Human Molecular Genetics, 2002, Vol. 11, No. 13 1517-1525
© 2002 Oxford University Press
Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension
1Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge CB2 2QQ, UK and 2Division of Medical Genetics, University of Leicester, Leicester LE1 7RH, UK
Received February 18, 2002; Accepted April 15, 2002
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ABSTRACT |
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A wide range of mutations in the type II receptor for bone morphogenetic protein (BMPR-II) have been shown to underlie primary pulmonary hypertension. To determine the mechanism of altered BMPR-II function, we employed transient transfection studies in cell lines and primary cultures of pulmonary vascular smooth muscle cells using green fluorescent protein (GFP)-tagged wild-type and mutant BMPR2 constructs and confocal microscopy to localize receptors. Substitution of cysteine residues in the ligand binding or kinase domain prevented trafficking of BMPR-II to the cell surface, and reduced binding of 125I-BMP4. In addition, transfection of cysteine-substituted BMPR-II markedly reduced basal and BMP4-stimulated transcriptional activity of a BMP/Smad responsive luciferase reporter gene (3GC2wt-Lux), compared with wild-type BMPR-II, suggesting a dominant-negative effect of these mutants on Smad signalling. In contrast, BMPR-II containing non-cysteine substitutions in the kinase domain were localized to the cell membrane, although these also suppressed the activity of 3GC2wt-Lux. Interestingly, BMPR-II mutations within the cytoplasmic tail trafficked to the cell surface, but retained the ability to activate 3GC2wt-Lux. Transfection of mutant, but not wild-type, constructs into a mouse epithelial cell line (NMuMG cells) led to activation of p38MAPK and increased serum-induced proliferation compared with the wild-type receptor, which was partly p38MAPK-dependent. We conclude that mutations in BMPR-II heterogeneously inhibit BMP/Smad-mediated signalling by diverse molecular mechanisms. However, all mutants studied demonstrate a gain of function involving upregulation of p38MAPK-dependent proproliferative pathways.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Primary pulmonary hypertension (PPH) is characterized by narrowing and obliteration of small pulmonary arteries leading to sustained elevation of pulmonary arterial pressure (1). Although uncommon (annual incidence 1–2 per 106), PPH is typically fatal, with a median survival from diagnosis of 2.8 years, death occurring from right ventricular failure (2). Recently, heterozygous germline mutations in the gene (BMPR2) encoding the bone morphogenetic protein type II receptor (BMPR-II), a member of the transforming growth factor ß (TGF-ß) superfamily of receptors, have been found to underlie familial primary pulmonary hypertension (FPPH) (3,4). Furthermore, in at least a quarter of apparently sporadic cases of PPH, either transmitted or de novo germline BMPR2 mutations may be detected (5). The 1038-amino-acid BMPR-II protein comprises ligand-binding, kinase and cytoplasmic domains, and mutations have been identified in all of these regions. To date, 46 unique BMPR2 mutations have been reported, including partial gene deletions, missense, splice-site, nonsense and frameshift mutation. The majority (58%) of these pathogenic mutations are predicted to cause premature truncation of the gene transcript through nonsense-mediated mRNA decay (6). In contrast, missense mutations occur at highly conserved and functionally important amino acid residues, and are likely to perturb ligand–receptor binding or disrupt the constitutively active serine–threonine kinase domain of BMPR-II.
In common with other TGF-ß receptors, BMPR-II transduces signals by forming heterodimers at the cell surface with a corresponding type I BMP receptor (BMPR-IA and BMPR-IB). In the presence of ligand, the serine–threonine kinase activity of the type I receptor initiates a signal transduction cascade that involves phosphorylation of a family of signalling proteins known as Smads (7–9). BMPs signal via a restricted set of receptor mediated Smads (R-Smads), Smads 1, 5 and 8, which, when complexed with the common partner Smad, Smad4, translocate to the nucleus and regulate target-gene transcription. Although signalling via Smads is well characterized, there is increasing evidence that MAP kinases, including ERK, JNK and p38MAPK, are activated by BMPs and TGF-ßs in certain cell types (10,11). The specific pathway activated by BMPR-II may depend on whether preformed type I/type II heterodimers are stimulated by ligand (Smad-dependent) or whether ligand leads to recruitment of type I and II receptors to the signalling complex (p38MAPK-dependent) (12).
