Báo cáo khoa học: Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin pot

Identification of tyrosine-phosphorylation sites in the
nuclear membrane protein emerin
Andreas Schlosser
1,
*, Ramars Amanchy
2,
* and Henning Otto
3
1 Charite
´
, Institut fu
¨
r Medizinische Immunologie, Berlin, Germany
2 McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD,
USA
3 Freie Universita
¨
t Berlin, Institut fu
¨
r Chemie und Biochemie, Germany
The nuclear envelope encloses the genetic material of a
eukaryotic cell and takes part in its structural and
functional organization. It consists of interconnected
membranes, an outer nuclear membrane (ONM) and
an inner nuclear membrane (INM). The ONM is part
of the rough endoplasmic reticulum and folds at the
nuclear pores into the INM, which is firmly attached
to the lamina by integral membrane proteins of the
INM. The INM proteins form complexes, transiently
or stably, with lamins, chromatin proteins and a vari-
ety of regulatory proteins, including transcriptional

regulators and splicing factors [1,2]. Attempts have
been made to identify and catalogue the complete rep-
ertoire of nuclear-envelope proteins by subcellular pro-
teomics. These approaches resulted in several novel
validated nuclear membrane proteins and also in long
lists of putative protein constituents of the nuclear
envelope awaiting their validation [3,4].
Such an inventory is just a first step that must be
followed by the analysis of molecular interactions of
the nuclear-envelope proteins. Well-characterized nuc-
lear-envelope proteins like the lamin B receptor, the
lamina-associated polypeptide 2 (LAP2) membrane iso-
forms, emerin or the lamins, evidently participate in
the formation of distinct complexes by the cell at the
right place and the right time [5–12]. To regulate such
complex interactions, cells use post-translational modi-
fications; their regulatory repertoire relies mostly on
the transient phosphorylation of either serine ⁄ threon-
ine or tyrosine residues [13,14]. The identification
of such post-translational modifications is efficiently
addressed by specialized mass spectrometric techniques
Keywords
Emerin; Emery–Dreifuss muscular
dystrophy; nuclear envelope;
phosphorylation; proteomics
Correspondence
H. Otto, Freie Universita
¨
t Berlin, Institut fu
¨

r
Chemie und Biochemie, Thielallee
63, D-14195 Berlin, Germany
Fax: +49 30 83853753
Tel: +49 30 83856425
E-mail:
*These authors contributed equally to this
work
(Received 17 January 2006, revised 27 April
2006, accepted 18 May 2006)
doi:10.1111/j.1742-4658.2006.05329.x
Although several proteins undergo tyrosine phosphorylation at the nuclear
envelope, we achieved, for the first time, the identification of tyrosine-phos-
phorylation sites of a nuclear-membrane protein, emerin, by applying two
mass spectrometry-based techniques. With a multiprotease approach com-
bined with highly specific phosphopeptide enrichment and nano liquid
chromatography tandem mass spectrometry analysis, we identified three
tyrosine-phosphorylation sites, Y-75, Y-95, and Y-106, in mouse emerin.
Stable isotope labeling with amino acids in cell culture revealed phospho-
tyrosines at Y-59, Y-74, Y-86, Y-161, and Y-167 of human emerin. The
phosphorylation sites Y-74 ⁄ Y-75 (human ⁄ mouse emerin), Y-85 ⁄ Y-86,
Y-94 ⁄ Y-95, and Y-105 ⁄ Y-106 are located in regions previously shown to
be critical for interactions of emerin with lamin A, actin or the transcrip-
tional regulators GCL and Btf, while the residues Y-161 and Y-167 are in
a region linked to binding lamin-A or actin. Tyrosine Y-94 ⁄ Y-95 is located
adjacent to a five-residue motif in human emerin, whose deletion has been
associated with X-linked Emery–Dreifuss muscle dystrophy.
Abbreviations
EDMD, Emery–Dreifuss muscle dystrophy; INM, inner nuclear membrane; LC, liquid chromatography; MS ⁄ MS, tandem mass spectrometry;
ONM, outer nuclear membrane; SILAC, stable isotope labeling with amino acids in cell culture.

3204 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
[15–17], which allowed for example the identification
of peptides from nuclear-envelope proteins phosphoryl-
ated at serine or threonine residues [18]. However,
tyrosine-phosphorylation sites have not been identified
so far.
While improving the search for phosphopeptides
containing phosphotyrosine, we identified one of the
well-defined integral INM proteins. Emerin is a type-II
integral membrane protein of 34 kDa, which is inser-
ted into the membrane with a single transmembrane
sequence near its carboxy-terminus and is targeted to
the inner nuclear membrane [19,20]. In humans, emerin
is the gene product of the EMD gene that is associated
with the X-chromosome-linked form of the inherited
Emery–Dreifuss muscular dystrophy (EDMD), leading
to slowly progressing muscle wasting and a cardiomy-
opathy with conduction defects [19,21]. On the cellular
level, EDMD is characterized by a mislocalization of
emerin that is caused by the loss of emerin binding to
lamin A (X- linked form) or by the loss of lamin A
(autosomal dominant form) [22,23].
Several emerin binding partners have been detected
and partial sequences required for their binding have
been mapped [10,11,24–29], which will probably form
different emerin complexes, whose formation may be
regulated by transient phosphorylation.
In this study, we describe the identification of emerin
as the first tyrosine-phosphorylated nuclear-envelope
protein. We have identified tyrosine phosphorylation

sites on human and mouse emerin using independently
two different strategies: (1) a multiprotease approach,
where we combined subcellular fractionation of mouse
N2a cells with in-gel digestion of emerin using a set of
different proteases followed by phosphopeptide enrich-
ment using the phosphopeptide affinity matrix titan-
sphere [30]; and (2) stable isotope labeling with amino
acids in cell culture (SILAC) in combination with
antiphosphotyrosine immunoprecipitation and tryptic
in-gel digestion to identify human emerin phosphory-
lation sites in HeLa cells. This led to the identification
of tyrosine phosphorylation sites of mouse and human
emerin.
Results
To identify tyrosine-phosphorylated nuclear-envelope
proteins and their phosphorylation sites, we used a mul-
tiprotease approach on mouse cells (Fig. 1A) and the
SILAC approach on human cells (Fig. 3A). The analy-
sis of phosphorylated cellular proteins requires an effi-
cient inhibition of endogenous protein phosphatases.
This is particularly important for studying tyrosine
phosphorylation, as it is highly transient due to very
A
B
Fig. 1. Multi-protease approach. (A) Scheme of the approach. Nuc-
lear envelopes were purified from BiPy-treated N2a cells (mouse
neuroblastoma), and the protein mixtures separated by SDS ⁄ PAGE.
An aliquot of the sample was used for western blot analysis. The
pattern of tyrosine-phosphorylated nuclear-envelope proteins, visu-
alized by using the phosphotyrosine-specific antibody PY99 (horse-

