Lamin A Ser404 Is a Nuclear Target of Akt Phosphorylation in C2C12
Vittoria Cenni,†,‡,#Jessika Bertacchini,†,#Francesca Beretti,†Giovanna Lattanzi,‡
Alberto Bavelloni,§Massimo Riccio,†Maria Ruzzene,|Oriano Marin,|Giorgio Arrigoni,|
Veena Parnaik,∇Manfred Wehnert,ONadir M. Maraldi,‡,§Anto de Pol,†Lucio Cocco,⊥and
Department of Anatomy and Histology and CIPro Proteomics Centre, University of Modena and Reggio Emilia,
Via Del Pozzo 71, I-41100 Modena, Italy, I.G.M.-CNR, Unit of Bologna, c/o IOR, via di Barbiano,
I-40125 Bologna, Italy, Laboratory of Cellular Biology and Electron Microscopy, IOR, I-40125 Bologna, Italy,
Department of Biological Chemistry, Viale Colombo, I-35121 Padova, Italy, Department of Anatomical
Sciences, Cellular Signalling Laboratory, Via Irnerio 48, I-40126 Bologna, Italy, Centre for Cellular & Molecular
Biology, Hyderabad, IN-500 007, India, and Institute of Human Genetics, Greifswald, D-17484, Germany
Received April 8, 2008
Akt/PKB is a central activator of multiple signaling pathways coupled with a large number of stimuli.
Although both localization and activity of Akt in the nuclear compartment are well-documented, most
Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained
elusive. A proteomic-based search for nuclear substrates of Akt was undertaken, exploiting 2D-
electrophoresis/MS in combination with an anti-Akt phosphosubstrate antibody. This analysis indicated
lamin A/C as a putative substrate of Akt in C2C12 cells. In vitro phosphorylation of endogenous lamin
A/C by recombinant Akt further validated this result. Moreover, by phosphopeptide analysis and point
mutation, we established that lamin A/C is phosphorylated by Akt at Ser404, in an evolutionary
conserved Akt motif. To delve deeper into this, we raised an antibody against the lamin A Ser404
phosphopeptide which allowed us to determine that phosphorylation of lamin A Ser404 is triggered
by the well-known Akt activator insulin, and is therefore to be regarded as a physiological response.
Remarkably, expression of S404A lamin A in primary cells from healthy tissue caused the nuclear
abnormalities that are a hallmark of Emery-Dreifuss muscular dystrophy (EDMD) cells. Indeed, it is
known that mutations at several sites in lamin A/C cause autosomal dominant EDMD. Very importantly,
we show here that Akt failed to phosphorylate lamin A/C in primary cells from an EDMD-2 patient with
lamin A/C mutated in the Akt consensus motif. Together, our data demonstrate that lamin A/C is a
novel signaling target of Akt, and implicate Akt phosphorylation of lamin A/C in the correct function of
the nuclear lamina.
Keywords: Akt/PKB • nucleus • Lamin A/C • proteomics • 2D-electrophoresis • phosphorylation
Since its discovery as an oncogene in the mouse leukemia
virus AKT81,2and as a homologue of protein kinase C,3the 63
kDa serine/threonine kinase Akt/PKB has been involved in
promotion of cell survival, proliferation and metabolic re-
sponses downstream the phosphoinositide-3-kinase (PI3 ki-
nase) signaling pathway.4-6In resting cells, Akt is a predomi-
nantly cytosolic enzyme; however, generation of PI3 kinase lipid
products recruits Akt to the plasma membrane,4,5,7resulting
in a conformational change which confers full enzymatic
activity through the phosphorylation of the membrane-bound
protein at two residues, Thr308 and Ser473. Thr308 lies within
the T loop of the catalytic domain, and is phosphorylated by
the 3-phosphoinositide-dependent-protein kinase 1, PDK-1.8
Conversely, the identity of the Ser473, in the C-terminal
hydrophobic motif, originally termed PDK-2, has remained a
mystery for several years, the results from various laboratory
being very controversial.9-12Nevertheless, the regulation of
Ser473 has been unravelled recently by the identification of
both the kinase and the phosphatase that specifically act on
this residue.13-15Indeed, a rapamycin-insensitive form of the
mTOR protein kinase complexed to Rictor:G?L (mTORC2) has
been shown to phosphorylate Ser473 directly in vivo, in
response to serum.13
†University of Modena and Reggio Emilia.
‡I.G.M.-CNR, Unit of Bologna.
#These authors contributed equally.
§Laboratory of Cellular Biology and Electron Microscopy.
|Department of Biological Chemistry.
∇Centre for Cellular & Molecular Biology.
OInstitute of Human Genetics.
⊥Cellular Signalling Laboratory.
