Báo cáo Y học: The Emery–Dreifuss muscular dystrophy associated-protein emerin is phosphorylated on serine 49 by protein kinase A pptx

The Emery–Dreifuss muscular dystrophy associated-protein emerin is phosphorylated on serine 49 by protein kinase A Rhys C. Roberts 1, * ,‡ , Andrew J. Sutherland-Smith 1,†,‡ , Matthew A. Wheeler 2 , Ole Norregaard Jensen 3 , Lindsay J. Emerson 2 , Ioannis I. Spiliotis 2 , Christopher G. Tate 1 , John Kendrick-Jones 1 and Juliet A. Ellis 2 1 MRC Laboratory of Molecular Biology, Cambridge, UK 2 The Randall Division of Cell and Molecular Biophysics, Kings College, London, UK 3 Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Keywords emerin; Emery–Dreifuss muscular dystrophy; mass spectrometry; phosphorylation; protein kinase A Correspondence J. A. Ellis, The Randall Division of Cell and Molecular Biophysics, Kings College, New Hunts House, Guy’s Campus, London SE1 1UL, UK Fax: + 44 20 78486435 Tel: + 44 20 78486498 E-mail: juliet.ellis@kcl.ac.uk Present address *The National Hospital for Neurology and Neurosurgery, London, UK † Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand ‡ These authors contributed equally to this work (Received 23 May 2006, revised 27 July 2006, accepted 14 August 2006) doi:10.1111/j.1742-4658.2006.05464.x Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X-linked Emery–Drei- fuss 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 phos- phorylation 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 em- erin 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 iden- tified 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. 4562 FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS Emerin is a ubiquitously expressed, single-membrane- spanning protein of the inner nuclear membrane [1,2]. It is encoded by the EMD gene on chromosome Xq28 and mutations within this gene give rise to X-linked Emery–Dreifuss muscular dystrophy (X-EDMD), a neuromuscular condition with an associated life-threat- ening cardiac conduction defect [3]. EDMD manifests in early childhood with progressive weakness and wast- ing of limb muscles and contractures at the elbows and ankles. A cardiac conduction defect develops in patients by their mid-30s associated with a substantial risk of sudden death. Molecular defects are character- ized by abnormalities in myonuclear architecture, inclu- ding marked condensation of chromatin. The majority of the mutations in the EMD gene produce the null phenotype, but 10% produce modified forms of emerin. Human emerin is a serine-rich protein of 254 amino acids (M r 28 994) with a C-terminal transmembrane region (residues 222–244) which spans the inner nuclear membrane and a hydrophilic N-terminal domain, which orientates towards the nucleoplasm [4]. Emerin requires the transmembrane domain and two regions in its nu- cleoplasmic domain to be post-translationally inserted into the endoplasmic reticulum (ER) membrane, where it diffuses through the ER network to the nuclear envel- ope [4–8]. Emerin is retained at the inner nuclear mem- brane by interacting with the intermediate filament proteins, nuclear lamin A and C [9–11]. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) studies [5,12] show that green fluorescent protein (GFP)-emerin has a decreased lateral mobility when in the inner nuclear membrane compared with when it is still in the ER [5]. This is con- sistent with the theory that it is stably anchored at the nuclear membrane through its interaction with lamin A ⁄ C. Interestingly, an autosomal dominant form of EDMD (AD-EDMD) also occurs due to mutations in the LMNA gene, which encodes for the alternatively spliced nuclear lamin A and C proteins [13]. Most mutations in the LMNA gene are missense, and there- fore modified forms of lamin A ⁄ C are produced which frequently exhibit aberrant nuclear localization. Emerin is re-distributed with the lamin A ⁄ C in AD-EDMD patients [14] and to the ER in the LMNA knock-out mouse [15], confirming that retention of emerin at the inner nuclear membrane is, at least partly, through its interaction with lamin A ⁄ C. The nucleoplasmic domain of emerin and the equiv- alent domains of two other inner nuclear membrane proteins termed MAN1 and the lamina-associated pro- tein isoforms (LAP1 and 2) contain a region of homol- ogy called the LAP-Emerin-MAN1 (LEM) domain; (residues 2–45 in human emerin). This domain is involved in binding to a dsDNA bridging protein termed BAF (barrier-to-autointegration factor), which functions in higher-order chromatin organization and nuclear assembly [16]. Other known binding partners of emerin include the nuclear lamins A, B and C, actin [9,17,18], the transcriptional repressors Btf [19] and GCL [20], BAF [21], the RNA-splicing factor YT521- B [22] and some members of the nesprin-1 and -2 nuc- lear membrane protein family [23,24]. Such a diverse array of binding partners suggests emerin may have a role