radish peroxidase conjugate) and ECL, was used for sample
selection on a Coomassie-stained reference gel. The protein bands
cut from the gel were divided into four aliquots and digested with
trypsin, elastase, proteinase K and thermolysin, respectively. The
extracted peptides of all four digests were mixed, phosphopeptides
were enriched on a titansphere column and analyzed by nanoLC-
MS ⁄ MS. (B) Immunoblot of tyrosine-phosphorylated nuclear
envelope proteins and the corresponding Coomassie-stained gel.
According to the pattern of phosphotyrosine immunostaining (ECL),
samples 1 and 2 were selected for further analysis.
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3205
active phosphotyrosine-specific phosphatases. There-
fore, dephosphorylation of phosphotyrosine was pre-
vented by the addition of very potent, cell-permeable,
highly specific tyrosine-phosphatase inhibitors to the
cells in culture.
In the case of the multiprotease approach (Fig. 1A),
we added 100 lm of the tyrosine-phosphatase inhibitor
BiPy [31] to the culture medium of mouse-neuroblast-
oma N2a cells 10 min before harvesting the cells
(BiPy-treated cells). This results in hyperphosphoryla-
tion of proteins [Fig. 1B, left panel and Fig. 2, left
panel (BiPy+)], which is required for a successful
phosphopeptide analysis.
Before starting with protein and phosphopeptide
identification, we compared tyrosine-phosphorylation
of nuclear-envelope proteins from control cells (no
BiPy added prior to homogenization) and from BiPy-
treated cells (Fig. 2). BiPy treatment should increase

the amount of tyrosine phosphorylation but should
not change the pattern of tyrosine-phosphorylated nuc-
lear envelope proteins. To prevent, as far as possible,
changes of tyrosine phosphorylation after breaking up
the cells, we simultaneously added 500 nm of the
broad-range protein kinase inhibitor staurosporine and
100 lm of phosphotyrosine phosphatase inhibitor BiPy
(in addition to sodium vanadate and sodium molyb-
date) to both control cells and BiPy-treated cells at the
beginning of homogenization. Both inhibitors were
then present throughout the preparation of nuclei and
nuclear envelopes, although, in contrast to the phos-
phatases, the kinases should not work efficiently
anymore due to a lack of ATP. Then, the nuclear-
envelope proteins were separated by SDS ⁄ PAGE, blot-
ted onto nitrocellulose and sequentially immunostained
for emerin and for phosphotyrosine.
Control cells already show a weak pattern of tyro-
sine-phosphorylated nuclear envelope proteins [Fig. 2,
left panel (BiPy–)], with some of the phosphotyrosine
immunostaining overlapping with emerin immuno-
staining (Fig. 2, arrows). For BiPy-treated cells, this
pattern of tyrosine-phosphorylated proteins increases
in intensity but does not considerably change other-
wise. This suggests that our approach of adding BiPy
before harvesting the cells enhances physiologically
relevant tyrosine phosphorylation of nuclear-envelope
proteins, as the interaction of tyrosine kinases and sub-
strate proteins is still restricted to their endogenous
compartments at that point.

An efficient hyperphosphorylation is achieved only,
when BiPy is added before homogenizing the cells. Sta-
urosporine, on the other hand, did not seem to have
much influence on the tyrosine phosphorylation pat-
tern after homogenization of the cells (data not
shown). Therefore, we omitted staurosporine during
preparation of hyperphosphorylated nuclear envelopes
intended for phosphopeptide analysis.
For the mass-spectrometric identification of phos-
phopeptides, we purified nuclei from BiPy-treated cells,
from which we obtained nuclear envelopes by digesting
nucleic acids under hypo-osmotic conditions (Fig. 1B).
Throughout the preparation, 100 lm BiPy and 1 mm
each of sodium vanadate and sodium molybdate were
present to preserve the phosphorylation obtained in
the living cell immediately before homogenization.
An aliquot of the protein mixture was then separ-
ated by one-dimensional SDS ⁄ PAGE, blotted on
nitrocellulose and immunostained with the phosphotyr-
osine-specific antibody PY99. Nuclear membrane frac-
tions were run in parallel on a second gel to separate
the protein mixtures for mass spectrometric analysis of
tyrosine-phosphorylated proteins. Figure 1B shows the
pattern of tyrosine-phosphorylation (PY) for nuclear-
envelope proteins (NE), and the corresponding Coo-
massie-stained gel. A complex pattern of putatively
tyrosine-phosphorylated proteins is visible. Regions of
Fig. 2. Enhancement and preservation of tyrosine phosphorylation
in nuclear envelopes from N2a cells. N2a cells in culture were
either treated with 100 l

M BiPy or left untreated as control cells.
Then, nuclear envelopes were prepared in the constant presence
of 500 n
M staurosporine and 100 lM BiPy in order to preserve the
phosphorylation status reached at the time of homogenization. The
proteins were separated by SDS ⁄ PAGE, blotted and immuno-
stained for emerin and for phosphotyrosine. In the absence of BiPy,
a weak pattern of proteins phosphorylated at tyrosine residues
appears, which is increased in intensity under hyperphosphorylating
conditions. Arrows indicate the phosphotyrosine bands correspond-
ing to the emerin bands on the left.
Tyrosine-phosphorylation of emerin A. Schlosser et al.
3206 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
the Coomassie-stained reference gel corresponding to
the strongest PY99-reactivity (samples 1 and 2;
Fig. 1B) were excised from the gel and further ana-
lyzed. In-gel digestion of these bands was done in par-
allel with four enzymes: trypsin, elastase, proteinase K,
and thermolysin. The four digests were pooled; phos-
phopeptides were enriched on a titansphere nano-col-
umn, eluted, and analyzed by nanoLC-MS ⁄ MS. Four
different proteins were detected in the two samples. In
sample 1 (apparent molecular weight 35–45 kDa),
LAP2, possibly the membrane isoform LAP 2 c
(38.5 kDa calculated, accession number AAH64677),
was identified.
In sample 2 (apparent molecular weight 25–35 kDa),
nucleophosmin-1 (nucleolar phosphoprotein B23, 28.4
kDa calculated, accession number NP_032748), cation-
dependent mannose-6-phosphate receptor (31.1 kDa