10.1021/pr800262g CCC: $40.75
2008 American Chemical Society
Journal of Proteome Research 2008, 7, 4727–4735 4727
Published on Web 09/23/2008
The mechanism by which Akt is inactivated was initially
suggested to depend on protein phosphatase 2A, PP2A. How-
ever, recent work has provided strong evidence that the PH
domain leucine-rich repeat protein phosphatases PHLPP1/2
dephosphorylate Ser473 directly and terminate Akt signaling,
thus, triggering apoptosis and suppressing tumor growth.14,15
Activated Akt redistributes to cytoplasm and nucleus, where
phosphorylation of specific substrates, such as GSK3, Bad,
Mdm2 and TSC2, occurs.6,16,17
Both the presence and the activity of Akt in the nucleus have
been described thoroughly.18,19In particular, a recent study
employed a genetically encoded fluorescent reporter for Akt
activity that allowed real-time imaging of phosphorylation
catalyzed by Akt.18By this technology, Akt signaling in the
nucleus was found to be more stable compared with that in
the cytosol, suggesting that Akt activity in these two compart-
ments is regulated by different mechanisms.18However, the
signal in the nucleus was also less rapid, probably reflecting
the time it takes for activated Akt to translocate into the nuclear
compartment, and thus raising the question of what mecha-
nism mediates Akt nuclear translocation. Our recent work
demonstrated that, in response to precise signals, Akt is
targeted to the cytoskeleton by direct interaction of its N-
terminal PH domain with actin, and that this process is
regulated by the small GTP-binding proteins cdc42 and Rac.20
We suggested that this mechanism might contribute to Akt
redistribution to diverse subcellular locations, such as the
nucleus. Another interesting mechanism that mediates nuclear
translocation of Akt has been described in human mature T-cell
leukemia: the product of TCL1 gene, Tcl1, interacts with the
PH domain of phosphorylated Akt, thus, driving Akt to the
nucleus.21In this context, Tcl1 may act as a direct transporter
of Akt or may contribute to the formation of a complex that
promotes the transport of active Akt to the nucleus, where it
can phosphorylate nuclear substrates. A well described nuclear
substrate is Foxo. IGF-1 triggers phosphorylation of Foxo by
Akt inside the nucleus, where phosphorylated Foxo associates
with 14.3.3 proteins that, in turn, promote its export to the
cytoplasm.22Remarkably, Foxo phosphorylation by Akt has
been shown to be a crucial event in Akt-dependent myogen-
esis.23In spite of these striking observations, most Akt nuclear
substrates, as well as nuclear Akt functions, have so far
remained elusive. This lack of information prompted us to
undertake a search of substrates of Akt in the nucleus. Reckon-
ing that functional proteomics is a very powerful approach to
the discovery of kinase targets, we decided to combine 2D-
separation/mass spectrometry with Western blotting by anti-
Akt-phosphosubstrate antibody and bioinformatic screening for
the Akt phosphorylation motif.24-26
Lamin A and lamin C, both products of the LMNA gene, are
type V intermediate filaments protein that participate in the
formation of the nuclear lamina, a protein meshwork lining
the nucleoplasmic surface of the inner nuclear membrane
thought to provide a framework for organizing nuclear envelope
structures and anchoring the interphase chromatin.27-29Lamin
A/C have essential roles in maintaining lamina stability, and
in regulating transcription factors. Our data establish lamin A/C
as new genuine signaling targets of Akt, and suggest that their
phosphorylation is involved in the correct function of the
Cell Culture and Transfections. Human embryonic kidney
epithelial (HEK) 293T cells and C2C12 mouse myoblasts were
grown in DMEM-HG (Dulbecco’s Modified Eagle’s Medium
High Glucose, Sigma-Aldrich) supplemented with 10% Fetal
Calf Serum (FCS, EuroClone Ltd., U.K.).
Transient transfections of 293T cells were performed by the
calcium-phosphate method.20After transfection, cells were
serum-starved for 24 h and used for experiments.
Plasmids. Wild-type, flag-tagged rat lamin A, subcloned in
pCI expression vector as described by Kumaran et al.,30was
used as template to generate S301A, S404A and S301A/S404A
mutants by means of the QuikChange strategy (Stratagene, La
Jolla, CA). The following mutation primers were used: S301A
mutant, forward primer CGAATCCGCATTGACGCCCTCTCAGC-
CCAG; S301A mutant, reverse primer CTGGGCTGAGAGGGCGT-
CAATGCGGATTCG; S404A mutant, forward primer GCCGCGC-
CTCCGCCCACTCCTCCC; S404A mutant, reverse primer GGGAG-
GAGTGGGCGGAGGCGCGGC. All constructs were verified by
DNA sequencing service (MWG Biotech Germany).
To transiently silence the expression of Akt1, C2C12 cells
were transfected with vectors that express Akt1 (or scramble,
where indicated) hairpin siRNAs under the control of the mouse
U6 promoter, as described previously,31with the Cell Line
Nucleofector kit V and Amaxa Nucleoporator (Instrumentation
Laboratory, Milano, Italy), following manufacturer’s instruc-
tions. To monitor transfection efficiency, pEGFPN1 was cotrans-
fected at a 1:10 ration with Akt vectors and 15 mm glass
coverslips were added to each plate before seeding. Count of
GFP-positive cells was 70% ( 5% in all samples.