BMP7 has been shown to inhibit proliferation of human aortic smooth muscle cell (13), and BMP2 inhibits vascular smooth muscle cell proliferation after balloon injury in rats (14). Furthermore, we have recently reported that BMPs suppress proliferation of pulmonary artery smooth muscle cells from normal subjects and patients with secondary forms of pulmonary hypertension, but fail to suppress proliferation of cells from patients with PPH (15). Thus, disruption of BMP signalling pathways may result in the failure of critical antiproliferative/differentiation programmes in the pulmonary vasculature.
In the present study, we sought to investigate the functional consequences of the range of BMPR2 mutations associated with PPH. Using heterologous transfection assays, we show that the mechanism for reduced BMP/Smad transcriptional activation is dependent upon the type of mutation. In contrast, all mutant constructs transfected into an epithelial cell line caused an increase in cell proliferation – a process associated with the activation of p38MAPK. Thus, we conclude that whilst inhibition of Smad-dependent pathways may occur, BMPR-II mutation causes a consistent gain of function involving activation of a Smad-independent pathway, which may represent a critical functional consequence of disease-associated mutations in PPH.
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RESULTS |
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Cysteine substitutions in the ligand-binding domain of BMPR-II prevent trafficking to the cell surface
All naturally occurring missense mutations identified in the extracellular ligand-binding domain of BMPR-II result in substitution of cysteine residues (6). Therefore, we determined the impact of a range of cysteine substitutions (C60Y, C117Y, C118W, C123R and C123S) (Fig. 1) on localization of and signalling via BMPR-II. Comparison of the subcellular localization of green fluorescent protein (GFP)-tagged ligand-binding mutants transiently expressed in HeLa and human pulmonary artery smooth muscle cells (PASMCs – not shown) clearly showed that these receptors failed to reach the cell membrane compared with wild-type GFP–BMPR-II (Fig. 2). The punctate perinuclear distribution of GFP is consistent with retention of the protein in the endoplasmic reticulum (ER) and supported by co-localization with the ER-specific protein conconavalin A (data not shown). Specific 125I-BMP4 binding was reduced in human coronary artery smooth muscle cells (CASMCs) transfected with ligand-binding-domain mutants compared with cells transfected with wild-type BMPR-II, or cells transfected with empty vector (Fig. 2). Using luciferase reporter assays, transfection of ligand-binding mutants into NMuMG cells consistently reduced basal and BMP4-stimulated transcriptional activity of 3GC2wt-Lux, compared with cells transfected with pcDNA3.0 vector or wild-type BMPR-II (Fig. 2).
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Missense mutations in the kinase domain of BMPR-II lead to reduced activation of a BMP/Smad responsive reporter gene by mutation-specific mechanisms
Missense mutations described within the kinase domain of BMPR-II include cysteine substitutions (C347Y, C420R and C483R) and substitutions of aspartate (D485G) and arginine (R491W and R491Q) (Fig. 1). Again, transfection of GFP-tagged cysteine-substituted mutants in PASMCs and HeLa cells demonstrated retention within the endoplasmic reticulum, whereas aspartate- and arginine-substituted mutations did localize to the cell membrane (Fig. 3). Consistent with these data, 125I-BMP4 binding was reduced in CASMCs transfected with cysteine-substituted but not other mutants (Fig. 3). However, transfection of all kinase-domain mutants into NMuMG cells led to reduced transcriptional activity of basal and BMP4-stimulated 3GC2wt-Lux (Fig. 3).
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Mutations in the cytoplasmic tail of BMPR-II reach the cell surface and support activation of a BMP/Smad responsive reporter gene
We next examined the localization and function of missense mutations (K512T and N519K) and truncating mutations (S532X and R899X) in the cytoplasmic tail of BMPR-II (Fig. 1). GFP-tagged K512T and N519K mutants demonstrated a cell surface distribution, similar to that of wild-type BMPR-II (Fig. 4). Cells transfected with cytoplasmic tail missense and truncating mutants showed no reduction in the density of 125I-BMP4-binding sites compared with wild-type transfected cells, consistent with cell membrane localization of mutant receptors, and furthermore showed no inhibition of basal or BMP4-stimulated activity of 3GC2wt-Lux (Fig. 4).
