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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.

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 helixloop-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 chromatinassociated 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 DNAbinding free energy of bHLH proteins that is necessary to accomplish their inhibitory effect.Id proteins function as global regulators of cell fate determination. They play a pivotal role in the coordinate regulation of gene expression during cell growth/cell cycle control, differentiation, and tumorigenesis (reviewed in Refs. 1 and 2). Recent studies have also highlighted their role in cellular senescence (3, 4) and in cell fate decisions in cells of specialized lineages such as lymphocytes (5) (reviewed in Ref. 6), vascular endothelial cells (7,8), and neuronal cells (7, 9). The four members of the Id protein family (Id1-Id4) function by directly associating with and modulating the activity of several families of transcriptional regulators (1, 10 -12). However, compelling biochemical and genetic data implicate members of the ubiquitously expressed Class A, basic helix-loop-helix (bHLH) 1 family of "E proteins" as the most important heterodimerization targets for Id proteins in the coordinate regulation of gene expression during cell fate determination (1, 2, 13, 14). In mammals, there are three E protein family genes, E2A (encoding three alternatively spliced variants, E12, E47, and E2-5), E2-2 (ITF2), and HEB (14). The E proteins bind to a consensus "E-box" recognition sequence, present in the transcriptional control regions of numerous cellular genes, either as a homodimer or, more commonly, as a heterodimeric partner with a member of the much larger family of tissue-specific, Class B bHLH proteins. Id proteins lack a basic, DNA-binding domain, and they heterodimerize avidly (via their HLH domain) with bHLH E proteins (13) to prevent the latter from binding to DNA (1,14). Since E proteins are obligate heterodimerization partners for tissue-specific bHLH proteins, this provides a common mechani...

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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-DSAYQSITHYRPV SASRSS-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.Abbreviations AD-EDMD, autosomal dominant Emery-Dreifuss muscular dystrophy; BAF, barrier-to-autointegration factor; ER, endoplasmic reticulum; Fe 3+ IMAC, immobilized metal affinity chromatography; PKA, protein kinase A; X-EDMD, X-linked Emery-Dreifuss muscular dystrophy.