All other constructs were as described previously.32,33
Preparation of Cell Extracts. Subconfluent cells were ex-
tracted by addition of RIPA buffer (20 mM Tris-Cl, pH 7.0, 1%
NP-40, 150 mM NaCl, 10% Glycerol, 10 mM EDTA, 20 mM NaF,
5 mM Sodium Pyrophosphate, 1 mM Na3VO4, and freshly
added Sigma-Aldrich Protease Inhibitor Cocktail) at 4 °C for
10 min. Lysates were cleared by centrifugation and used for
immunoprecipitation experiments, as described below. A total
of 50 µL of the total lysate was immediately boiled in SDS
sample buffer, resolved, and immunoblotted with the indicated
antibodies. Separation of nuclei was obtained by hypotonic
shock and shearing; nuclei were obtained as pellet by a 300g
centrifugation at 4 °C. The purity of nuclear fraction was
checked by evaluating the presence of cytoplasmic contami-
nants (tubulin, not shown).
Immunoprecipitation and Electrophoresis. Equal amounts
of precleared lysates, whose protein concentration was deter-
mined by the Bradford method, were incubated for 3 h with
anti-flag (M2 clone, Sigma-Aldrich), anti-lamin A/C (Santa
Cruz) (3 µg all) or mouse IgG to provide a negative control,
plus 30 µL of 50% (v/v) of protein A/G agarose slurry (Santa
Cruz Biotech.) at 4 °C with gentle rocking. Pellets were washed
twice with PBS plus 1% NP-40, twice in TNE (10 mM Tris-Cl,
pH 7.5, 100 mM NaCl, 1 mM EDTA), once with 10 mM Tris-Cl,
pH 7.4, boiled in Laemmli sample buffer, and centrifuged.
Supernatants were loaded onto SDS-polyacrylamide gel, blotted
on Immobilon-P membranes (Millipore, MA), and processed
by Western blot with the indicated antibody, detected by
Supersignal substrate chemiluminescence detection kit (Pierce,
U.K.). Quantitation of the signal was obtained by chemilumi-
nescence detection on a Kodak Image Station 440CF and
analysis with the Kodak 1D Image software.
Cenni et al.
4728Journal of Proteome Research • Vol. 7, No. 11, 2008
For 2D-electrophoresis, a total volume of 120 µL of TSU
(Urea 7M, Thiourea 2M, CHAPS 4%, DTT 1% Tris-Cl, pH 7.4,
20 mM, Ampholines 0.2% plus fresh protease inhibitor cocktail,
DNase and RNase) containing nuclear lysates (120 µg) or anti-
M2 immunoprecipitates (eluted with the 3× FLAG peptide,
Sigma-Aldrich, following manufacturer’s instructions) was
loaded onto 3-10 or 5-8 IEF strips (Bio-Rad) and left focused
in a Protean IEF cell (from Bio-Rad) until a final VoltHour of
12000 was reached. Strips were then reduced for 15 min with
125 mM DTT in EB (equilibration buffer: Tris-Cl, pH 8.6, 5 mM,
Urea 6M, Glycerol 30%, SDS 2%) and alkylated with 250 mM
iodacetamide in EB for 8 min. Strips and samples were resolved
on SDS-polyacrylamide gel, blotted onto nitrocellulose, probed
with specific primary antibodies and HRP-conjugated second-
ary antibodies (Pierce, U.K.), and developed with Pierce Su-
persignal substrate chemiluminescence detection kit. The
following antibodies were used: anti-flag, clone M2, from
Sigma-Aldrich, anti-lamin A/C (N-terminal), anti-lamin A /C
(C-terminal), anti-actin, anti-tubulin, anti-parp, anti-Akt, from
Santa Cruz Biotechnology; anti-Akt phosphoserine 473 (pAkt
S473) and anti-Akt phosphosubstrate, from Cell Signalling
Technology (MA). Anti-phospholamin A (S404) and its unphos-
phorylated counterpart were raised in New Zealand rabbits
against two synthetic peptides corresponding to amino acids
[400-413] of the Akt protein, and phosphorylated in Ser404 or
not, respectively; the antisera were purified using an im-
mobilized peptide affinity resin (Sulfo Link Coupling Gel,
Pierce, U.K.) according to the manufacturer’s instructions.
In-Gel Digestion and LC-MS/MS Analysis. The spots were
excised from the gel with the help of an Ettan spot picker (GE
Healthcare), and the protein digestion was performed in-gel
using sequencing grade modified trypsin (Promega, Madison,
WI). Briefly, gel spots were washed with acetonitrile, dried
under vacuum and treated with 12.5 ng/µL of trypsin at 37 °C
overnight. After digestion, the peptides were extracted by 3
changes of 50% acetonitrile/0.1% formic acid (20 min between
changes), dried under vacuum, resuspended with 10 µL of 0.1%
formic acid and analyzed by LC-MS/MS.