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Expression of mutant BMPR-II promotes p38MAPK-dependent proliferation of NMuMG cells
p38MAPK has recently been shown to be an alternative Smad-independent signalling pathway downstream of BMPR-II (12) and other TGF-ß superfamily receptors (16,17). Therefore, we questioned whether p38MAPK intracellular signalling was altered in the presence of mutant BMPR-II receptors. Overexpression of wild-type BMPR-II was associated with phosphorylation of p38MAPK only when cells were incubated with BMP4 (50 ng/ml) (Fig. 5). In contrast, transfection of mutant receptors consistently led to activation of phospho-p38MAPK even in the absence of ligand, although the addition of BMP4 often led to a further increase (Fig. 5), probably due of the presence of untransfected NMuMG cells. In addition, wild-type transfected cells formed confluent monolayers, whereas mutant BMPR-II transfectants reached confluence more rapidly, and by 3 days were post-confluent with cells overgrowing each other and lifting from the plate (Fig. 6). [3H]-thymidine incorporation studies confirmed that NMuMG cells transfected with mutant receptors demonstrated increased DNA synthesis compared with cells transfected with wild-type BMPR-II or empty vector (Fig. 6). Pretreatment of cells with a specific inhibitor of p38MAPK, SB203580, markedly inhibited the proliferation of cells induced by transfection with mutant BMPR-II.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The identification of heterozygous germline mutations in the BMPR2 gene underlying familial and many apparently sporadic cases of PPH has provided the starting point from which to advance our understanding of the pathogenesis of this often-fatal disease. The pathogenic mutations identified within the coding sequence of the BMPR2 gene, are characterized by significant molecular heterogeneity. We therefore sought to determine the impact of these defects on BMPR-II signalling and function, and to explore the molecular mechanisms involved. Our findings demonstrate that disease-associated mutations in BMPR2 disrupt BMP/Smad signalling by a range of mechanisms. The combined use of 125I-BMP-4-binding studies, subcellular localization of GFP-tagged transfected receptors, and functional studies using a BMP/Smad-responsive reporter gene construct allowed us to identify the molecular mechanisms for distinct classes of mutation. Thus, mutations leading to substitution of cysteine residues in the ligand-binding and kinase domains disrupt trafficking of BMPR-II to the cell surface, with retention in the endoplasmic reticulum. In contrast, BMPR-II mutants containing non-cysteine missense mutations in the kinase domain attain the cell surface, as evidenced by localization of GFP-tagged protein and 125I-BMP-4 binding, but fail to activate a BMP/Smad-responsive reporter gene. The latter is consistent with disruption of the serine–threonine kinase activity of BMPR-II (18), or the inability to form preformed complexes with type I receptors (12).
Interestingly, missense mutations in the cytoplasmic region of BMPR-II distal to the kinase domain reached the cell surface and retained the ability to activate a BMP/Smad-responsive reporter gene. In addition, the introduction of nonsense mutations predicted to cause premature truncation of the cytoplasmic tail of BMPR-II (S532X and R899X) also demonstrated the ability to activate the reporter gene. The failure of the cytoplasmic tail mutants to inhibit BMP-4-induced activation of the BMP/Smad reporter gene differentiates them from mutations involving the kinase and ligand-binding domains. Recent evidence suggests that alternative pathways, independent of Smads, are critical to the context-specific nature of TGF-ß superfamily signalling. These alternative pathways include kinases such as ERK1/2, JNK and p38MAPK (10,19). The presence of these alternative pathways and the balance between Smad and Smad-independent pathways may explain the complex and often cell-specific actions of the TGF-ß superfamily.