in regulating nuclear envelope structure and gene expression. Indeed, it has been shown that cell cycle timing and Rb-MyoD myogenic transcriptional path- ways are perturbed in myoblasts isolated from EDMD patients or emerin null mice [25,26]. Prediction analysis identifies 36 potential phosphory- lation sites (28 Ser, one Thr and seven Tyr as predicted by the netphos 2.0 prediction programme) spanning human emerin residues 1–199. They cover a wide range of kinases, including protein kinases A and C, casein kinase II and tyrosine kinases. We have previously shown that four different phosphorylated forms of emerin (phospho-emerin species) can be immunoprecipitated from phospholabelled human lymphoblastoid cell lines [4]. Three of these phospho- emerin species appear to be associated with cell cycle- dependent events, although the connection with the cell cycle remains to be directly determined. Lympho- blastoid cell lines derived from X-EDMD patients expressing either D95-99 or 1–218(238) mutant forms of emerin exhibited aberrant emerin phosphorylation [4], whereas in HeLa cells, in vivo phosphorylation of emer- in resulted in multiple phospho-emerin species, which were enhanced in the presence of the phosphatase 1 and 2a inhibitor, okadaic acid [27]. Consistent with these findings, five residues in human emerin have recently been identified as undergoing mitotic phospho- rylation in a M-phase Xenopus egg cell-free system [28]. In this paper, we have shown by protein mass spectr- ometry that recombinant human emerin expressed in insect cells contains at least four phosphorylation sites, in agreement with our previous findings in human lymphoblastoid cell lines. We have isolated by immobi- lized metal affinity chromatography (Fe 3+ IMAC) two phosphopeptides and identified residue S49 as being a phosphorylated residue by tandem mass spectrometry. Structural prediction analysis identifies S49 as the resi- due most likely to be phosphorylated by cAMP-depend- ent protein kinase A (PKA) and we have confirmed this using an in vitro protein kinase assay. Potential signal transduction pathways that could be involved in phos- phorylating emerin on residue S49 and how they might influence emerin function are discussed. R. C. Roberts et al. PKA phosphorylates emerin FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS 4563 Results Expression of recombinant human emerin The two fusion proteins expressing human emerin fragments spanning residues 1–176 and 1–220 both expressed at high levels in bacteria. The emerin fusion protein 1–176 expressed in the pET-29b vector with a C-terminal 6 · His-tag was 50% soluble after treat- ment with 1% (v ⁄ v) Triton X-100 and sonication (Fig. 1A, lane 4). This soluble protein was purified by Ni 2+ -NTA (nitrilotriacetic acid) and it migrated as a single band of 30 kDa (predicted molecular mass of 20.114 kDa) on 13% SDS ⁄ PAGE (Fig. 1A, lane 5). Immunoblotting this purified protein with our antiem- erin AP8 antibody also produced one band (Fig. 1A, lane 6). However, the same emerin construct expressed with an N-terminal 6 · His-tag in insect Sf9 cells after partial purification by Ni 2+ -NTA migrated on 12.5% SDS ⁄ PAGE as three major and three minor protein bands as visualized by Coomassie blue staining (Fig. 1B; lane Co). These bands spanned an approximate molecular mass range of 30–33 kDa. Immunoblotting with the antiemerin antibody AP8 confirmed that all of the extra bands were emerin and two to three higher molecular mass bands repre- senting possible further minor emerin species were also identified (Fig. 1B, lane IB). None of these bands were present in uninfected control Sf9 insect cells (data not shown). The additional forms of emerin observed in the eukaryotic expression system are likely to result from the addition of post-translational modifications. The pattern of post-translational modi- fications produced from baculovirus-expressed systems has been shown to reflect those characterized in mam- malian systems [29]. Our previous studies had indica- ted that human emerin does not undergo any proteolytic cleavage events, nor is it glycosylated, but it is phosphorylated in mammalian cells [4,28]. As the emerin protein bands shown in Fig. 1B are evenly spaced, and within the limits of gel resolution differ by the weight of a phosphate group (80 Da), this pat- tern of bands suggests phosphorylated emerin species. Indeed the three lowest molecular mass emerin species cross-reacted with an antiphosphotyrosine antibody but, perhaps not surprisingly, the commercial anti- phosphoserine and threonine antibodies used did not cross-react with any of the emerin bands on the blot (data not shown). We have observed that these latter antibodies do not cross-react with any of the phos- phorylated cytoskeletal proteins (known to contain phospho-serine and phospho-threonine residues) we have tested (Kendrick-Jones, unpublished results). The human