The samples were analyzed using a LCQ XP (Thermo
Electron, San Jose ´, CA) interfaced with a nano-LC system 1100
series (Agilent Technologies, Santa Clara, CA) and a capillary
column Zorbax 300SB C18, 3.5 µm, 150 mm × 75 µm (Agilent),
Journal of Proteome Research • Vol. 7, No. 11, 2008
Figure 1. Lamin A/C is a nuclear substrate of Akt. Nuclear lysates
from C2C12 myoblasts were purified and subjected to 2D-
electrophoresis on a pH 3-10 isoelectric focusing (IEF) gradient
and a 10% polyacrilammide gel. (a) Nuclear proteins were
visualized by Coomassie blue staining; asterisks indicate proteins
identified as A-type lamins; (b) a replicate gel was blotted and
probed with anti-Akt phosphosubstrate (Akt psubstrate) antibody
(1:1000, Cell Signaling Technology); (c) the filter was stripped
and reprobed with an anti-lamin A/C antibody (1:500, Santa Cruz
Biotech). Arrows indicate lamin A (72 kDa) and lamin C (68 kDa).
Table 1. Tryptic Peptides Derived from Lamin A/C and
Identified by LC-MS/MS Analysisa
sequence Mascot scoreexp. value
aThe MS/MS spectra were searched with Mascot search engine.
Peptide sequence, Mascot score, and expectation value are reported.
Lamin A Ser404 Is a Target of Akt Phosphorylation
using a linear gradient of acetonitrile/0.1% formic acid from
5% to 40% in 30 min. Data were acquired in a data dependent
mode: a full MS scan was followed by a Zoom scan and a MS/
MS scan on the 3 most intense peaks. MS/MS data were
searched using Mascot (Matrix Science, London, U.K.) against
the human section of the Swiss-Prot database (version 51.5,
149 052 entries). Enzyme specificity was set to trypsin with 1
missed cleavage using carbamidomethylcysteine as fixed modi-
fication. The tolerance of the precursor ion was set to 1 mass
unit for both peptide and fragment ion matches.
Akt Kinase Assay and Phosphopeptide Analysis. Lamin A/C
was immunoprecipiatetd as described above, then phospho-
rylated in vitro with 3 µg of recombinant, active Akt1 (∆PH,
S473D, kind gift of Dario Alessi, University of Dundee, U.K.) in
a buffer comprising 20 mM Tris-Cl, pH 7.5, 25 mM MgCl2, 80
mM ATP and 5 µCi/assay (γ-32P)ATP (3000 Ci/mmol), (Amer-
sham-GE Healthcare), and carried out for 20 min at 25 °C.
Pellets were then washed and treated as described above.
Radioactive bands were localized by autoradiography (Cyclone
storage phosphor system, Packard). For phosphopeptide analy-
sis, nitrocellulose membranes from a duplicate experiment
were cut and digested by trypsin. The tryptic peptides were
subjected to acidic hydrolysis (6 M HCl for 4 h at 110 °C) and
the phospho-amino acids were detected by high voltage paper
electrophoresis at pH 1.9. As migration reference, nonradioac-
tive phospho-amino acids were added to each sample and
detected by ninhydrin reaction.
Lamin A/C Is a Nuclear Substrate of Akt. Nuclear lysates
from C2C12 cells were resolved by 2D-electrophoresis in
dupicates, using a 3-10 IEF strip for the first dimension. Then,
one gel was stained with MS-compatible Coomassie blue
(Figure 1a), while the replicate gel was blotted and revealed
with an anti-Akt phosphosubstrate antibody24to detect putative
substrates of Akt (Figure 1, panel b). This approach has been
validated by previous studies.26,34,35The overlapping spots were
excised from the Coomassie-blue stained gel and peptides were
extracted by in-gel digestion for MS analysis. Our attention was
drawn by two trains of spots (Figure 1b, arrows and asterisks)
which, according to MS analysis, corresponded to A-type lamins
(Table 1). Consequently, the filter was probed with an anti-
lamin A/C antibody which recognizes an N-terminal epitope,
common to both lamin A and its splicing product lamin C, that
differs from lamin A by a short stretch of amino acids at the
C-terminus. The antibody reacted with a doublet at the
expected isoelectric point (6.5-6) and molecular weight of
lamin A (72 kDa) and lamin C (68 kDa), confirming the identity
of the protein (Figure 1c). Interestingly, a recent study also
reported lamin A/C as a candidate Akt substrate identified by
mass spectrometry upon in vitro phosphorylation of mesangial
cell lysate with active recombinant Akt.34
This prompted us to investigate whether Akt and lamin A
interact in vivo. Therefore, lysates from C2C12 cells were
immunoprecipitated with anti-lamin A/C, and the pellets
probed with anti-Akt. The presence of endogenous Akt in the
IP suggested that the two proteins interact in vivo (Figure 2a).