To define abnormalities in alternative pathways specific to mutant BMPR-II, we studied the activation of p38MAPK, which was recently shown to be an important mediator of BMP signalling (12,16). Overexpression of mutant BMPR-II receptors, including cytoplasmic tail mutants, in NMuMG cells led to activation of p38MAPK in the absence of exogenous ligand, whereas overexpression of the wild-type receptor required the presence of BMP-4 to activate p38MAPK. These findings are consistent with our observation that transfection of mutant receptors caused increased serum-stimulated proliferation of NMuMG cells – an effect that was inhibited by SB203580. How then does overexpression of mutant receptors lead to activation of p38MAPK in the absence of exogenous ligand, particularly when some receptors fail to reach the cell surface? Recent data have shown that the state of receptor oligomerization at the cell surface determines different BMP signalling pathways (12), and that the state of BMP receptor oligomerization is highly flexible, allowing considerable variation in response to ligand (20). In addition, it has been shown that BMP type I/type II receptor heterocomplexes permit ligand-independent signalling in transfected cells, supporting our finding that transfection of mutant receptor in the absence of exogenous ligand increased p38MAPK activation (20). These data strongly suggest that disruption of Smad-dependent signalling may not be the only, or indeed the most important, pathway, downstream of BMPR-II, involved in the pathogenesis of PPH. It is plausible that alterations in the critical balance of wild-type receptor availability at the cell surface leads to the suppression of certain signalling pathways (e.g. Smads) with preservation or activation of alternative pathways (e.g. p38MAPK). All heterozygous mutations in BMPR-II are likely to cause a deficiency or disruption of normally functioning receptor at the cell surface. Thus, alterations in the critical balance of BMP receptor availability at the cell surface, which determines the pattern of ligand-induced and ligand-independent signalling, may be an important mechanism contributing to the abnormal cellular proliferation observed in NMuMG cells transfected with mutant BMPR2 constructs.
Our recent observations in cells and lung tissue from patients with PPH support the hypothesis that the vascular cell dysfunction in PPH may depend on the density of functional cell surface receptors. Immunohistochemical studies using an antibody to human BMPR-II demonstrated that the level of BMPR-II protein expression is markedly reduced to about 10% of normal levels in PPH patients harbouring a mutation in BMPR2 (21). These findings are consistent with the hypothesis that the initial germline mutation is not sufficient, without additional genetic or environmental events, to cause disease progression as observed in PPH patients with end-stage disease.
Our data suggest a dominant inhibition of wild-type receptor function (at least with regard to Smad signalling) as a consequence of mutation in the kinase domain of BMPR-II. Previously, the introduction of non-cysteine amino acid substitutions in the kinase domain of BMPR-II had been shown to exert a dominant inhibitory effect on Smad signalling (12). Our data extend these observations to pathogenic mutations, but also suggest that cysteine-substituted kinase-domain mutants, which are retained intracellularly, exhibit a dominant-negative effect on wild-type receptor function. We established by RT–PCR that the cell lines used in these experiments expressed endogenous BMPR-II, BMPR-IA and BMPR-IB (data not shown). One mechanism that may explain this effect is potential interference with trafficking of the endogenous wild-type receptor. This hypothesis will require further experimental studies of the effect of the presence of mutant receptors on wild-type receptor trafficking and dimerization. Although one might expect a dominant-negative effect to be associated with a more severe disease phenotype, at present there is no evidence for such a genotype–phenotype correlation in clinical genetic studies (6).
Of note, these experimental observations suggest that although BMP/Smad pathways may be inhibited in the presence of mutant BMPR-II, a more consistent feature is the gain of function associated with increased p38MAPK signalling. Since familial primary pulmonary hypertension is a condition with low penetrance, this gain of function via Smad-independent pathways correlates better with the clinical genetic findings than the dominant-negative effect exerted by some BMPR-II mutants on BMP/Smad signalling, which would be expected to be fully penetrant.
In summary, we have demonstrated that disease-associated mutations in BMPR-II lead to disruption of downstream signalling pathways, involving in some cases suppression of Smad signalling or more uniformly potentiation of p38MAPK signalling. The molecular mechanism for these effects varies between classes of mutation, including failure of receptor trafficking or loss of kinase function. However, all classes of mutation will lead to changes in the relative amounts of wild-type and mutant receptors at the cell surface, which will impact on selection of specific signalling pathways, as a consequence of the complex patterns of BMPR-II heteromerization. These alterations in receptor function, in combination with additional genetic (22) and/or environmental (23) insults occurring in the pulmonary vascular cells of individuals harboring germline mutations in BMPR-II, may provide the trigger for the abnormal vascular remodelling that characterizes primary pulmonary hypertension.