calculated, accession number NP_034879), and emerin
(29.4 kDa calculated, accession number NP_031953)
were present in addition to LAP2.
First, surprisingly neither a phosphotyrosine-contain-
ing peptide nor an emerin peptide could be detected in
sample 1, although this sample should correspond to a
region of phosphotyrosine-immunostaining stronger
than that corresponding to sample 2. Secondly, sample
1 should also contain the upper emerin band, which
most likely reflects a different, not yet characterized
emerin phosphorylation state [20].
Both samples, cut from the gel, contain more than
one protein. Also, it is not possible to exactly control
the protein composition in such an excised gel piece.
One explanation for this lack could therefore be that
the phosphotyrosine immunostaining, despite overlap-
ping with emerin immunostaining, may be caused by
another protein. Another explanation could be a low
content of tyrosine-phosphorylated peptides, for
example, due to a high amount of other proteins in
the sample. Also, the strength of the phosphotyrosine
immunosignal may be misleading, since the affinity of
phosphotyrosine-specific antibodies is always influ-
enced by amino acid residues surrounding the phos-
photyrosine. Finally, emerin in sample 1 could carry
different phosphotyrosines that, despite using four
different proteases, might be located in a sequence
not suitable for mass spectrometric analysis.
The method applied facilitates the identification of
phosphopeptides in general. As most regulatory

phosphorylation events occur at serine and threonine
residues and persist longer in the cells than the
highly transient tyrosine phosphorylation, their detec-
tion is much more likely. It is therefore not surpri-
sing that we detected in all identified proteins serine-
and threonine phosphorylation sites. We found the
following Ser ⁄ Thr-phosphorylation sites. Three new
sites for LAP 2: (1) S-183, (2) T-316 or T-319, and
(3) one in the region between T-153 and S-158) in
addition to the previously identified sites [18]; for
nucleophosmin, S-4, S-10, S-70, and S-125; and for
A
B
Fig. 3. Stable isotope labeling with amino acids in cell culture
(SILAC) approach. (A) Scheme for the identification of emerin phos-
phorylation sites. HeLa cells were grown in two different popula-
tions, one in normal medium and the other in medium containing
arginine and lysine labeled with stable isotopes (described in meth-
ods). The cells growing in heavy isotope medium were treated to
1m
M sodium pervanadate. The cell lysates were mixed after deter-
gent lysis of the cells, followed by the immunoprecipitation of
tyrosine-phosphorylated proteins. The proteins were separated by
SDS ⁄ PAGE. A protein band corresponding to 30 kDa was excised
and digested with trypsin before analyzing the peptides by LC-
MS ⁄ MS. (B) MS spectrum showing the doubly charged peptide
pair (light and heavy isotope pair) with a mass shift of 6 Da, which
corresponds to the unphosphorylated emerin peptide KIFEYETQR
(aa residues 37–45, with and without one
13

C
6
-Arg and one
13
C
6
-
Lys). The heavy peptide from the pervanadate-treated cells shows
an increased intensity due to the increase of tyrosine-phosphorylat-
ed emerin in these cells.
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3207
mouse emerin, one Ser-phosphorylation site was
detected on S-72.
Tyrosine phosphorylation sites could only be detec-
ted for emerin, which colocalizes in western blotting
with a weak phosphotyrosine immunosignal in control
cells and with a strong immunosignal under hyper-
phosphorylating conditions (Fig. 2). The identified
peptides from mouse emerin (including the S-72 phos-
phorylation) are listed in Table 1. In total, 12 peptides
were assigned to emerin, eight phosphopeptides and
four acidic peptides. Although the phosphopeptide
affinity material titansphere shows excellent selectivity
for phosphopeptides, peptides with five or more acidic
residues are sometimes coenriched under the applied
conditions. Three tyrosine phosphorylation sites were
identified: Y-75, Y-95, and Y-106. Figure 4A shows
the MS ⁄ MS spectrum of the peptide DYNDD-pY-YE-
ESYLTTK (aa residue 90–104, mouse emerin) as an

example. Although the peptide contains four tyrosine
residues, the phosphorylated tyrosine can be clearly
located on Y-95 (mass difference of a phosphotyrosine
residue (243.03 Da) between carboxy-terminal frag-
ment ions y
9
and y
10
, which comprise the 9 and 10
carboxy-terminal amino-acid residues, respectively).
The SILAC approach (Fig. 3A) is based on in vivo
labeling of all the cellular proteins by isotope-coded
amino acids. In addition, we used the determination of
relative ratios of peptide abundance obtained from
proteins isolated from hyperphosphorylated and refer-
ence cells to distinguish between nonspecifically cap-
tured vs. true IP-captured tyrosine-phosphorylated
proteins.
To achieve labeling, we added a mixture of argin-
ine and lysine, each containing six
13
C atoms (
13
C
6
-
Arg and
13
C
6

-Lys), to HeLa cells in culture. The two
amino acids were chosen because the tryptic protein
digestion applied later in the procedure would gener-
ate peptides ending with arginine and lysine residues.
This ensures the generation of labeled peptides and
nonlabeled but otherwise identical reference peptides.
As reference, HeLa cells were grown with unlabeled
arginine and lysine (Fig. 3A). The cells first were
serum-starved for 12 h, prior to 1 mm sodium per-
vanadate treatment for 30 min. Sodium pervanadate
treatment of HeLa cells (20 large dishes) grown in
the presence of the heavy amino acids
13
C
6
-arginine
and
13
C
6
-lysine created a state of hyperphosphoryla-
tion of all cellular proteins. For comparison, HeLa
cells (20 large dishes) were grown with unlabeled
arginine and lysine. In cells growing in five (i.e. one
quarter) of these reference dishes, tyrosine phosphory-
lation was also stimulated to generate a certain
amount of tyrosine phosphorylation necessary for the
final comparison of tyrosine-phosphorylated proteins
Table 1. Phosphorylation sites of mouse and of human emerin.
Phosphorylation

site
Number of
phosphorylated residues Peptide sequence Residues
Additional
modifications Species
S-72 or Y-75 1 AVDSDMYDLPKKEDAL 69–85 Oxidation (M) Mouse
Y-75 1 AVDSDMYDLPKKEDA 69–84 Mouse
S-72 and Y-75 2 AVDSDMYDLPKKE 69–82 Mouse
S-72 and Y-75 2 AVDSDMYDLPKKEDAL 69–85 Oxidation (M) Mouse
Y-75 1 MYDLPKKE 74–81 Mouse
0 DYNDDYYEE 90–98 Mouse
0 DYNDDYYEESY 90–100 Mouse
0 DYNDDYYEESYLTTK 90–104 Mouse
Y-95 1 DYNDDYYEESYLTTK 90–104 Mouse
Y-106 1 LTTKTYGEPES 101–111 Mouse
Y-106 1 LTTKTYGEPESVGMSKS 101–117 Oxidation (M) Mouse
0 DDIFSSLEEEGKDR 138–150 Mouse
0 RYNIPHGPVVGSTR 17–31 2
13
C
6
-Arg Human
0 YNIPHGPVVGSTR 18–31
13
C
6
-Arg Human
0 KIFEYETQR 37–45
13
C