emerin fusion protein spanning residues 1–220 expressed only as insoluble inclusion bodies under all the conditions tested in our bacterial expres- sion systems and attempts to re-fold this protein were Fig. 1. Expression of recombinant emerin 1–176 in (A) the bacterial cell line BL21 (DE3) and in (B) the insect cell line Sf9 (B). (A) 13% SDS ⁄ PAGE of pET-29b emerin 1–176 bacterial expression (Coo- massie blue staining): lane 1 is the un-induced cell lysate, lane 2, the induced cell lysate, lane 3, the insoluble pellet, lane 4, the sol- uble fraction, lane 5, the purified fraction from Ni 2+ -NTA chromato- graphy after elution with 0.5 M imidazole treatment and lane 6 the purified fraction immunoblotted with antiemerin antibody AP8 at a dilution of 1:3000. Bacterially expressed emerin migrates as a sin- gle band on SDS ⁄ PAGE (arrow). (B) FastBacHTb-emerin 1–176 expressed in the Sf9 insect cell line and purified by Ni 2+ -NTA chro- matography and subjected to 12.5% SDS ⁄ PAGE. The recombinant emerin 1–176 expressed in the insect FastBacHTb vector had an additional 29 residues which adds an extra 3.53 kDa to this emerin fusion protein compared with the same protein expressed in the bacterial pET-29b vector. The left hand panel (Co) is Coomassie blue stained, and the right hand panel was immunoblotted with the antiemerin antibody AP8 (IB) at 1:3000. Baculoviral expressed emerin migrated as a ladder of 6–9 bands. The area marked by a boxed arrow represents the gel bands individually excised from a Coomassie blue stained gel for the tryptic digestion. PKA phosphorylates emerin R. C. Roberts et al. 4564 FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS unsuccessful (data not shown). Expressing emerin 1– 220 in our insect cell expression system resulted in very low yields with 50% of the protein insoluble, but mul- tiple bands were apparent from immunoblotting with the antiemerin antibody AP8. It was therefore decided to use the more soluble purified emerin fusion protein 1–176 for mass spectrometry analysis since it still con- tains 88% of the predicted phosphorylated sites. Mass spectrometry analysis of recombinant emerin 1–176 Recombinant human emerin 1–176 taken directly after purification on the Ni 2+ -NTA column was rapidly de- salted using a high-flow HPLC and then immediately analyzed by ESI Q-TOF mass spectrometry (Fig. 2A). The mass spectrum exhibited clusters of multiply charged ions, suggesting that more than one species of emerin was present. The raw mass spectrum was de- convoluted using the MaxEnt algorithm (masslynx, Waters Micromass, St Quentin, France) to reveal the intact molecular mass of the various emerin species (Fig. 2B). Interestingly, this revealed five different molecular species of emerin differing by multiples of 80 Da, which is the molecular mass of a single phos- phate group (HPO 3 ). Therefore, we hypothesized that the mass spectral peaks at 23 625, 23 705, 23 785, 23 864 and 23 944 Da corresponded to recombinant emerin 1–176 with no phosphate and with one, two, three and four phosphate groups attached, respectively. Phosphorylation was confirmed by analysis of the recombinant emerin by electrospray ionisation mass Fig. 2. Baculovirus expressed 1–176 emerin analyzed by ESI Q-TOF mass spectrometry. (A) The raw spectrum illustrating the mul- tiple charged ions corresponding to emerin 1–176. (B) The deconvoluted data of the raw spectrum using MaxEnt algorithm, revealing emerin as five distinct species at 23 625, 23 705, 23 785, 23 864 and 23 944 Da. The peak at 23 625 Da is non- phosphorylated and each subsequent peak increases by an increment of 80 Da corres- ponding with the molecular mass of an addi- tional phosphate group. The y axis is an arbitrary scale and represents relative inten- sity where 100% is taken as the most intense peak. R. C. Roberts et al. PKA phosphorylates emerin FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS 4565 spectrometry (ESI-MS) following alkaline phosphatase (ALP treatment). Now, following deconvolution of the mass spectrum (Fig. 3A), only one peak at 23 625 Da corresponding to the nonphosphorylated form of emer- in was detected (Fig. 3B). Our previous studies had indicated that human emerin does not undergo any proteolytic cleavage events, nor is it glycosylated [4]. In agreement with these findings, the only post-transla- tional modification we identified on human recombin- ant emerin 1–176 by tandem mass spectrometry was phosphorylation. As a first step to identify the residues phosphorylat- ed in emerin, candidate phosphopeptides were identi- fied in tryptic digests of emerin using the MALDI-MS. Purified recombinant emerin 1–176 was run as several gel tracks on 12.5% SDS ⁄ PAGE, where one track was immunoblotted for emerin and the others stained with Coomassie blue. From the immunoblotting pattern for emerin, a region in the adjacent Coomassie blue- stained portion of the gel was identified