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MATERIALS AND METHODS |
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Cell culture and growth assays
Both cell lines and primary cultured cells were used in these studies. Normal mouse mammary gland epithelial cells (NMuMG, European Collection of Cell Cultures no. 94081121), constitutively competent for TGF-ß superfamily signalling (24) were used for signal transduction experiments. HeLa cells were used to study subcellular localization of GFP-tagged receptors because they are readily transfected, but were not used for signalling studies because this immortalized cell line possesses abnormally upregulated growth pathways. Immortalized human coronary artery smooth muscle cells (CASMCs) were a generous gift from Professor Martin Bennett (University of Cambridge, UK) (25), and were found to possess high levels of specific binding sites for 125I-BMP-4, and thus were used for binding studies. In addition, primary cultures of human pulmonary smooth muscle cells (PASMCs) were obtained from peripheral pulmonary arteries (<2 mm diameter) of normal lung, as previously described (26). We determined by RT–PCR that all cell lines used in these studies constitutively express mRNA for BMPR-II, BMPR-IA and BMPR-IB (data not shown). Cells were grown in Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. To determine the effect of BMPR2 mutations on cell proliferation, cells were seeded in 12-well plates and transiently transfected with pcDNA3.0–BMPR2 wild-type or mutant plasmids, as described below. Cells were placed into fresh DMEM/10% FBS 24 hours post transfection, and cell numbers were counted with a haemocytometer at days 0, 3 and 5 post transfection. For [3H]thymidine incorporation studies, cells were grown in DMEM/10% FBS for 48 hours and then quiesced in DMEM/0.1% FBS for 48 hours. The medium was then exchanged for DMEM/10% FCS for a further 24 hours, with 0.5 µCi/well [methyl-3H]thymidine added for the final 6 hours. In additional experiments, cells were pre-incubated with a specific inhibitor of p38MAPK, SB203580 (1 µM), before addition of serum.
Preparation of BMPR2 constructs
The entire coding sequence of wild-type BMPR2 cloned into pcDNA3.0 was a generous gift from Professor K. Miyazono (Tokyo, Japan). A wild-type GFP C-terminally tagged BMPR2 construct was generated by removing the stop codon and subcloning into pEGFP–N1 (Clontech). Using pcDNA3–BMPR2 as a template, forward primer 5'-AAC TCC CTA TTC TCT TAA GC-3' (which contains a BMPR2 unique AflII site) and reverse primer 5'-GAT CCT CGA GGC CAG ACA GTT CAT TCC TAT ATC-3' (which contains an XhoI site) were used to generate a 200 bp fragment by PCR amplification. Following AflII/XhoI digestion, the PCR product was ligated into AflII/XhoI-digested pcDNA3–BMPR2. The new construct was then cleaved with HindIII and XhoI, and the BMPR2 fragment was subcloned into HindIII/SalI-digested pEGFPN1. Mutant BMPR2 constructs were generated by subjecting the wild-type construct to site-directed mutagenesis using the Stratagene QuikChange protocol. The sequences of the wild-type and mutant constructs were confirmed by DNA sequencing of the entire receptor and, where appropriate, the GFP tag using an ABI 377 sequencer with the Applied Biosystems Big Dye terminator kit. The range of mutant receptors generated are shown in Figure 1.
Subcellular localization of BMPR-II
HeLa cells, NMuMG cells or PASMCs were seeded on glass coverslips at 1x105 cells per well in 12-well plates. Cells were then transiently transfected as described below with pEGFP–N1–BMPR2 plasmids, either wild-type or mutants, using a total of 2 µg of plasmid per well. Transfectants were grown for 48 hours in DMEM/10% FBS and then fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS). Coverslips were washed in PBS and mounted on microscope slides in glycerol/PBS solution. Cells were viewed and photographed using an ultraviolet confocal microscope (TCSSB, Leica), or with a fluorescent microscope (Zeiss), and images were captured using SmartCapture 2 software. Transfection efficiency was approximately 30–40% in HeLa cells, 20–30% in NMuMG cells and approximately 5% in PASMCs.