Lamin A/C is also clearly detectable in anti-Akt immunopre-
cipitates. Besides this, the filter was probed with the anti-Akt
phosphosubstrate antibody showing that lamin A/C co-immu-
noprecipitated with Akt is phosphorylated at a putative Akt
motif (Figure 2a).
293T cells were therefore transiently transfected with a pCI-
flagLMNA plasmid, which we have already described to express
both lamin A (72 kDa) and its precursor prelamin A (74 kDa).30
Then lysates were immunoprecipitated with anti-flag antibody
and the pellets were phosphorylated in vitro in the presence
of rAkt or buffer alone. Pellets were run on SDS-polyacrylamide
gel and stained with Coomassie blue. In the sample in which
lamin A has been phosphorylated by rAkt, an additional band
with a retarded mobility is clearly visible, compatible with the
appearance of a phosphorylated form (Figure 2b). Therefore,
replicate pellets were run on 2DE, using 5-8 IEF strips,
transferred to nitrocellulose and probed with anti-flag. In
agreement with the previous result, the spots indicated as 1 of
prelamin A and 2, 3 of lamin A, upon phosphorylation by Akt
are shifted to the left side of the gel, indicating a more acidic
pI compatible with a phosphorylation (Figure 2c, asteriks).
Figure 2. Lamin A/C co-immunoprecipitates with Akt. (a) Endog-
enous lamin A/C immunoprecipitates (R-lamin A/C IP), and
endogenous Akt immunoprecipitates (R-Akt IP), obtained from
C2C12 nuclear lysates, were probed with anti-Akt phosphosub-
strate (R-Akt psubstrate WB), then checked for the presence of
Akt (R-Akt WB) and of lamin A/C (R-lamin A/C WB). The result is
representative of three independent experiments. (b) 293T cells
were transfected with pCI flag-lamin A, expressing prelamin A
(74 kDa) and lamin A (72 kDa), then pellets from anti-flag
immunoprecipitation were phosphorylated in vitro in the pres-
ence (+) or not (-) of recombinant Akt (rAkt). Proteins were
resolved by SDS-polyacrylamide gel and stained with colloidal
Coomassie blue. An asterisk indicates the band with retarded
mobility in the sample phosphorylated by rAkt. (c) Duplicate
pellets from the above experiment were resolved by 2D-electro-
phoresis on pH 5-8 IEF gradient and an 8% polyacrilammide gel,
blotted onto nitrocellulose and incubated with an anti-flag
antibody. In the sample phosphorylated by rAkt, spots 1, 2, and
3 are clearly shifted toward the acidic side of the gel (marked by
Cenni et al.
4730 Journal of Proteome Research • Vol. 7, No. 11, 2008
Akt Phosphorylates Lamin A at Ser404. On the basis of the
above findings, we then asked whether Akt phosphorylates
A-type lamins directly. Therefore, cells were transfected with
flag-lamin A, and the anti-flag immunoprecipitate was com-
bined with recombinant, active Akt in the presence of γ32P-
ATP. The result shows that lamin A can indeed be phospho-
rylated by Akt in vitro (Figure 3a). A weak phosphorylation of
lamin A is detected in the control sample, most probably due
to endogenous Akt which, as shown above, co-immunopre-
cipitates with lamin A. A replicate sample was digested with
trypsin for phosphopeptide analysis. The result plainly indicates
that Akt phosphorylates lamin A at a serine, while threonine
phosphorylation is undetectable (Figure 3b).
In good agreement with the above result, a lamin A sequence
analysis at high stringency with the scansite algorithm (http://
scansite.mit.edu) produced two putative Akt phosphorylation
sites, serine 301 and serine 404. Therefore, the corresponding
peptides were synthesized and phosphorylated in vitro with
recombinant Akt. It is clear that both peptides can be phos-
phorylated in vitro, though the peptide containing serine 404
to a greater extent (Figure 3c). On this basis, both serines were
mutated to alanine, then wild-type, S301A or S404A flag-lamin
A were expressed into cells, and anti-flag pellets were phos-
phorylated in vitro with active, recombinant Akt. The result
clearly shows that lamin A S301A is phosphorylated by Akt to
the same extent as wild-type lamin A. Conversely, Akt failed to
phosphorylate lamin A S404A, indicating Ser404 as a genuine
Akt phosphorylation site (Figure 3d). It is worth noting that
the S404-containing Akt motif is evolutionary conserved in
vertebrate A-type lamins.
Phosphorylation of Lamin A by Akt Is Elicited by Insulin.
To delve deeper into lamin A phosphorylation by Akt, we
investigated whether this event can be evoked in vivo in
response to physiological stimuli known to activate Akt. To this
goal, we raised a phosphoantibody against the lamin A GRASp-
SHSSQTQGGC peptide phosphorylated at Ser 404. Lamin A
S404 phosphorylation was then probed in cells in which Akt
had been activated by insulin treatment. As shown in Figure
4a, insulin-dependent Akt activation correlated with phospho-
rylation of lamin A/C. That phosphorylation of lamin A/C is
Figure 3. Akt phosphorylates lamin A at Ser404. (a) Flag-lamin A overexpressed in 293T cells was immunoprecipitated with an anti-
flag antibody, then phosphorylated in vitro in the presence (+) or not (-) of recombinant, active Akt (rAkt) and γ32P-ATP. One sample
was incubated with heat inactivated rAkt. Pellets were resolved on SDS-polyacrylamide gel and blotted, and the radioactive bands
were detected and quantified by autoradiography (upper panel) (Cyclone storage phosphor system, Packard). Next, the filter was probed
with anti-Akt and anti-flag antibodies (lower panel). (b) A duplicate filter was digested with trypsin for phosphopeptide analysis and
resolved by high voltage paper electrophoresis at pH 1.9. A phospho-amino acid mixture was run as control. (c) The 0.1 or 0.5 mM
peptides containing either the Ser301 [QSRIRIDSLSAQLSQ(NH2)] peptide, the Ser404 [RSRGRASSHSSQTQG(NH2)] peptide, or the
Ser404 peptide modified with the mutation R401C [RSRGCASSHSSQTQG(NH2)] were phosphorylated in vitro with rAkt. Crosstide
substrate peptide (Sigma-Aldrich) was used as positive control. Values are expressed as cpm. (d) Flag-tagged lamin A wild-type (wt),
or the lamin mutants S301A and S404A were expressed in 293T cells, immunoprecipitated and subjected to an in vitro kinase assay
with rAkt as described for panel a. As control, filters were probed with anti-flag and anti-Akt antibodies. The data shown are representative
of three independent experiments.
Lamin A Ser404 Is a Target of Akt Phosphorylation
Journal of Proteome Research • Vol. 7, No. 11, 2008
due to Akt was revealed using siRNA against Akt1, the Akt
isoform expressed in growing myoblast.36Reduction of endog-
enous Akt1 abrogated the increase of lamin phosphorylation
observed in the presence of insulin alone. The filter was then
probed with antipoly(ADP-ribose)polymerase (parp), anti-
caspase 9 and anti-caspase 3. The absence in all samples of
the 80 kDa form cleaved parp and 17/19 kDa cleaved caspase
3, as well as equal amounts of full-length caspase 9 demonstrate
cell viability. Moreover, Figure 4b shows that the anti-phos-
pholamin A S404 antibody recognizes both endogenous lamin
A and flag-lamin A, but not lamin A S404A, immunoprecipitated
from cells overexpressing Akt, thus, confirming the antibody
Expression of Lamin A S404A Affects the Nuclear Morphol-
ogy. To gain insight into the physiological function of lamin A
phosphorylation by Akt, human primary cells were transfected
with wild-type or S404A-mutated lamin A (Figure 5a,b) and the
nuclear lamina was analyzed by immunofluorescence. Com-
pared to primary cells transfected with wild-type lamin A
(Figure 5, panel a), cells transfected with lamin A S404A (Figure
5, panel b) are featured by the presence of blebs and honey-
comb lamina structures reminiscent of the phenotype of the
nuclear lamina observed in Emery-Dreifuss muscular dystrophy
(EDMD).37-39Indeed, it is well-known that mutations at several
sites in lamin A cause autosomal dominant EDMD (EDMD-
2).33To investigate the extent of lamin A S404 phosphorylation
in EDMD-2 cells, we obtained primary fibroblasts from an
EDMD-2 patient carrying a mutation at R401 (R401C lamin
A/C), in the Akt phosphorylation motif. It is worth noting that
arginine at -3 position is regarded as a minimal requirement
for Akt phosphorylation.16Observed by immunofluorescence,
these cells exhibit disorganization of the nuclear lamina typical
of the EDMD-2 cells reported above in 10% of nuclei (Figure
5c), as well as focal loss of peripheral heterochromatin (our
Therefore, endogenous lamin A/C was immunoprecipitated
from either EDMD-2 cells or from control primary cells, then
phosphorylated in vitro by recombinant Akt. Clearly, the extent
of Akt phosphorylation of lamin A/C R401C was greatly reduced
(Figure 5d). To rule out that the low level of phosphorylation
of lamin A/C is due to down-regulation of Akt itself in these
cells, EDMD-2 cells were treated with insulin and Akt activity
was detected by anti-Akt phosphoserine-473 antibody (Figure
5e). Moreover, the R401C peptide was phosphorylated in vitro
with recombinant Akt (Figure 3c). As expected, in conditions
in which both S301 and S404 peptides were phosphorylated
by Akt, phosphorylation of the R401C peptide was undetectable.