6
-Arg and
13
C
6
-Lys Human
0 IFEYETQR 38–45 Human
S-49 and Y-59 2 RLSPPSSSAASSYSFSDLNSTR 47–68 2
13
C
6
-Arg Human
Y-74 1 GDADMYDLPKKEDALLYQSK 69–88 Oxidation (M)
3
13
C
6
-Lys
Human
0 KEDALLYQSK 79–88 Human
0 KEDALLYQSK 79–88 2
13
C
6
-Lys Human
Y-85 1 KEDALLYQSK 79–88 2
13
C
6
-Lys Human

Y-161 and Y-167 2 DSAYQSITHYRPVSASR 158–174 2
13
C
6
-Arg Human
Tyrosine-phosphorylation of emerin A. Schlosser et al.
3208 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
in both cell populations. The cells were lyzed, the
lysates from the two states were mixed, and tyrosine-
phosphorylated proteins were extracted by immuno-
precipitation applying the phosphotyrosine-specific
antibodies 4G10 and RC20. Proteins were eluted
from the precipitated immune complexes with phenyl-
phosphate, separated by SDS ⁄ PAGE and stained with
colloidal Coomassie blue. Bands of stained proteins
were then excised from the gel. The proteins were
reduced, alkylated, and digested with trypsin within
the gel matrix. The peptides extracted from the gel
matrix were finally analyzed by reversed-phase liquid
chromatography tandem mass spectrometry (LC-
MS ⁄ MS). The sequences obtained from MS ⁄ MS spec-
tra were analyzed and potential phosphopeptides con-
taining tyrosine were scanned by plotting the relevant
extracted ion chromatograms for the corresponding
unphosphorylated peptide (80 Da mass difference for
single-charged peptides). Unphosphorylated peptides
could be detected for all identified tyrosine-phosphor-
ylated peptides giving an additional confirmation for
the correct assignment.
In comparison to the reference cells, tryptic peptides

from the tyrosine-phosphorylated cells labeled with
13
C
6
-Arg and
13
C
6
-Lys show up with a mass difference
of 6 or multiples of 6. As a quarter of the reference
Fig. 4. Identification of emerin and of tyro-
sine-phosphorylation sites. (A) MS ⁄ MS
spectrum of the mouse-emerin peptide
DYNDD-pY-YEESYLTTK (aa residues 90–
104), phosphorylated at Y-95, which was
obtained by the multiprotease approach.
(B) MS ⁄ MS spectrum of the human-emerin
peptide RL-pS-PPSSSAASS-pY-SFSDLNSTR
(aa residues 47–68), which is phosphorylat-
ed at S-49 and Y-59, obtained by using the
SILAC approach. In both figure parts, the
phosphotyrosine-specific mass difference
between the appropriate carboxy-terminal
y-ions is indicated by a bar labeled ‘pY’.
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3209
cells were also stimulated to undergo tyrosine-phos-
phorylation, peptide pairs that are tyrosine-phosphor-
ylated are expected to show such a mass difference.
Peptides derived from proteins that undergo tyrosine-

phosphorylation due to pervanadate treatment but are
not tyrosine-phosphorylated in control cells appear as
pairs, which show an ion ratio (peptides from hyper-
phosphorylated cells (pervanadate treatment) ⁄ peptides
from control cells) close to 4. In contrast, all proteo-
lytic peptides from nonspecifically captured proteins
show a ratio close to 1.
For the identification of tyrosine phosphorylation
sites, only such peptide pairs were used, where the
quantification showed a significantly increased amount
of the tyrosine-phosphorylated peptides obtained from
the pervanadate-treated population of cells [32]. As an
example, the peptide pair corresponding to the non-
phosphorylated peptide KIFEYETQR (aa residues 37–
45) of human emerin is shown in Fig. 3B. Since tyro-
sine-phosphorylated emerin has been enriched by the
phosphotyrosine affinity-purification step, this partic-
ular peptide shows a 3.5-fold increase in signal inten-
sity between the peptide from the control cells and
from the pervanadate-treated cells. The heavy peptide
contains one
13
C
6
-lysine and one
13
C
6
-arginine, which
result in a m ⁄ z-difference of +6 for the doubly

charged peptide.
Analyzing all Coomassie-stained bands of the
SDS ⁄ PAGE, several tyrosine-phosphorylated proteins
have been identified. However, emerin (human emerin,
accession number NP_000108) was the only identified
protein with known localization at the inner nuclear
membrane.
For emerin, we identified the tyrosine residues Y-59,
Y-74, Y-85, Y-161 and Y-167 as phosphorylation sites
of human emerin. The observed ratios between heavy
and light emerin peptides range from 2.5 to 6.5. Sim-
ilar values are obtained for other tyrosine-phosphoryl-
ated proteins. This fluctuation is greater than typically
observed for classic SILAC experiments. However, this
is not particularly relevant for our approach, since we
only have to be able to distinguish between ratios close
to 1 (nonspecific impurities) and ratios close to 4 (pro-
teins that are tyrosine-phosphorylated upon pervana-
date treatment). Ratios smaller than 4 are expected, if
a protein is already partially tyrosine-phosphorylated
before treatment with pervanadate.
All of these tyrosines are conserved between human
and mouse emerin (equivalent positions of mouse
emerin are the aa residues Y-60, Y-75, Y-86, Y-161
and Y-167). Figure 4B, for example, shows the emerin
peptide RL-pS-PPSSSAASS-pY-SFSDLNSTR (aa res-
idues 47–68), which is phosphorylated at S-49 and
Y-59. The serine phosphorylation site, unique for
human emerin, reflects probably a basal phosphoryla-
tion not attributed to the pervanadate treatment.