as containing the different emerin species. This region spanned an approximate molecular mass range of 30–35 kDa (shown as boxed arrows on the left-hand side lane Co; Fig. 1B). The gel bands containing emerin were cut out individually as far as was technically feasible, as the bands were very close together and digested in-gel with trypsin and the resulting peptides extracted. Each individual emerin band cut out from the gel gave the same tryptic digestion pattern upon MALDI-MS ana- lysis of peptides. All the tryptic peptides were identified Fig. 3. Baculovirus 1–176 emerin analyzed by ESI Q-TOF mass spectrometry following treatment with alkaline phosphatase (ALP). Following incubation of emerin 1–176 with ALP, peaks corresponding to a single un- phosphorylated emerin species were detec- ted. The raw data are shown in (A), while the deconvoluted spectrum showing the intact unphosphorylated protein peak is shown in (B). The four emerin species lost (compared with the peaks of 23 705.0, 23 785, 23 864, 23 944 Da on Fig. 2) repre- sent the removal of four phosphate groups. The y axis is an arbitrary scale and repre- sents relative intensity where 100% is taken as the most intense peak. PKA phosphorylates emerin R. C. Roberts et al. 4566 FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS to be emerin as ascertained by molecular mass deter- mination by MALDI-MS and amino acid sequencing by ESI tandem mass spectrometry. Phosphopeptides were enriched by micropurification using Fe 3+ IMAC [30] and analyzed by MALDI-MS. We identified three candidate emerin tryptic phosphopeptides (m ⁄ z 2191.9, 2271.7 and 2396.9 on Fig. 4A,B) from one digested band. The upper traces in both Figs 4A and 4B repre- sent the MALDI mass spectra without prior purifica- tion by Fe 3+ IMAC, illustrating that this purification technique is extremely useful for enriching the phos- phopeptides. The peptides at m ⁄ z 2191.9 and 2271.1 correspond to the peptide masses of singly and doubly phosphorylated emerin peptide 158-DSAYQSITHYR PVSASRSS-176 and the peptide at a m ⁄ z 2396.9 corresponds to a single phosphorylated emerin peptide 47-RLSPPSSSAASSYSFSDLNSTR-68. These phos- pho-peptides include four of the residues reported to be phosphorylated on emerin under mitotic conditions (S49, S66, T67 and S175 [28]); and two reported to be phosphorylated on emerin in human T cells (S163 and Y167 [31]). To pinpoint the specific phosphorylated residues, we analyzed the tryptic peptide mixture by ESI-MS, which produces the complex peptide mass spectrum (Fig. 5A). The triply protonated peptide of m ⁄ z 799.791 corresponded to the potentially phosphorylat- ed emerin peptide 47-RLSPPSSSAASSYSFSDLN STR-68 (Fig. 5B). Tandem mass spectrometry of this phospho-peptide candidate confirmed the identity of the peptide and determined that the phosphorylated residue was serine 49. The presence of a doubly charged C-terminal fragment ion of 19 residues with the emerin sequence (PPSSSAASSYSFSDLNSTR; m ⁄ z 980.95; labelled as y19 in Fig. 5B) demonstrated that this peptide fragment carried no phosphate group. We also detected an N-terminal fragment ion correspond- ing to the sequence RLS (labelled as b3 in Fig. 5B) of Fig. 4. Identification of candidate phospho- peptides from 1 to 176 emerin by MALDI- TOF MS peptide mass mapping. The tryptic peptides from 1 to 176 emerin were extrac- ted from the gel slices, micropurified by Fe 3+ IMAC and introduced into the MALDI- TOF mass spectrometer. All the protein bands extracted had a tryptic peptide finger- print consistent with emerin. We show the spectrum from one peptide digest of one individual gel slice which is the lower trace (labelled as + IMAC) in (A) and (B). This trace is shown in comparison with one obtained from a tryptic peptides which were not micropurified by Fe 3+ IMAC (upper trace labelled –IMAC in A and B) illustrating this purification step was necessary to selec- tively purify the phosphorylated peptides for detection by mass spectrometry. (A) repre- sents the full view of all the peptides detec- ted by MALDI mass spectrometry from m ⁄ z 600–4000 and (B) shows a magnified image of the spectrum between m ⁄ z 2150 and 2400. Three candidate tryptic phosphopep- tides were identified of m ⁄ z 2191.9, 2271.7 and 2396.9. The y axis is an arbitrary scale and represents relative intensity. R. C. Roberts et al. PKA phosphorylates emerin FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS 4567 m ⁄ z 339.23. This fragment had lost 98 Da in molecular mass by elimination of a phosphate group (80 Da) and a water molecule (18 Da). Thus the detection of the y19 and b3 ions allowed us to conclude that serine 49 was a phosphorylation site in emerin. Therefore, we can confirm that residue S49 in human emerin is phos- phorylated and, furthermore, as Sf9 cells do not divide after baculovirus infection, this residue is likely to be phosphorylated in interphase. Does