RESUMO
In-frame mutations in nuclear lamin A/C lead to a multitude of tissue-specific degenerative diseases known as the 'laminopathies'. Previous studies have demonstrated that lamin A/C-null mouse fibroblasts have defects in cell polarisation, suggesting a role for lamin A/C in nucleo-cytoskeletal-cell surface cross-talk. However, this has not been examined in patient fibroblasts expressing modified forms of lamin A/C. Here, we analysed skin fibroblasts from 3 patients with Emery-Dreifuss muscular dystrophy and from 1 with dilated cardiomyopathy. The emerin-lamin A/C interaction was impaired in each mutant cell line. Mutant cells exhibited enhanced cell proliferation, collagen-dependent adhesion, larger numbers of filopodia and smaller cell spread size, compared with control cells. Furthermore, cell migration, speed and polarization were elevated. Mutant cells also showed an enhanced ability to contract collagen gels at early time points, compared with control cells. Phosphotyrosine measurements during cell spreading indicated an initial temporal lag in ERK1/2 activation in our mutant cells, followed by hyper-activation of ERK1/2 at 2 h post cell attachment. Deregulated ERK1/2 activation is linked with cardiomyopathy, cell spreading and proliferation defects. We conclude that a functional emerin-lamin A/C complex is required for cell spreading and proliferation, possibly acting through ERK1/2 signalling.
Assuntos
Fibroblastos/fisiologia , Lamina Tipo A/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Adolescente , Adulto , Animais , Adesão Celular , Ciclo Celular , Movimento Celular , Proliferação de Células , Ativação Enzimática , Feminino , Fibroblastos/citologia , Humanos , Lamina Tipo A/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X-linked Emery-Dreifuss muscular dystrophy (X-EDMD), a neuromuscular condition with an associated life-threatening cardiomyopathy. We have previously reported that emerin is phosphorylated in a cell cycle-dependent manner in human lymphoblastoid cell lines [Ellis et al. (1998) Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the EDMD phenotype. J. Cell Sci. 111, 781-792]. Recently, five residues in human emerin were identified as undergoing cell cycle-dependent phosphorylation using a Xenopus egg mitotic cytosol model system (Hirano et al. (2005) Dissociation of emerin from BAF is regulated through mitotic phosphorylation of emerin in a Xenopus egg cell-free system. J. Biol. Chem.280, 39 925-39 933). In the present paper, recombinant human emerin was purified from a baculovirus-Sf9 heterogeneous expression system, analyzed by protein mass spectrometry and shown to exist in at least four different phosphorylated species, each of which could be dephosphorylated by treatment with alkaline phosphatase. Further analysis identified three phosphopeptides with m/z values of 2191.9 and 2271.7 corresponding to the singly and doubly phosphorylated peptide 158-DSAYQSITHYRPVSASRSS-176, and a m/z of 2396.9 corresponding to the phosphopeptide 47-RLSPPSSSAASSYSFSDLNSTR-68. Sequence analysis confirmed that residue S49 was phosphorylated and also demonstrated that this residue was phosphorylated in interphase. Using an in vitro protein kinase A assay, we observed two phospho-emerin species, one of which was phosphorylated at residue S49. Protein kinase A is thus the first kinase that has been identified to specifically phosphorylate emerin. These results improve our understanding of the molecular mechanisms underlying X-EDMD and point towards possible signalling pathways involved in regulating emerin's functions.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Membrana/química , Proteínas Nucleares/química , Proteínas de Ancoragem à Quinase A , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Serina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mutations in the LMNA gene, which encodes nuclear lamins A and C by alternative splicing, can give rise to Emery-Dreifuss muscular dystrophy. The mechanism by which lamins A and C separately contribute to this molecular phenotype is unknown. To address this question we examined ten LMNA mutations exogenously expressed as lamins A and C in COS-7 cells. Eight of the mutations when expressed in lamin A, exhibited a range of nuclear mislocalisation patterns. However, two mutations (T150P and delQ355) almost completely relocated exogenous lamin A from the nuclear envelope to the cytoplasm, disrupted nuclear envelope reassembly following cell division and altered the protein composition of the mid-body. In contrast, exogenously expressed DsRed2-tagged mutant lamin C constructs were only inserted into the nuclear lamina if co-expressed with any EGFP-tagged lamin A construct, except with one carrying the T150P mutation. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data identify specific functional roles for the emerin-lamin C- and emerin-lamin A- containing protein complexes and is the first report to suggest that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.
Assuntos
Lamina Tipo A/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Animais , Células COS , DNA Complementar/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Lâmina Nuclear/metabolismo , Proteínas Nucleares , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Timopoietinas/genética , Timopoietinas/metabolismo , TransfecçãoRESUMO
Emerin and specific isoforms of nesprin-1 and -2 are nuclear membrane proteins which are binding partners in multi-protein complexes spanning the nuclear envelope. We report here the characterisation of the residues both in emerin and in nesprin-1alpha and -2beta which are involved in their interaction and show that emerin requires nesprin-1 or -2 to retain it at the nuclear membrane. Using several protein-protein interaction methods, we show that residues 368 to 627 of nesprin-1alpha and residues 126 to 219 of nesprin-2beta, which show high homology to one another, both mediate binding to emerin residues 140-176. This region has previously been implicated in binding to F-actin, beta-catenin and lamin A/C suggesting that it is critical for emerin function. Confirmation that these protein domains interact in vivo was shown using GFP-dominant negative assays. Exogenous expression of either of these nesprin fragments in mouse myoblast C2C12 cells displaced endogenous emerin from the nuclear envelope and reduced the targeting of newly synthesised emerin. Furthermore, we are the first to report that emerin mutations which give rise to X-linked Emery-Dreifuss muscular dystrophy, disrupt binding to both nesprin-1alpha and -2beta isoforms, further indicating a role of nesprins in the pathology of Emery-Dreifuss muscular dystrophy.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Proteínas do Citoesqueleto , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Deleção de Sequência , TransfecçãoRESUMO
Id helix-loop-helix (HLH) proteins act as global regulators of metazoan cell fate, cell growth, and differentiation. They heterodimerize with and inhibit the DNA-binding function of members of the basic helix-loop-helix (bHLH) family of transcription factors. Using real time fluorescence microscopy techniques in single living cells, we show here that nuclear pools of chromatin-associated bHLH transcription factor are freely exchangeable and in constant flux. The existence of a dynamic equilibrium between DNA-bound and free bHLH protein is also directly demonstrable in vitro. By contrast, Id protein is not associated with any subcellular, macromolecular structures and displays a more highly mobile, diffuse nuclear-cytoplasmic distribution. When co-expressed with antagonist Id protein, the chromatin-associated sublocalization of bHLH protein is abolished, and there is an accompanying 100-fold increase in its nuclear mobility to a level expected for freely diffusible Id-bHLH heterodimer. These results suggest that nuclear Id protein acts by sequestering pools of transiently diffusing bHLH protein to prevent reassociation with chromatin domains. Such a mechanism would explain how Id proteins are able to overcome the large DNA-binding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect.