mutação , Fosfoproteínas/genética , Ligação ProteicaRESUMO
Dystrophin and the dystrophin-associated protein complex (DAPC) have recently been implicated in cell signalling events. These proteins are ideally placed to transduce signals from the extracellular matrix (ECM) to the cytoskeleton. Here we show that beta-dystroglycan is tyrosine-phosphorylated in C2/C4 mouse myotubes. Tyrosine phosphorylation was detected by mobility shifts on SDS-polyacrylamide gels (SDS-PAGE) and confirmed by immunoprecipitation and two-dimensional gel electrophoresis. The potential functional significance of this tyrosine phosphorylation was investigated using peptide 'SPOTs' assays. Phosphorylation of tyrosine in the 15 most C-terminal amino acids of beta-dystroglycan disrupts its interaction with dystrophin. The tyrosine residue in beta-dystroglycan's WW-binding motif PPPY appears to be the most crucial in disrupting the beta-dystroglycan-dystrophin interaction. beta-dystroglycan forms the essential link between dystrophin and the rest of the DAPC. This regulation by tyrosine phosphorylation may have implications in the pathogenesis and treatment of Duchenne's muscular dystrophy (DMD).
Assuntos
Proteínas do Citoesqueleto/metabolismo , Distrofina/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Diferenciação Celular , Linhagem Celular , Distroglicanas , Distrofina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Desenvolvimento Muscular , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Fosforilação , Fosfotirosina/metabolismo , Ligação ProteicaRESUMO
Integrins play an important role in tumour progression by influencing cellular responses and matrix-dependent adhesion. However, the regulation of matrix-dependent adhesion assembly in epithelial cells is poorly understood. We have investigated the integrin and signalling requirements of cell-matrix adhesion assembly in colon carcinoma cells after plating on fibronectin. Adhesion assembly in these, and in the adenoma cells from which they were derived, was largely dependent on alpha v beta 6 integrin and required phosphorylation of FAK on tyrosine-397. The rate of fibronectin-induced adhesion assembly and the expression of both alpha v beta 6 integrin and FAK were increased during the adenoma-to-carcinoma transition. The matrix-dependent adhesion assembly process, particularly the final stages of complex protrusion that is required for optimal cell spreading, required the activity of extracellular signal-regulated kinase (ERK). Furthermore, phosphorylated ERK was targeted to newly forming cell--matrix adhesions in the carcinoma cells but not the adenoma cells, and inhibition of FAK--tyrosine-397 phosphorylation or MEK suppressed the appearance of phosphorylated ERK at peripheral sites. In addition, inhibition of MEK--ERK activation blocked the formation of peripheral actin microspikes that were necessary for the protrusive phase of cell-matrix adhesion assembly. Thus, MEK--ERK--dependent peripheral actin re-organization is required for the full development of integrin-induced adhesions and this pathway is stimulated in an in vitro model of colon cancer progression.
Assuntos
Adenoma/patologia , Antígenos de Neoplasias , Moléculas de Adesão Celular/fisiologia , Neoplasias do Colo/patologia , Células Epiteliais/patologia , Integrinas/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Proteínas Tirosina Quinases/fisiologia , Actinas/metabolismo , Adenoma/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Transformada , Neoplasias do Colo/metabolismo , Progressão da Doença , Células Epiteliais/metabolismo , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/fisiologia , Humanos , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The notion of dystroglycan as a simple laminin-binding receptor is increasingly being challenged. New roles and new binding partners are continually emerging. Recent structural advances have provided exciting new insights into the precise molecular interactions between dystroglycan and other key components of the dystroglycan complex. Coupled with an increasing understanding of dystroglycan function at the molecular level, we are finally beginning to probe the complexities of dystroglycan, not only in disease, but in development, adhesion and signalling.
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Animais , Adesão Celular , Divisão Celular , Citoesqueleto/metabolismo , Distroglicanas , Laminina/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Integrin engagement generates cellular signals leading to the recruitment of structural and signalling molecules which, in concert with rearrangements of the actin cytoskeleton, leads to the formation of focal adhesion complexes. Using antisera reactive either with total ERK or with phosphorylated/activated forms of ERK, in rat embryo fibroblasts and embryonic avian cells that express v-Src, we found that active ERK is targeted to newly forming focal adhesions after integrin engagement or activation of v-Src. UO126, an inhibitor of MAP kinase kinase 1 (MEK1), suppressed focal adhesion targeting of active ERK and cell spreading. Also, integrin engagement and v-Src induced myosin light chain kinase (MLCK)-dependent phosphorylation of myosin light chain downstream of the MEK/ERK pathway, and MLCK and myosin activities are required for the focal adhesion targeting of ERK. The translocation of active ERK to newly forming focal adhesions may direct specificity towards appropriate downstream targets that influence adhesion assembly. These findings support a role for ERK in the regulation of the adhesion/cytoskeletal network and provide an explanation for the role of ERK in cell motility.