phosphorylation at residue S49 regulate its ability to target to the nuclear envelope? S49A and S49E mutant forms of full-length human emerin were generated by site-directed mutagenesis and cloned directly into the pEGFP-C2 enhanced green fluorescent protein vector that adds the green auto-fluorescent GFP tag to the N-terminus of emerin. We have previously shown that the attachment of the EGFP tag to the N-terminus of wild-type emerin does not affect its ability to locate to the nuclear envelope [32]. The S49A substitution generated an emerin mutant that could not be phosphorylated at this resi- due, whereas the S49E substitution mimicked constitu- tive phosphorylation. These GFP-emerin constructs were transiently expressed in an asynchronous popula- tion of C2C12 myoblasts and wild-type GFP-emerin correctly localized to the nuclear envelope of a cell in interphase after 20 h which was used as the optimum expression time (Fig. 6). The level of EGFP-emerin expression was optimized to be similar to the level of endogenous wild-type emerin expression, by comparing Fig. 5. Identification of residue S49 as a phosphorylated site in human emerin. The tryptic peptides generated from the diges- tion of any single Coomassie blue stained band of Sf9 insect cell expressed 1–176 emerin (isolated from the boxed region labelled on the LH side of Fig. 1b), were analyzed to identify the specific residues phosphorylated. Analysis of the tryptic peptide mixture by ESI MS is shown in (A). The peptide ion at m ⁄ z 799.79 was sequenced by MS ⁄ MS (B) and identified the phosphopeptides 47-RLSPPSSSAASSYSF SDLNSTR-68. The presence of the phos- phopeptides fragment ion y19 and b3 in this MS ⁄ MS spectrum allowed us to identify serine 49 as a site of phosphorylation in emerin (B). PKA phosphorylates emerin R. C. Roberts et al. 4568 FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS immunofluorescence intensities of endogenous emerin stained with our AP8 antiemerin antibody with EGFP- emerin stained with an anti-GFP antibody (data not shown). The transfection efficiency (10%) and level of EGFP-protein expression was similar for all three EGFP constructs. Since the nuclear-trafficking route for emerin involves prior transit through the ER net- work [7,8], some emerin is always seen in the ER of interphase cells, as shown by the staining for the ER with an anti-PDI antibody (merge panel; Fig. 6). C2C12 cells expressing the EGFP-emerin S49A mutant exhibited a wild-type distribution, although some pro- tein aggregation was observed in the perinuclear region (Fig. 6). Similar aggregates were seen in cells expres- sing the EGFP-emerin S49E mutant. The aggregates of exogenous mutant emerin protein did not colocalize with staining for Golgi-associated protein (GM130), coat associated protein of non-clathrin-coated vesicles (b-COP) (data not shown) and protein disulfide iso- merase (PDI) (Fig. 6), suggesting they were located in the cytoplasm and therefore excluded from the ER and thus unable to be transported to the nuclear envel- ope. These aggregates were most likely the result of misfolding of the mutant fusion proteins. Endogenous lamin A ⁄ C staining was normal in all our transfected cells and we saw no evidence for structural alterations to the nuclear envelope or rearrangement of hetero- chromatin (data not shown). Interestingly, in 90% of the myoblasts expressing the EGFP-emerin S49E mutant, consistently slightly less exogenous emerin was expressed at the nuclear membrane (Fig. 6) compared with wild-type EGFP-emerin in interphase cells. How- ever, if we allowed our transfected cells to undergo one round of cell division, both mutants targeted directly to the nuclear envelope in a similar manner to wild-type emerin (data not shown). These results sug- gest that newly synthesized endogenous emerin is either unphosphorylated at residue S49 and ⁄ or that phosphorylation at this site is not crucial for its nuc- lear envelope targeting. It is therefore probable that phosphorylation on residue S49 regulates its interac- tions with one or more of its nuclear binding partners, Fig. 6. Intracellular localization of wild-type, S49A and S49E emerin tagged with EGFP in C2C12 cells. An asynchronous population of C2C12 myoblasts were transfected with our pEGFP-C2 emerin constructs as described in the Experimental procedures. The cells were fixed )20 °C methanol and the intra- cellular location of emerin was monitored by EGFP (green) fluorescence, the endoplasmic reticulum staining was monitored by PDI staining (red) and chromatin with DAPI (blue). The merged panel illustrates some emerin being present in the ER (yellow). Bar ¼ 10 lm. R. C. Roberts et al. PKA phosphorylates emerin FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS 4569 whilst at the nuclear envelope. We therefore examined the effect of phosphorylation at residue S49 on emer- in’s ability to interact with lamin A and C. Using our coimmunoprecipitation ⁄ in vitro transcription-trans- lation assay system described previously [33] we dem- onstrate that both the S49A and S49E- mutant forms of emerin appear to bind to either lamin A or lamin C with the same relative affinity as wild-type emerin (data not shown). Therefore, phosphorylation at resi- due S49 is unlikely to directly regulate emerin-lamin A ⁄ C interaction in vivo. Protein kinase A-dependent phosphorylation of serine 49 Computer prediction analysis using the scansite 2.0 motif scan computer programme [34] set at the high- est stringency setting (score falls within the top 0.2% of known motifs) identified emerin residue S49 as being the most likely residue in emerin to be phosphor- ylated by cAMP-dependent PKA [28]. In addition, no other kinase was predicted to phosphorylate residue S49, using the scansite 2.0 programme. We experi- mentally verified this to be the case using an in vitro PKA-dependent assay on purified wild type and S49A mutant (1–176) 6 · His-tagged emerin fusion proteins. The purified S49A fusion protein expressed in bacteria migrated at a slightly lower molecular mass than wild type upon Coomassie blue staining on SDS ⁄ PAGE (data not shown). When we performed the in vitro PKA assay on both the purified wild-type and S49A mutant emerin 1–176 fusion proteins, we observed two and one distinct phospho-emerin bands, respectively, when separated by 1D SDS ⁄ PAGE (Fig. 7). When this experiment was repeated in the presence of the PKA inhibitor, H-89, all PKA phosphorylated forms of em- erin were absent (Fig. 7). These results provide good evidence that S49 is phosphorylated by PKA and that there is probably at least one other residue within em- erin which is also phosphorylated by a PKA-dependent mechanism. Possible candidates for this second residue are S66, T67, S120, S163 and S175, with residue S120 giving the highest score using the scansite 2.0 pro- gramme set at a medium stringency setting. Discussion We have previously shown that emerin can occur in at least four differently phosphorylated forms in human lymphoblastoid cells, three of which are asso- ciated with cell cycle-dependent events [4]. In this paper, we have used mass spectrometry to detect five different species of human recombinant emerin expressed in insect cells, and identified serine 49 as being phosphorylated by a PKA-dependent mechan- ism. Whilst this manuscript was in preparation, Hirano et al. [28] reported the identification of five residues in human emerin which undergo cell cycle- dependent phosphorylation. This included residue S49, as well as S66, T67, S120 and S175, all of which, except S120, are contained within the two phosphopeptides we isolated. Similarly, neither they nor we identified any phosphorylated residues in the LEM domain. In addition, both groups present evi- dence that there remain many more phosphorylation sites to be identified. Indeed, whilst carrying out a phospho-proteomic profiling screen of tyrosine phos- phorylation sites in proteins of human T cells, Brill et al. [31] reported two additional residues, S163 and Y167, which are phosphorylated in human emerin. These too are within our second phosphopeptide. The identification of residue Y167 as being a site of phos- phorylation is in agreement with our findings of immunoblotting our baculovirus-Sf9 expressed emerin protein with a phospho-tyrosine antibody, which recognized one emerin species. Thus three different scientific approaches to elucidate the phosphorylated sites in human emerin have produced similar results. Residue S49 has been shown to be surface exposed and therefore accessible for protein–protein interac- tions and to both protein kinases and phosphatases [35]. It is also conserved in emerin from Rattus norve- gicus, Bos taurus and Caenorhabditis elegans and there- Fig. 7. Residue S49 is phosphorylated by a protein kinase A- dependent mechanism. Approximately 5 lg of purified wild-type and S49A mutant (1–176) 6 · His tagged emerin fusion proteins were incubated in a PKA-dependent assay as described in Experi- mental procedures. The purified 1–176 S49A emerin fusion protein migrates at a lower molecular mass than its wild type counterpart by Coomassie blue stained SDS ⁄ PAGE gel (data not shown). Fusion proteins were incubated under three different experimental conditions: (i) without either PKA or H-89; (ii) with PKA and (iii) with PKA and H89. All protein samples were separated on a 12% SDS ⁄ PAGE gel and autoradiography performed. Two phospho-em- erin species were observed with wild-type emerin, which was reduced to one for the S49A mutant. Addition of the PKA inhibitor H-89 disrupted phosphorylation at both of these sites. PKA phosphorylates emerin R. C. Roberts et al. 4570 FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS fore phosphorylation at this site is likely to have func- tional consequences. Although we were unable to show a direct effect