The identification of genuine substrates of kinases is re-
garded as an important issue to the complete dissection of any
signaling pathway. Given the important roles of Akt in both
cell metabolism, proliferation and survival, and its potential
as therapeutic target, we have undertaken a search for new
substrates of Akt by a functional proteomic approach, com-
bining high-throughput 2DE/MS with anti-phosphomotif an-
tibodies and bioinformatic search with algorithms for the
detection of phosphomotifs associated with specific kinases.
Here we present evidence of lamin A/C as a novel nuclear
substrate of Akt. By coimmunoprecipitation, we demonstrated
that endogenous lamin A/C and Akt proteins interact, and that
lamin A/C is phosphorylated by Akt both in vitro and in vivo.
Moreover, by phospho-amino acid analysis and mutagenesis,
we further demonstrated that Akt phosphorylates lamin A at
Ser404, in the evolutionary conserved RSRGRASSH Akt motif.
Lamin phosphorylation is a key event in the mitotic breakdown
of the nuclear lamina.40In cells undergoing mitosis, cyclin-
dependent kinases mediate phosphorylation of lamins A, B and
C.41Moreover, interphase phosphorylation of lamins A and C
was reported following insulin treatment of quiescent fibro-
blasts.42Phosphorylation of lamins by PKC in interphase cells
has also been described, and the site of molecular interaction
Figure 4. Lamin A S404 phosphorylation is triggered by insulin.
(a) C2C12 cells were transfected for 48 h with either pU6-Akt1
siRNA, pU6-scramble siRNA or pU6 empty vector (ev), together
with pEGFP to monitor tansfection, using the Cell Line Nucleo-
fector kit V and Amaxa Nucleoporator, serum starved for 16 h
then treated for 1 h with 0.4 µM insulin. Endogenous lamin A/C
was immunoprecipitated from whole cell lysates and the filter
revealed with anti-phospholamin S404 (pS404), then with anti-
lamin A/C. Whole lysates were probed with anti-phosphoAkt
Ser473 (pAkt) and anti-Akt1 to confirm siRNA efficacy. Equivalent
amounts of uncleaved poly(ADP-ribose)polymerase (parp, 113
kDa, Santa Cruz Biotech) and caspase 9 (49 kDa, Cell Signaling
#9504) demonstrate cell viability. Since anti-caspase 3 (Cell
Signalling #9661) detects only the large fragment (17/19 kDa) of
cleaved caspse-3, Jurkat cells treated with 5 µM sodium arsenite
(As2O3) for 1 day were loaded as positive control (As2O3, lane
5); (b) Cells were co-transfected with caax-Akt and either empty
vector (ev), lamin A wild-type (wt) or lamin A S404A, serum
starved for 16 h then treated for 1 h with 0.4 µM insulin and equal
amounts of proteins were probed with anti-phospholamin S404
(pS404). The absence of labeling at the expected molecular
weight in the lamin A S404A sample confirms the antibody
Cenni et al.
4732Journal of Proteome Research • Vol. 7, No. 11, 2008
between lamin A and protein kinase C alpha has been mapped
to the C-terminal tail of lamin A.43In addition, modulation of
lamin A/C phosphorylation has been reported in cells infected
by murine cytomegalovirus.44In our previous work, we ana-
lyzed lamin A/C phosphorylation in muscle cells, using an
antibody directed against an N-terminal phosphorylated
epitope.33Our results show that lamin A N-terminal phospho-
rylation is decreased in quiescent myoblasts to almost unde-
tectable levels, while a high level of phosphorylated lamin A
can be detected in cycling myoblasts and in differentiating
myotubes. Moreover, lamin A N-terminus phosphorylation was
induced by insulin, which conversely did not affect lamin C
As this work was performed in C2C12 cells, which originate
from activated satellite cells, we also assume that it might be
relevant to the process of skeletal muscle growth. This process
implicates functional interactions between intrinsic muscle
restricted genetic programs and extracellular-regulated signal-
ing pathways. It has been determined that an active PI
3-kinase/Akt pathway is needed for myocyte maturation. IGF-
stimulated activation of PI3 kinase and Akt promotes myoblast
survival during the initial stages of differentiation by inducing
expression of the cyclin-dependent kinase inhibitor p21,45and
sustained activation of PI3 kinase and Akt is required for IGF-
mediated initiation of differentiation, in part acting to stimulate
myogenin accumulation.46Notably, forced expression of active
versions of either of the above molecules substitutes for IGF
signaling,46and enhances the rate and extent of both bio-
chemical and morphological differentiation.47-50Similar con-
clusions were reached using PI3 kinase pharmacological in-
hibitors such as LY294002 or wortmannin,48as well as a
dominant-negative regulatory subunit of PI3 kinase.51In
particular, expression of active Akt sustains transcription of
muscle-specific genes, resulting in myoblast differentiation.36
Thus, a role of Akt in muscle cell growth and differentiation is
well-defined. Interestingly, lamin A mutations at several sites
cause autosomal dominant Emery Dreifuss muscular dystrophy
(EDMD-2).37-39,52,53In particular, we have recently observed
that, in cells from an EDMD-2 patient carrying a mutation at
Arg401, lamin phosphorylation is dramatically reduced (not
shown). Notably, Arg401 lies at -3 position of the Akt consen-
sus motif, and a large body of evidence has demonstrated that
arginine at -3 is a requisite of Akt phosphorylation. This
correlates well with our data that lamin A/C from primary
EDMD-2 cells carrying the above mutation is not phosphory-
lated in vitro by recombinant Akt. Moreover, in primary cells
transfected with lamin S404A, we observed the presence of
misshapen nuclei and nuclear abnormalities mostly consisting
of concentrated foci of lamin A in the nucleoplasm, nuclear
envelope breaches, blebs and honeycomb lamina structures,
which are a hallmark of the EDMD-2 phenotype.