Radioligand-binding studies
Human recombinant BMP-4 (R&D Systems) was iodinated using the chloramines-T method, as previously described for TGF-ß (27). Preliminary studies demonstrated that the highest specific-binding 125I-BMP4 density was found in immortalized CASMCs, which were used for subsequent studies of the effect of specific mutations on binding. Cells were grown to confluence in DMEM/10%FCS in 24-well plates. 125I-BMP4 competition binding was performed on confluent cell populations. Cells were pre-incubated with DMEM containing 0.5%BSA for 1 hour at 37°C and then incubated at 4°C for 2 hours with DMEM/0.5%BSA contain-ing 125I-BMP4 in the absence or presence of increasing concentrations of unlabelled BMP-4 (0.1–100 ng/ml). To investigate the effect of BMPR-II mutation on 125I-BMP4 binding, CASMCs were transiently transfected with wild-type or mutant pcDNA3.0–BMPR2 constructs, as described below. Following transfection, cells were incubated for 48 hours in DMEM/10% FBS, prior to performing 125I-BMP-4 competition binding as described above.
Luciferase reporter gene assays
NMuMG cells were seeded in 12-well plates at 3x105 cells per well and incubated overnight. Transient transfections were performed on cells at 90% confluence using Lipofectamine 2000 transfection reagent (Invitrogen) complexed with a total of 2 µg of plasmid diluted in OptiMem I (Life Technologies). Preparation of the transfection solution was carried out according to the manufacturer's protocol, and 200 µl was added to each well containing 750 µl DMEM/10%FBS. Cells were co-transfected with wild-type or mutant pcDNA3.0–BMPR2 and a luciferase reporter plasmid, 3GC2wt-Lux (a kind gift from W. Ishida), which contains a BMP-responsive element derived from the mouse Smad6 promoter (28). After 24 hours, the medium was replaced with DMEM containing 0.1% FBS with or without 50 ng/ml of BMP-4 (R&D Systems). After a further 24 hours, cell supernatant was assayed for luciferase activity according to the manufacturer's protocol (Roche). In order to control for transfection efficiency, cells were co-transfected with alkaline phosphatase under the control of a cytomegalovirus promoter. Secreted alkaline phosphatase activity was measured by spectrophotometry.
p38MAPK immunoblotting
NMuMG cells were plated onto 10 cm Petri dishes, grown to confluence, and then transfected with pcDNA3.0, wild-type or mutant constructs, as described above. Following transfection, cells were placed in fresh DMEM/10%FBS for 48 hours, and then quiesced for 24 hours in serum-free medium. BMP-4 (50 ng/ml) was then added for 60 minutes. Protein was harvested by washing cells in cold PBS and immediately freezing in an ethanol/dry-ice bath. Cells were scraped into 200 µl of protein loading buffer containing protease inhibitors, antipain, leupeptin, pepstatin (all at 1 µg/ml), PMSF (1 mM), sodium vanadate (2 mM) and sodium fluoride (2 mM). Samples were then boiled for 5 minutes prior to centrifugation and storage at -20°C. For western blotting, samples (30 µg) were electrophoresed by SDS–PAGE (12%) and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk powder/0.1% Tween in TBS for 1 hour at room temperature. After washing, blots were incubated with anti-phospho-p38MAPK (1 : 1000) (Cell Signalling Technology, Inc., MA) in 5% BSA/0.1%Tween/TBS overnight at 4°C. Blots were then washed and incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 2500, DAKO) in 2% milk powder/0.1%Tween/TBS for 1 hour at room temperature. Bands were visualized by chemiluminescence (Amersham, UK). Blots were the stripped and reprobed using an antibody to total p38MAPK (Cell Signalling Technology, Inc.).
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ACKNOWLEDGEMENT |
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This study was supported by a grant from the British Heart Foundation (Programme Grant RG/2000012 to R.C.T. and N.W.M.).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Hills Road, Cambridge CB2 2QQ, UK. Tel: +44 1223 336744; Fax: +44 1223 762007; E-mail: nwm23{at}cam.ac.uk
Correspondence may also be addressed to Professor Richard C. Trembath, Division of Medical Genetics, Departments of Medicine and Genetics, Adrian Building, University of Leicester, Leicester LE1 7RH, UK. Tel: +44 116 2522263; E-mail: rtrembat{at}hgmp.mrc.ac.uk
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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