of phosphorylation on emerin’s interac- tion with lamin A or C, it may affect emerin’s binding to its other binding partners. In the human emerin sequence, residue S49 is located just outside the glob- ular LEM domain [35] and phosphorylation at this site could therefore regulate emerin’s interaction with BAF. However, emerin requires BAF for its assembly into a reforming nuclear envelope [36] and our data suggests that phosphorylation at residue S49 does not have a significant effect on emerin’s targeting to a reas- sembling nuclear envelope. Phosphorylation at S49 may also regulate emerin’s interaction directly with Btf, GCL or YT521-B, since their binding sites all encompasses this residue [37]. If this proves to be the case, then the X-EDMD patient with the mutation S54F (which is in the Btf binding region), where emer- in has been shown to bind with a reduced affinity to Btf [19], may also be affected by phosphorylation at residue S49. In addition, emerin phosphorylation maybe involved in regulating the formation of ternary or higher multiprotein complexes containing emerin. For instance, it is known that BAF and GCL compete to form either emerin–BAF–lamin A or emerin–GCL– lamin A complexes [20] and phosphorylation of any of these proteins could regulate these interactions. We have shown that at least two residues in emerin are phosphorylated in a PKA-dependent manner, one of which we identified as S49. This is consistent with earlier results describing the isolation of a large protein complex consisting of PKA, the A-kinase anchoring protein AKAP149, protein phosphatase 1, nuclear lamins, emerin and actin from both differentiating myoblasts and mature myotubes [17,38]. Indeed these results suggested that PKA might regulate the emerin– actin interaction [17], with dephosphorylation enhan- cing their interaction, although it was not clear whether this was directly due to the effect of PKA phosphorylation on emerin. However, our results sug- gest that PKA phosphorylation could provide a mech- anism for regulating the role of emerin in organizing a nuclear actin cortical network [18]. Furthermore, nesprin-1a has been shown to be the receptor for AKAP149 at the nuclear envelope in neonatal rat car- diomyocytes [39] and it has the tightest affinity for emerin of any of its known binding partners. This sug- gests that nesprin, in addition to lamin A ⁄ C, anchors emerin to the nuclear envelope and is also involved in localizing PKA signalling at the nuclear envelope. Indeed, PKA-mediated phosphorylation of emerin con- taining complexes would explain the apparent link between emerin phosphorylation and cell cycle-depend- ent events, since PKA is activated as a downstream consequence of cdc2 kinase activity [40]. In conclusion, our results suggest that emerin is potentially a multiphosphorylated protein and phos- phorylation may be crucial in regulating its binding interactions with its large number of binding partners. Apart from PKA it is likely that several other known Ser-Thr ⁄ Tyr kinases or even an emerin-specific kinase may be present as components of the emerin nuclear protein complex, as is observed for the lamin B recep- tor [41]. The identification of a specific kinase that phosphorylates emerin will allow us to target specific signalling pathways to elucidate the molecular mecha- nisms underlying X-EDMD. Experimental procedures Recombinant expression and purification of human emerin The human emerin constructs encoding amino acids 1–176 and 1–220 were generated by PCR using full length emerin in pBluescript KS- as the template [4]. The amplified sequences were confirmed by sequencing and subcloned 5¢-BamH1-Sal1–3¢ into the bacterial expression vectors pET-29b (Novagen; C-terminal 6 · His tag) and pGEX- 4T-3 (Pharmacia Biotech Inc, Piscataway, NJ, USA; N-ter- minal tagged glutathione S-transferase) and into the insect cell expression vector, FastBacHTb (Bac-to-Bac expression system, Invitrogen, Ltd., Paisley, UK encoding an N-ter- minal 6x His tag). Recombinant baculovirus was prepared as described in the Bac-to-Bac manual (Invitrogen). Sf9 cells were infected with recombinant virus to express emerin and cell pellets harvested by centrifugation at 4000 g and frozen in liquid nitrogen. Infected insect cell pellets expressing emerin 1–176 were thawed and resuspended in 20 mm Tris pH 8.0, 25% (w ⁄ v) sucrose, 1 mm EDTA, 2 mm MgCl 2 and complete pro- tease inhibitor cocktail (Roche, Basel, Switzerland). Samples were refrozen in liquid nitrogen and allowed to defrost at room temperature before sonication to induce cell lysis. The samples were spun at 174 000 g for 15 min at 4 °C, with the resulting supernatant applied to Ni 2+ -NTA spin columns (Qiagen, Dorking, UK) previously washed with 20 mm Tris pH 8.0, 300 mm NaCl, 3 mm 2-mercaptoethanol. Histidine tagged emerin 1–176 bound to the Ni 2+ -NTA spin column during centrifugation at 700 g for 4 min whilst unbound protein was eluted. Contaminating proteins were further removed by centrifugation wash steps of 20 mm Tris pH 8.0, 300 mm NaCl, 3 mm 2-mercaptoethanol, followed by further washes with the same buffer augmented with 10 mm imidaz- ole. Emerin 1–176 was eluted from the Ni 2+ -NTA spin column with 20 mm Tris pH 8.0, 300 mm NaCl, 250 mm imidazole, and 3 mm 2-mercaptoethanol. Any aggregated R. C. Roberts et al. PKA phosphorylates emerin FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation ª 2006 FEBS 4571 [...]