All identified phosphopeptides are summarized in
Table 1 and the phosphorylation sites are indicated in
the emerin alignment (Fig. 5A).
Fig. 5. Scheme of emerin interactions and EDMD mutations. (A)
Alignment of human and mouse emerin. ‘P’ indicates identified
tyrosine-phosphorylation sites. (B) Schematic representation of the
binding interactions mapped onto the emerin structure. Black lines
indicate the different phosphorylation sites, grey bars and a star the
EDMD mutations S-54 F, Del95–99, and P-183 H ⁄ T. The numbers
shown are based on the human emerin sequence. Equivalent
sequence positions (human ⁄ mouse emerin) are Y-59 ⁄ Y-60,
Y-74 ⁄ Y-75, Y-86 ⁄ Y-87, Y-94 ⁄ Y-95, Y-105 ⁄ Y-106, Y-161, and Y-167.
LEM, LEM domain; TM, membrane-spanning sequence.
Tyrosine-phosphorylation of emerin A. Schlosser et al.
3210 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
Discussion
By applying two different approaches to either lysates
from human cells or to isolated mouse nuclear enve-
lopes, we identified emerin as a tyrosine-phosphoryla-
ted protein of the inner-nuclear membrane, which
seems to be a key protein in building different com-
plexes with other proteins at the nuclear envelope. For
both human and mouse emerin, we were able to deter-
mine a few sites that are targets of tyrosine kinase
activity. In this study, the multiprotease approach and
the SILAC approach complement each other. While
the phosphorylation site Y-74 ⁄ 75 (human ⁄ mouse
emerin) has been identified with both methods, the
phosphorylation sites Y-94 ⁄ 95 and Y-105⁄ 106 have
been detected only in mouse emerin and the sites

Y-59 ⁄ Y-60, Y-161 and Y-167 only in human emerin.
Although a different phosphorylation may result from
species-specific differences of the phosphorylation
machinery, it seems more likely that the different cell
types with their specific kinase and phosphatase equip-
ment and their differing regulatory properties account
for the differences in the usage of the emerin phos-
phorylation sites in mouse N2a and human HeLa cells.
In addition, the different methods applied may also
contribute to the differences in identified phosphoryla-
tion sites. The multiprotease approach was used in a
subcellular-proteomics background. As it does not rely
on the comparison of emerin from differently treated
cells but on the analysis of isolated emerin from a
quasi homogeneous source, this approach should
enable the identification of all tyrosine-phosphorylated
emerin peptides provided that their quantity and their
affinity to the titansphere column are sufficient to pass
a detectable amount to the mass spectrometer. The
SILAC approach, on the other hand, uses two differ-
ent filters for identifying phosphorylation sites. First,
binding to phosphotyrosine-specific antibodies is used
to enrich tyrosine-phosphorylated proteins. As residues
surrounding the phosphotyrosine also influence bind-
ing to such antibodies, this step might favor sub-
populations of differentially phosphorylated emerin.
Secondly, this method filters for such peptides, which
show an increase in quantity of tyrosine-phosphorylat-
ed peptides from the unlabeled reference sample to the
labeled tyrosine-phosphorylation sample. Although this

might discriminate against peptides that may be con-
siderably phosphorylated in the unlabeled reference
cells, this filter was applied to prevent false positives
due to nonspecific binders that would appear with the
same intensity in both samples. As all identified tyro-
sine-phosphorylation sites seem to be conserved in
mammalian emerin, they could as well be used simi-
larly in all species for differentially regulating the
diverse interactions demonstrated for emerin.
Emerin is the product of a gene linked to EDMD
[19,21]. An integral membrane protein specifically loca-
ted at the inner nuclear membrane, emerin, like other
INM proteins, binds to lamins. It is linked to EDMD
by its interaction with lamin A [20]. In EDMD this
interaction is weakened or lost either by mutations in
emerin itself, which leads to the X-linked form of
EDMD [20,25,33,34], or by a loss of lamin A, which
causes the autosomal-dominant form of EDMD
[22,23,35]. In both forms, emerin is mislocalized [36]
and cannot efficiently accumulate at the inner nuclear
membrane [37].
For several proteins shown to interact with emerin,
binding regions were mapped onto the emerin
sequence (Fig. 5B). The best characterized of these
interactions occurs between the DNA ⁄ protein com-
plexes of the heterochromatin protein BAF and emer-
in’s amino-terminal LEM domain (aa residues 2–44)
[38], which similarly exists in the INM proteins MAN1
and LAP2 [24,39]. This interaction is also necessary
for a correct localization after mitosis [37]. Recently,

Hirano and coworkers identified with a similar
approach four serine and one threonine residues of
human emerin that in vitro are strongly phosphoryla-
ted in M-phase extracts prepared from Xenopus eggs.
They showed that phosphorylation of one site, S-175,
causes BAF dissociation from emerin. Remarkably,
this site is at the primary structure level far away from
the N-terminal LEM-domain that is thought to medi-
ate this interaction [40]. Lamin-A binding has been
mapped to the central region of emerin (aa residues
70–178) [38], which overlaps with a region capping
actin filaments [10,26]. Binding of transcriptional regu-
lators like GCL and the death-promoting transcrip-
tional repressor Btf or the splicing factor YT521-B
involves emerin sequences on both sides of the central
region that interacts with cytoskeletal elements, parti-
ally overlapping with the LEM domain as well as with
the lamin-A binding region [11,27,41]. Not as well
characterized but important in the context of a muscle
dystrophy is a muscle-specific interaction with the
actin-binding spectrin-repeat proteins nesprin 1a and
nesprin 2 [28,29].
Since the different binding regions overlap at least
partially, a simultaneous binding of some binding part-
ners may be excluded, giving rise to distinct emerin
complexes [41]. Cells may be able to control the forma-
tion of such complexes by phosphorylating critical resi-
dues of emerin. In fact, the disruption of BAF binding
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3211

to emerin in mitosis seems to be mediated by phos-
phorylation of S-175 of emerin [40].
Interestingly, the identified tyrosine phosphorylation
sites all are located near regions that have been proven
as disease-related. Several mutations in human emerin
have been linked to EDMD. Among these are the mis-
sense mutations S54F, P183H, P183T, and a deletion
of five amino acids (Del 95–99, YEESY), which hint
at regions critical for regulated complex formation
[34,37]. S54F and Del 95–99 disrupt indeed the Btf
binding to emerin. Del 95–99 also disrupts the interac-
tion with lamin A and GCL [27].
The tyrosine-phosphorylation sites Y-59 ⁄ Y-60
(human ⁄ mouse emerin) and Y-74 ⁄ Y-75 are exactly
positioned in a region of overlapping binding sites and
could help to control interactions with lamin A, actin
and the transcriptional regulators GCL and Btf. For
comparing mouse and human emerin sequences, note
that the sequence alignment shown for mouse emerin
has an additional amino acid residue after residue 57
and a gap after residue 138. Thus, equivalent amino
acid residues of human and mouse emerin differ in
numbering by one between these sequence positions
(Fig. 5A). The phosphorylation sites Y-94 ⁄ Y-95
(human ⁄ mouse emerin) and Y-105 ⁄ Y-106 are even
more directly linked to an EDMD mutation. Y-94 ⁄ 95
is almost directly affected by the EDMD-linked dele-
tion mutation Del 95–99 of human emerin, while
Y105 ⁄ 106 seems near enough to this region to influ-
ence emerin binding to other proteins. Phosphorylation