Assuntos
Integrinas/metabolismo , Junções Intercelulares/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Fosfoproteínas/metabolismo , Animais , Transporte Biológico , Adesão Celular , Compartimento Celular , Embrião de Galinha , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , MAP Quinase Quinase 1 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , RatosRESUMO
Many cell adhesion-dependent processes are regulated by tyrosine phosphorylation. In order to investigate the role of tyrosine phosphorylation of the utrophin-dystroglycan complex we treated suspended or adherent cultures of HeLa cells with peroxyvanadate and immunoprecipitated (beta)-dystroglycan and utrophin from cell extracts. Western blotting of (&bgr;)-dystroglycan and utrophin revealed adhesion- and peroxyvanadate-dependent mobility shifts which were recognised by anti-phospho-tyrosine antibodies. Using maltose binding protein fusion constructs to the carboxy-terminal domains of utrophin we were able to demonstrate specific interactions between the WW, EF and ZZ domains of utrophin and (beta)-dystroglycan by co-immunoprecipitation with endogenous (beta)-dystroglycan. In extracts from cells treated with peroxyvanadate, where endogenous (beta)-dystroglycan was tyrosine phosphorylated, (beta)-dystroglycan was no longer co-immunoprecipitated with utrophin fusion constructs. Peptide 'SPOTs' assays confirmed that tyrosine phosphorylation of (beta)-dystroglycan regulated the binding of utrophin. The phosphorylated tyrosine was identified as Y(892) in the (beta)-dystroglycan WW domain binding motif PPxY thus demonstrating the physiological regulation of the (beta)-dystroglycan/utrophin interaction by adhesion-dependent tyrosine phosphorylation.
Assuntos
Adesão Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/efeitos dos fármacos , Distroglicanas , Células HeLa , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Fosforilação , Estrutura Terciária de Proteína/fisiologia , Utrofina , Vanadatos/farmacologiaRESUMO
Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J(CC) = 28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1J(N,CO) and 1,2J(N,CA) coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.
Assuntos
Magnetismo , Ressonância Magnética Nuclear Biomolecular/métodos , Algoritmos , Animais , Proteínas de Ligação ao Cálcio/química , Isótopos de Carbono , Processamento Eletrônico de Dados , Humanos , Proteínas dos Microfilamentos , Isótopos de Nitrogênio , Estrutura Terciária de Proteína , Fatores de Tempo , CalponinasRESUMO
Utrophin is a close homologue of dystrophin, the protein defective in Duchenne muscular dystrophy. Like dystrophin, it is composed of three regions: an N-terminal region that binds actin filaments, a large central region with triple coiled-coil repeats, and a C-terminal region that interacts with components in the dystroglycan protein complex at the plasma membrane. The N-terminal actin-binding region consists of two calponin homology domains and is related to the actin-binding domains of a superfamily of proteins including alpha-actinin, spectrin and fimbrin. Here, we present the 2.0 A structure of the second calponin homology domain of utrophin solved by X-ray crystallography, and compare it to the other calponin homology domains previously determined from spectrin and fimbrin.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas do Citoesqueleto/química , Distrofina/análogos & derivados , Proteínas de Membrana/química , Actinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Filogenia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Alinhamento de Sequência , Espectrina/química , Utrofina , CalponinasRESUMO
BACKGROUND: Utrophin is a large multidomain protein that belongs to a superfamily of actin-binding proteins, which includes dystrophin, alpha-actinin, beta-spectrin, fimbrin, filamin and plectin. All the members of this family contain a common actin-binding region at their N termini and perform a wide variety of roles associated with the actin cytoskeleton. Utrophin is the autosomal homologue of dystrophin, the protein defective in the X-linked Duchenne and Becker muscular dystrophies, and upregulation of utrophin has been suggested as a potential therapy for muscular dystrophy patients. RESULTS: The structure of the actin-binding region of utrophin, consisting of two calponin-homology (CH) domains, has been solved at 3.0 A resolution. It is composed of an antiparallel dimer with each of the monomers being present in an extended dumbell shape and the two CH domains being separated by a long central helix. This extended conformation is in sharp contrast to the compact monomer structure of the N-terminal actin-binding region of fimbrin. CONCLUSIONS: The crystal structure of the actin-binding region of utrophin suggests that these actin-binding domains may be more flexible than was previously thought and that this flexibility may allow domain reorganisation and play a role in the actin-binding mechanism. Thus utrophin could possibly bind to actin in an extended conformation so that the sites previously identified as being important for actin binding may be directly involved in this interaction.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Distrofina/química , Distrofina/genética , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Distrofias Musculares/genética , Distrofias Musculares/terapia , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Software , UtrofinaRESUMO
Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin-F-actin binding and was shown to recognize a predicted helical region at residues 13-22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.
Assuntos
Actinas/metabolismo , Anticorpos Monoclonais/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Proteínas do Citoesqueleto/química , Mapeamento de Epitopos , Proteínas de Membrana/química , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Coelhos , UtrofinaAssuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ciclo Celular , Estrutura Terciária de Proteína , Animais , Sítios de Ligação , Proteínas Ativadoras de GTPase , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , CalponinasRESUMO
A sequence motif of about 100 amino acids, termed the 'calponin homology domain' has been suggested to confer actin binding to a variety of cytoskeletal and signalling molecules. Here we analyse and compare the sequences of all calponin homology domain-containing proteins identified to date. We propose that single calponin homology domains do not confer actin-binding per se and that the actin-binding motifs of cross-linking proteins, which comprise two disparate calponin homology domains, represent a unique protein module.