We have identified tyrosine phosphorylation sites on human and mouse emerin using independently two different strategies: 1 a multiprotease approach, where we combined subcellular fractio
Trang 1nuclear membrane protein emerin
Andreas Schlosser1,*, Ramars Amanchy2,* and Henning Otto3
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 tyrosthreon-ine 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: hotto@chemie.fu-berlin.de
*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.
Trang 2[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.
Trang 3active 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 l M 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.
Trang 4the 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
1 m 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 C6-Arg and one 13 C6
-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.
Trang 5mouse 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 y9 and y10, 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-capcap-tured tyrosine-phosphorylated proteins
To achieve labeling, we added a mixture of argin-ine and lysargin-ine, each containing six 13C atoms (13C6 -Arg and 13C6-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 perper-vanadate treatment of HeLa cells (20 large dishes) grown in the presence of the heavy amino acids 13C6-arginine and 13C6-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
Additional
3 13 C6-Lys
Human
Trang 6in 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
13C6-Arg and13C6-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, obtainphosphorylat-ed 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’.
Trang 7cells 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 13C6-lysine and one 13C6-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.
Trang 8By 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 iddiffer-entifying 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 unlabeltyrosine-phosphorylat-ed 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
Trang 9to 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 mutadele-tion 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 Phoshyperphos-phorylation, 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 37C in a humidified atmosphere with 5% CO2 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 13C6-arginine and 13C6-lysine supplemented with 10% dialyzed fetal bovine serum plus antibiotics [32] The cells were grown for five passages in the above medium
Trang 10prior 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 4C
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 37C 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 NH4HCO3
(pH 8) at 30C 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 NH4HCO3 (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 uscorrespond-ing 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