of both tyrosine residues may differentially influence
emerin binding to actin and lamin A.
The phosphorylated tyrosine residues Y-161 and
Y-167, identified in human emerin, are located within
a region of emerin that has been linked to lamin A ⁄ ac-
tin or to lamin-A binding, respectively, which is likely
to be regulated by such phosphorylation. Ellis et al.
[20] have shown that different phosphorylation states
of emerin are linked to EDMD [20]. Four different cell
cycle-dependent phosphorylation states have been iden-
tified. In patients, mutated emerin forms, which show
a changed solubility or extractability compared with
wild-type emerin, undergo also aberrant phosphoryla-
tion. The corresponding phosphorylation sites, how-
ever, remained elusive.
If and how the phosphorylation sites determined
here may be correlated to those aberrant phosphoryla-
tion states described for EDMD patients and how
these sites take part in intermolecular interactions,
remains to be investigated. The analysis of phosphory-
lation sites that we present here requires hyperphos-
phorylation conditions. Phosphorylation, however, was
initiated in both approaches by already adding a
cell-permeable tyrosine-phosphatase inhibitor based on
pervanadate before opening up the cells. The compar-
ison of the tyrosine-phosphorylation status of nuclear
envelopes from BiPy-treated and control cells (Fig. 2)
indicates that the regular pattern of tyrosine phos-
phorylation of the control cells is just enhanced in the
presence of the pervanadate compounds. Also, the

ATP concentration drops after homogenization to lev-
els that do not allow any further phosphorylation.
Therefore, the phosphorylation at the determined sites
occurred already in the living cells, while emerin was
still in its native environment, involved in its normal
molecular interactions. Thus, the determined sites most
likely reflect sites that are used under physiological
conditions.
Experimental procedures
Multi-protease approach: sample preparation
Mouse-neuroblastoma N2a cells were cultured in Dulbec-
co’s modified Eagle’s medium containing 10% fetal bovine
serum, 100 mgÆmL
)1
streptomycin, and 100 mgÆmL
)1
peni-
cillin at 37 °C in a humidified atmosphere with 5% CO
2
.
Nuclei and nuclear envelopes were prepared from N2a
cells [42]. Ten minutes before the cells were harvested for
nuclear preparation, the selective tyrosine-phosphatase
inhibitor potassium-(2,2¢-bipyridine)-oxobisperoxovanadate
(BiPy) [31] was added to the culture medium at a concen-
tration of 100 lm. Throughout the purification, BiPy
(100 lm), as well as the phosphatase inhibitors sodium
vanadate (1 mm) and sodium molybdate (1 mm), were
present to prevent dephosphorylation of the proteins.
Tyrosine-phosphorylated proteins were separated by

SDS ⁄ PAGE, blotted onto a nitrocellulose blot membrane
[43,44] and visualized on the membrane by enhanced chemi-
luminescence (ECL), using the phosphotyrosine-specific
antibody PY99 conjugated to horseradish peroxidase (BD
Biosciences-Pharmingen, Heidelberg, Germany) [42]. Like-
wise, blotted protein mixtures were probed for emerin by
applying the antibody Emerin (FL-254) (Santa Cruz Bio-
technology, Inc., Heidelberg, Germany).
When staurosporine was used, 500 nm were added at the
time of homogenization together with the 100 lm BiPy.
Staurosporine and BiPy were then kept present throughout
the preparation.
SILAC: sample preparation
Human cervical carcinoma (HeLa) cells were grown in Dul-
becco’s modified Eagle’s medium containing ‘light’ arginine
and lysine or
13
C
6
-arginine and
13
C
6
-lysine supplemented
with 10% dialyzed fetal bovine serum plus antibiotics [32].
The cells were grown for five passages in the above medium
Tyrosine-phosphorylation of emerin A. Schlosser et al.
3212 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
prior to initiating these experiments. HeLa cells were serum
starved for 12 h before treatment with 1 mm pervanadate

for 30 min and were subsequently lysed in a modified RIPA
buffer (50 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl, 1 mm
EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate,
and 1 mm sodium orthovanadate in the presence of prote-
ase inhibitors). Light and heavy cell lysates were precleared
with protein A-agarose, mixed and incubated with 400 lg
of 4G10 monoclonal antibodies coupled to agarose beads
and 75 lg of RC20 antibodies, overnight at 4 °C. Precipita-
ted immune complexes were washed three times with lysis
buffer and then eluted three times with 100 mm phenyl-
phosphate in lysis buffer at 37 °C. The eluted phosphopro-
teins were dialyzed and resolved by 10% SDS ⁄ PAGE. The
gels were stained using colloidal Coomassie stain.
Proteolytic digestion for MS analysis
Proteins were excised from the gel. In the multiprotease
approach, the excision was guided by the pattern of the
immunoblot of an identical reference gel. The gel pieces
were destained with 30% acetonitrile. After reduction and
alkylation of the proteins, the gel pieces were dehydrated
with 100% acetonitrile and dried in a vacuum centrifuge.
In the multiprotease approach, the proteins were digested
in parallel with trypsin, elastase, proteinase K, and thermo-
lysin (about 0.1 lg of each protease) in 0.1 m NH
4
HCO
3
(pH 8) at 30 °C overnight. Peptides were then extracted
from the gel slices with 5% formic acid. All supernatants
and extracts were combined, dried in a vacuum centrifuge,
and redissolved in 10 lL of 30% acetonitrile and 2% for-

mic acid. Phosphopeptides were enriched on nano-columns
(inner diameter: 50 lm; length: 1.5 cm) packed with titan-
sphere (5 lm particles, GL Sciences Inc., Tokyo, Japan),
which were washed with 30% acetonitrile, 2% formic acid,
and were eluted with 10 lL of 0.1 m NH
4
HCO
3
(pH 9).
The eluate was acidified by mixing with formic acid and
was analyzed with nanoLC-MS ⁄ MS using a reversed-phase
column with an inner diameter of 25 lm [30].
In the SILAC experiment, the 30 kDa band correspond-
ing to emerin was digested by trypsin using an in-gel diges-
tion protocol. The peptides were extracted as described
above and the peptide mixture was analyzed by reversed-
phase LC-MS ⁄ MS [32].
Mass spectrometry
All mass spectra were recorded on a quadrupole time-of-
flight tandem mass spectrometer, type Q-TOF (Waters
Micromass, Manchester, UK), equipped with a nanoESI
source. The parameters for data-dependent MS ⁄ MS were
set to fragment up to three precursors at a time (charge
states +1 to +4). The intensity threshold for precursor
selection was set to 20 countsÆs
)1
, which indicates sufficient
ion intensity for recording MS ⁄ MS spectra. The scan time
for MS ⁄ MS spectra was set to 2 s using two different colli-
sion offsets. A Mascot Server (Matrix Science, London,