... This work was funded by an MRC studentship and Marie Curie Fellowship awarded to RCR and Muscular Dystrophy Campaign project grants awarded to AJS-S (RA3 ⁄ 593) and JAE (RA3 ⁄ 577 and RA3 ⁄ 655) and a grant from the Danish Natural Sciences Research Council (ONJ) ONJ is a Lundbeck Foundation Research Professor 12 13 References 1 Nagano A, Koga R, Ogawa M, Kurano Y, Kawada J, Okada R, Hayashi YK, Tsukahara... double labelling in our immunofluorescence experiments A rabbit polyclonal affinitypurified antiemerin antibody AP8 was used at a dilution of 1:2000 for immunoblotting emerin fusion proteins [4] An antiphosphotyrosine antibody PY20 (Zymed Laboratories, San Francisco, CA, USA) and an antithreonine antibody 4572 In vitro synthesis of radiolabelled emerin and immunoprecipitation For binding assays and subsequent... Chalfont, UK) in a final volume of 25 lL Protein products were separated on 10% SDS ⁄ PAGE gels and radiolabelled proteins visualized by autoradiography with Kodak BIOMAX-MR film (Kodak, Rochester, NY, USA) The in vitro binding assays were performed as described previously [33] Protein kinase A assay Approximately 5 lg of either wild-type or S4 9A mutant emerin (1–176) 6 · His-tagged purified fusion protein. .. CA, USA) and sequenced in their entirety by Oswel Research Products Ltd (Southampton, UK) They were then subcloned into pcDNA3.1 (Invitrogen), 5¢ HindIII-BamHI- for the in vitro transcription-translation assays Full-length wild-type human lamin A and lamin C cDNAs in pcDNA3.1 (Stratagene) were generated [33] For the PKA assay, C-terminal His-tagged wild-type and S4 9A mutant emerin (1–176) fusion proteins... Tsukahara T & Arahata K (1996) Emerin deficiency at the nuclear membrane in 14 patients with Emery–Dreifuss muscular dystrophy Nat Genet 12, 254–259 Manilal S, thi Man N, Sewry CA & Morris GE (1996) The Emery–Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein Hum Mol Genet 5, 801–808 Bione S, Maestrini E, Rivella S, Nancini M, Regis S, Romeo G & Toniolo D (1994) Identification of a. ..PKA phosphorylates emerin R C Roberts et al material was removed by centrifugation at 174 000 g for 15 min and the imidazole was removed by extensive dialysis against 20 mm Tris, pH 8.0, 300 mm NaCl, 3 mm 2-mercaptoethanol Post-translational modification of expressed emerin 1–176 was examined by 12.5% SDS ⁄ PAGE and immunoblotting with the antiemerin antibody AP8 [4] (Cell Signalling Technology, Danvers,... (Sigma) for 30 min at 4 °C Each fusion protein was then incubated with protein kinase A and [32P]ATP[cP] as described above To rule out autophosphorylation, each fusion protein was incubated with [32P]ATP[cP] in the absence of protein kinase All protein samples were separated on a 12% SDS ⁄ PAGE gel and autoradiography performed FEBS Journal 273 (2006) 4562–4575 ª 2006 The Authors Journal compilation... phosphorylation sites from human T cells using immobilised metal affinity chromatography and tandem mass spectrometry Anal Chem 76, 2763–2772 Fairley EAL, Riddell A, Ellis JA & Kendrick-Jones J (2002) The cell cycle dependent mislocalisation of emerin may contribute to the EDMD phenotype J Cell Sci 115, 341–354 Motsch I, Kaluarachchi M, Emerson LJ, Brown CA, Brown SC, Dabauvalle M-C & Ellis JA (2005) Lamin... overlap extension PCR protocols using Pfu DNA polymerase (Stratagene, Amsterdam, the Netherlands) from the full-length human emerin wild-type cDNA clone in pBluescript KS- to synthesis the emerin S4 9A and S49E mutants [9] The restriction enzyme sites HindIII and BamHI were incorporated at the 5¢- and 3¢-ends, respectively The PCR fragments generated were cloned into the pEGFP-C2 (Clontech, Palo Alto, CA,... immunoprecipitation of the bound proteins, wild-type emerin, lamin A and C as well as the mutant emerin S4 9A and S49E cDNA constructs in pcDNA3.1 were synthesized in vitro using the coupled transcription-translation TNT T7 polymerase coupled Reticulocyte Lysate System (Promega, Southampton, UK), as described previously [4,33] Synthesized proteins were labelled with l-[35S] methionine (Amersham Biosciences, . Nagano A, Koga R, Ogawa M, Kurano Y, Kawada J, Okada R, Hayashi YK, Tsukahara T & Arahata K (1996) Emerin deficiency at the nuclear membrane in patients. two, three and four phosphate groups attached, respectively. Phosphorylation was confirmed by analysis of the recombinant emerin by electrospray ionisation mass Fig.