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

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.

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-

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.

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

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

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

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

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

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

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

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,

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

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

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-

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.
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