UK) was used for database searching, as follows. (1) Multi-
protease approach: the mass tolerance was set to ± 0.1 Da
for both precursor mass and fragment ion mass. Searches
were performed in SwissProt without protease specificity
and without any taxonomic restrictions [30]. (2) SILAC:
searches with tryptic peptides were done in RefSeq (http://
www.ncbi.nlm.nih.gov/RefSeq/) with a mass tolerance of
0.3 Da and up to two missed tryptic cleavages [32].
Phosphopeptides identified by the search engine Mascot
have been verified by manual inspection of the MS ⁄ MS
spectra.
Acknowledgements
RA would like to thank Dr Akhilesh Pandey and Dr
Dario Kalume, Institute of Genetic Medicine, Johns
Hopkins University, Baltimore, MD, USA for finan-
cial support and help on ESI-qTOF mass spectro-
meter, respectively, and for fruitful scientific
discussions. HO is grateful for all the support provided
by Dr Ferdinand Hucho and his laboratory at the
Freie Universita
¨
t Berlin, Germany.
References
1 Holaska JM, Wilson KL & Mansharamani M (2002)
The nuclear envelope, lamins and nuclear assembly.
Curr Opin Cell Biol 14, 357–364.
2 Gant TM & Wilson KL (1997) Nuclear assembly. Annu
Rev Cell Dev Biol 13, 669–695.
3 Dreger M, Bengtsson L, Schoneberg T, Otto H &
Hucho F (2001) Nuclear envelope proteomics: novel

integral membrane proteins of the inner nuclear mem-
brane. Proc Natl Acad Sci USA 98, 11943–11948.
4 Schirmer EC, Florens L, Guan T, Yates JR 3rd & Ger-
ace L (2003) Nuclear membrane proteins with potential
disease links found by subtractive proteomics. Science
301, 1380–1382.
5 Nikolakaki E, Simos G, Georgatos SD & Giannakouros
T (1996) A nuclear envelope-associated kinase phos-
phorylates arginine-serine motifs and modulates interac-
tions between the lamin B receptor and other nuclear
proteins. J Biol Chem 271, 8365–8372.
6 Polioudaki H, Kourmouli N, Drosou V, Bakou A,
Theodoropoulos PA, Singh PB, Giannakouros T &
Georgatos SD (2001) Histones H3 ⁄ H4 form a tight
complex with the inner nuclear membrane protein
LBR and heterochromatin protein 1. EMBO Rep 2,
920–925.
7 Nili E, Cojocaru GS, Kalma Y, Ginsberg D, Copeland
NG, Gilbert DJ, Jenkins NA, Berger R, Shaklai S,
Amariglio N et al. (2001) Nuclear membrane protein
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3213
LAP2beta mediates transcriptional repression alone and
together with its binding partner GCL (germ-cell-less).
J Cell Sci 114, 3297–3307.
8 Lang C & Krohne G (2003) Lamina-associated polypep-
tide 2beta (LAP2beta) is contained in a protein complex
together with A- and B-type lamins. Eur J Cell Biol 82,
143–153.
9 Martins S, Eikvar S, Furukawa K & Collas P (2003)

HA95 and LAP2beta mediate a novel chromatin–nuc-
lear envelope interaction implicated in initiation of
DNA replication. J Cell Biol 160, 177–188.
10 Holaska JM, Kowalski AK & Wilson KL (2004)
Emerin caps the pointed end of actin filaments: evidence
for an actin cortical network at the nuclear inner mem-
brane. Plos Biol 2, E231.
11 Wilkinson FL, Holaska JM, Zhang Z, Sharma A,
Manilal S, Holt I, Stamm S, Wilson KL & Morris
GE (2003) Emerin interacts in vitro with the splicing-
associated factor, YT521-B. Eur J Biochem 270, 2459–
2466.
12 Lee KK, Starr D, Cohen M, Liu J, Han M, Wilson KL
& Gruenbaum Y (2002) Lamin-dependent localization
of UNC-84, a protein required for nuclear migration in
Caenorhabditis elegans. Mol Biol Cell 13, 892–901.
13 Hunter T (1995) Protein kinases and phosphatases: the
yin and yang of protein phosphorylation and signaling.
Cell 80, 225–236.
14 Hunter T (1998) The role of tyrosine phosphorylation
in cell growth and disease. Harvey Lect 94, 81–119.
15 Kalume DE, Molina H & Pandey A (2003) Tackling the
phosphoproteome: tools and strategies. Curr Opin Chem
Biol 7, 64–69.
16 McLachlin D & Chait BT (2001) Analysis of phos-
phorylated proteins and peptides by mass spectrometry.
Curr Opin Chem Biol 5, 591–602.
17 Loyet KM, Stults JT & Arnott D (2005) mass spectro-
metric contributions to the practice of phosphorylation
site mapping through 2003: a literature review. Mol Cell

Proteomics 4, 235–245.
18 Dreger M, Otto H, Neubauer G, Mann M & Hucho F
(1999) Identification of phosphorylation sites in native
lamina-associated polypeptide 2 beta. Biochemistry 38,
9426–9434.
19 Cartegni L, di Barletta MR, Barresi R, Squarzoni S,
Sabatelli P, Maraldi N, Mora M, Di Blasi C, Cornelio
F, Merlini L et al. (1997) Heart-specific localization of
emerin: new insights into Emery–Dreifuss muscular
dystrophy. Hum Mol Genet 6, 2257–2264.
20 Ellis JA, Craxton M, Yates JR & Kendrick-Jones J
(1998) Aberrant intracellular targeting and cell cycle-
dependent phosphorylation of emerin contribute to the
Emery–Dreifuss muscular dystrophy phenotype. J Cell
Sci 111, 781–792.
21 Bione S, Maestrini E, Rivella S, Mancini M, Regis S,
Romeo G & Toniolo D (1994) Identification of a novel
X-linked gene responsible for Emery–Dreifuss muscular
dystrophy. Nat Genet 8, 323–327.
22 Bonne G, Di Barletta MR, Varnous S, Becane HM,
Hammouda EH, Merlini L, Muntoni F, Greenberg CR,
Gary F, Urtizberea JA et al. (1999) Mutations in the
gene encoding lamin A ⁄ C cause autosomal dominant
Emery–Dreifuss muscular dystrophy. Nat Genet 21,
285–288.
23 Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M,
Bhat N, Nagashima K, Stewart CL & Burke B (1999)
Loss of A-type lamin expression compromises nuclear
envelope integrity leading to muscular dystrophy. J Cell
Biol 147, 913–920.