Phosphorylation of a substrate by Akt can result in its
subsequent binding to a member of the 14.3.3 family. In turn,
association to 14.3.3 may drive the intracellular redistribution
or proteolytic cleavage of the substrate, modify the enzymatic
activity or shield the phosphorylated residue.54-56It is worth
Figure 5. The morphology of nuclei expressing lamin A S404A mutant is reminiscent of that of EDMD-2 nuclei. Human primary fibroblasts
were transfected with wild-type (a) or S404A (b) lamin A and detected by immunofluorescence labeling with anti-lamin A/C 24 h after
transfection. Alterations of lamin A distribution, nuclear lamina thickening (asterisks) and nuclear blebbing (arrowheads) in lamin A
S404, compared with wt-transfected cells, are observed. (c) Immunofluorescence labeling of lamin A/C in fibroblasts from an EDMD-2
patient carrying a lamin A/C mutation at R401, at -3 position in the Akt phosphorylation motif (R401C EDMD-2). (d) Endogenous lamin
A/C was immunoprecipitated from primary cells from a control patient with wild-type lamin A/C (control) or from an EDMD-2 patient
carrying a lamin A/C mutation at R401 (R401C EDMD-2), using an anti-lamin A/C antibody. Pellets were then phosphorylated in vitro
by recombinant Akt (rAkt), and resolved and autoradiographed as described for Figure 3a. To confirm equivalent immunoprecipitation,
the filter was next incubated with anti-lamin A/C antibody. The data shown are representative of three independent experiments. (e)
Primary fibroblasts from a control patient and from an EDMD-2 patient carrying a lamin A/C mutation at R401 (R401C EDMD-2) were
treated (+) or not (-) with 0.4 µM insulin for 1 h to check the viability of the Akt pathway. The same amount of protein lysate from 293T
cells was loaded as positive control. Anti-Akt and anti-tubulin show equal loading.
Lamin A Ser404 Is a Target of Akt Phosphorylation
Journal of Proteome Research • Vol. 7, No. 11, 2008
noting that, according to the scansite algorithm, phospho-
Ser404 represents a good 14.3.3 phosphomotif, suggesting that
Akt phosphorylation of lamin A at Ser404 creates putative
binding sites for 14.3.3 proteins. Remarkably, a recently pub-
lished proteomic analysis of 14.3.3 binding partner demon-
strates that lamin A associates directly with 14.3.3 proteins in
mitotic cells.57Thus, it is conceivable that failure to phospho-
rylate Ser404 might prevent binding to 14.3.3, thus, affecting
either stability or localization of lamin A. Though we have no
evidence at present that 14.3.3 binding can affect the correct
intranuclear location of lamin A, it has been previously shown
that phosphorylation at Ser403 and Ser404, which lie in
proximity to the nuclear localization signal, can regulate the
correct positioning of human lamin A/C into the nucleus.58On
the other hand, Ser404 phosphorylation might control lamin
A stability, hence affecting its ability to form a functional
scaffold or a correct compartmentalization of chromatin. In this
regard, it is worth noting that a disorganization of the nuclear
lamina as well as focal loss of peripheral heterochromatin in
the cells carrying the Arg401 mutant lamin has been described
recently. Moreover, we have shown previously that in muscle
biopsies from EDMD-2 patients the extent of lamin phospho-
rylation is dramatically reduced compared to the health tis-
In summary, the results presented here establish lamin A as
a genuine nuclear substrate of Akt, phosphorylated in response
to physiological stimuli at a precise residue, and pave the way
for a functional link between a central nuclear structural
component, lamin A, and an essential regulator of multiple
signaling pathways, Akt, for the first time implicating a phos-
phorylation defect of the nuclear lamina in muscle disease
Acknowledgment. This work was supported by the
following grants: FAR-University of Modena and Reggio
Emilia; MIUR (PRIN 2005 grant 2005055737, FIRB inter-
Human Proteome Net.); EU FP6“Euro-laminopathies” grant
n 018690; Italian ISS “Rare diseases Italy-USA program”
grant n 526/D30; Fondazione Cassa Risparmio Modena. We
thank Dario Alessi for providing reagents.
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