24 Cai M, Huang Y, Ghirlando R, Wilson KL, Craigie R
& Clore GM (2001) Solution structure of the constant
region of nuclear envelope protein LAP2 reveals two
LEM-domain structures: one binds BAF and the other
binds DNA. EMBO J 20, 4399–4407.
25 Fairley EA, Kendrick-Jones J & Ellis JA (1999) The
Emery–Dreifuss muscular dystrophy phenotype arises
from aberrant targeting and binding of emerin at the
inner nuclear membrane. J Cell Sci 112, 2571–2582.
26 Lattanzi G, Cenni V, Marmiroli S, Capanni C, Mattioli
E, Merlini L, Squarzoni S & Maraldi NM (2003) Asso-
ciation of emerin with nuclear and cytoplasmic actin is
regulated in differentiating myoblasts. Biochem Biophys
Res Commun 303, 764–770.
27 Haraguchi T, Holaska JM, Yamane M, Koujin T,
Hashiguchi N, Mori C, Wilson KL & Hiraoka Y (2004)
Emerin binding to Btf, a death-promoting transcrip-
tional repressor, is disrupted by a missense mutation
that causes Emery–Dreifuss muscular dystrophy. Eur J
Biochem 271, 1035–1045.
28 Mislow JM, Holaska JM, Kim MS, Lee KK, Segura-
Totten M, Wilson KL & McNally EM (2002) Nesprin-
1alpha self-associates and binds directly to emerin and
lamin A in vitro. FEBS Lett 525, 135–140.
29 Zhang Q, Ragnauth CD, Skepper JN, Worth NF, War-
ren DT, Roberts RG, Weissberg PL, Ellis JA & Shana-
han CM (2005) Nesprin-2 is a multi-isomeric protein
that binds lamin and emerin at the nuclear envelope
and forms a subcellular network in skeletal muscle.
J Cell Sci 118, 673–687.

30 Schlosser A, Vanselow JT & Kramer A (2005) Mapping
of phosphorylation sites by a multi-protease approach
with specific phosphopeptide enrichment and NanoLC-
MS ⁄ MS analysis. Anal Chem 77, 5243–5250.
31 Posner BI, Faure R, Burgess JW, Bevan AP, La-
chance D, Zhang-Sun G, Fantus IG, Ng JB, Hall
DA, Lum BS et al. (1994) Peroxovanadium com-
pounds: a new class of potent phosphotyrosine phos-
phatase inhibitors which are insulin mimetics. J Biol
Chem 269, 4596–4604.
32 Amanchy R, Kalume DE & Pandey A (2005) Stable iso-
tope labeling with amino acids in cell culture (SILAC)
Tyrosine-phosphorylation of emerin A. Schlosser et al.
3214 FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS
for studying dynamics of protein abundance and post-
translational modifications. Sci STKE 2005, 267, pl2.
33 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 with Emery–Dreifuss muscular dystrophy. Nat
Genet 12, 254–259.
34 Ellis JA, Yates JR, Kendrick-Jones J & Brown CA
(1999) Changes at P183 of emerin weaken its protein–
protein interactions resulting in X-linked Emery–Drei-
fuss muscular dystrophy. Hum Genet 104, 262–268.
35 Clements L, Manilal S, Love DR & Morris GE (2000)
Direct interaction between emerin and lamin A. Bio-
chem Biophys Res Comm 267, 709–714.
36 Vaughan A, Alvarez-Reyes M, Bridger JM, Broers JL,
Ramaekers FC, Wehnert M, Morris GE, Whitfield

WGF & Hutchison CJ (2001) Both emerin and lamin C
depend on lamin A for localization at the nuclear envel-
ope. J Cell Sci 114, 2577–2590.
37 Fairley EA, Riddell A, Ellis JA & Kendrick-Jones J
(2002) The cell cycle dependent mislocalisation of
emerin may contribute to the Emery–Dreifuss muscular
dystrophy phenotype. J Cell Sci 115, 341–354.
38 Lee KK, Haraguchi T, Lee RS, Koujin T, Hiraoka Y &
Wilson KL (2001) Distinct functional domains in
emerin bind lamin A and DNA-bridging protein BAF.
J Cell Sci 114, 4567–4573.
39 Lin F, Blake DL, Callebaut I, Skerjanc IS, Holmer L,
McBurney MW, Paulin-Levasseur M & Worman HJ
(2000) MAN1, an inner nuclear membrane protein that
shares the LEM domain with lamina-associated poly-
peptide 2 and emerin. J Biol Chem 275, 4840–4847.
40 Hirano Y, Segawa M, Ouchi FS, Yamakawa Y, Fur-
ukawa K, Takeyasu K & Horigome T (2005) Dissocia-
tion of emerin from barrier-to-autointegration factor is
regulated through mitotic phosphorylation of emerin in
a xenopus egg cell-free system. J Biol Chem 280, 39925–
39933.
41 Holaska JM, Lee KK, Kowalski AK & Wilson KL
(2003) Transcriptional repressor germ cell-less (GCL)
and barrier to autointegration factor (BAF) compete
for binding to emerin in vitro. J Biol Chem 278, 6969–
6975.
42 Otto H, Dreger M, Bengtsson L & Hucho F (2001)
Identification of tyrosine-phosphorylated proteins asso-
ciated with the nuclear envelope. Eur J Biochem 268,

420–428.
43 Towbin H, Staehelin T & Gordon J (1979) Electro-
phoretic transfer of proteins from polyacrylamide gels
to nitrocellulose sheets: procedure and some applica-
tions. Proc Natl Acad Sci USA 76, 4350–4354.
44 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
A. Schlosser et al. Tyrosine-phosphorylation of emerin
FEBS Journal 273 (2006) 3204–3215 ª 2006 The Authors Journal compilation ª 2006 FEBS 3215