Báo cáo khoa học: Proteomic and biochemical analysis of 14-3-3-binding proteins during C2-ceramide-induced apoptosis pot

Proteomic and biochemical analysis of 14-3-3-binding
proteins during C2-ceramide-induced apoptosis
Mercedes Pozuelo-Rubio
Centro Andaluz de Biologı
´
a Molecular y Medicina Regenerativa, Consejo Superior de Investigaciones Cientı
´
ficas, Sevilla, Spain
Keywords
14-3-3; apoptosis; C2-ceramide; Hela cells;
proteomics
Correspondence
M. Pozuelo-Rubio, CABIMER (SC4),
Americo Vespucio s ⁄ n, Sevilla 41092, Spain
Fax: +34 954 461664
Tel: +34 600826730
E-mail:
(Received 14 March 2010, revised 28 May
2010, accepted 3 June 2010)
doi:10.1111/j.1742-4658.2010.07730.x
14-3-3 is a family of proteins comprising several isoforms that, in many cases,
promote cell survival by association with proapoptotic proteins. This study
was designed to obtain further understanding of the 14-3-3 role in apoptosis
regulation, by analyzing apoptosis-related protein–14-3-3 interactions.
Western blot analysis of an eluted fraction from the 14-3-3-affinity chroma-
tography column identified proapoptotic proteins as receptor-interacting
protein 3 and Bcl-2-antagonist ⁄ killer as new phophorylation-dependent
14-3-3-binding proteins under physiological conditions. The apoptosis indu-
cer C2-ceramide promoted decay of the 14-3-3-binding signal of protein cell
extracts. Investigation of the role of 14-3-3 in C2-ceramide-induced apoptosis
showed that depletion of the 14-3-3f isoform sensitized to cell death, whereas

overexpression of this isoform delayed cell death. A combination of tandem
affinity purification and liquid chromatography–tandem MS techniques
identified 15 proteins involved in cell survival processes whose 14-3-3-binding
status changed during C2-ceramide-induced apoptosis. Under physiological
conditions, desmin was clearly identified as a new 14-3-3-interactor protein,
and vasodilator-stimulated phosphoprotein, nucleophosmin and calmodulin,
whose 14-3-3 binding was suggested by others on the basis of MS analysis,
were confirmed here as phosphorylation-dependent 14-3-3-associated pro-
teins. Interestingly, proteins related to the regulation of DNA double-strand
break repair in the early stages of apoptosis, such as DNA-dependent protein
kinase, or the regulation of cell shrinkage during apoptosis, such as vasodila-
tor-stimulated phosphoprotein and death promoters like receptor-interacting
protein 3, were identified as 14-3-3-associated proteins whose 14-3-3-binding
status changed when apoptosis was initiated. The functional diversity of
these identified proteins suggests that 14-3-3 may regulate the apoptotic pro-
cess through new mechanisms, in addition to others previously characterized.
Structured digital abstract
l
A list of the large number of protein–protein interactions described in this article is available
via the MINT article ID
MINT-7899808
Abbreviations
CAN, acetonitrile; ASK1, apoptosis signal-regulating kinase 1; B23, nucleophosmin; BAD, Bcl-xL ⁄ Bcl-2-associated death promoter; BAK,
Bcl2-antagonist ⁄ killer; BAX, Bcl2-associated X protein; BMH1 ⁄ 2, yeast 14-3-3 homolog; CaM, calmodulin; COX IV, cytochrome c oxidase
subunit IV; DIG, digoxigenin; DNA-PK, DNA-dependent protein kinase; FADD, Fas-associated death domain; FOXO, forkhead box protein;
G418, geneticin; GADPH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HIP-55, hematopoietic progenitor
kinase 1-interacting protein of 55 kDa; JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide; LC-MS ⁄ MS, liquid
chromatography-tandem MS; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor-jB; RIP1, receptor-interacting protein 1;
RIP3, receptor-interacting protein 3; siRNA, small interfering RNA; Smac, second mitochondrial-derived activator of caspase; STAT3, signal
transducer and activator of transcription 3; TAP, tandem affinity purification; TNF-a, tumor necrosis factor-a; TSC2, tuberous sclerosis

protein 2; VASP, vasodilator-stimulated phosphoprotein.
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3321
Introduction
The term 14-3-3 denotes a large family of acidic pro-
teins that exist primarily as homodimers and heterodi-
mers within all eukaryotic cells [1,2]. In mammals,
there are seven 14-3-3 isoforms, designated by Greek
letters (a ⁄ b, g, e, c, s ⁄ h, f ⁄ d, and r) and encoded by
seven different genes [3,4]. 14-3-3 proteins play central
regulatory roles in eukaryotic cells by binding to
diverse target proteins, thereby modulating the function
of the associated partners [5]. In most cases, 14-3-3
proteins regulate cellular processes by binding to spe-
cific phosphoserine and phosphothreonine motifs
within target proteins [6]. Two optimal 14-3-3 phos-
phopeptide ligands with the consensus sequences
RSX(pS ⁄ T)XP and RX(Y ⁄ F)X(pS ⁄ T)XP (where pS ⁄ T
represents phosphoserine or phosphothreonine, and X
is any amino acid) have been defined [7]. Alternatively,
some 14-3-3 proteins bind to phosphorylated motifs
that are completely different to the consensus sites
described above [8], or even bind to unphosphorylated
motifs [9].
14-3-3 binding can alter the enzymatic activity, sub-
cellular localization, protein–protein interactions,
dephosphorylation and proteolysis of individual target
proteins [10]. Many 14-3-3 target proteins have been
shown to be involved in cancers, diabetes, Parkinson’s
disease, and other neurological diseases [11]. More-
over, 14-3-3 proteins have been shown to be key regu-

lators of a large number of processes, such as control
of cell proliferation, the cell cycle, regulation of human
metabolism, and apoptosis in mammalian cells [12–20].
In a number of cases, interaction of 14-3-3 proteins
with their target proteins promotes events that support
cell survival, mediating an essential antiapoptotic
signal in cells [21].
Apoptosis is an active process of cell death that
plays a critical role in normal development, mainte-
nance of tissue homoeostasis and elimination of dam-
aged or unwanted cells through a balance of
antiapoptotic and proapoptotic factors, which may be
shifted by extracellular signals [22]. It has been
reported that 14-3-3 binds members of the Bcl-2 fam-
ily, named Bcl-xL ⁄ Bcl-2-associated death promoter
(BAD) and Bcl-2-associated X protein (BAX), inhibit-
ing their proapoptotic activities [23,24]. 14-3-3 inhibits
cell death caused by other death promoters, such as
apoptosis signal-regulating kinase 1 (ASK1) [25]. Fur-
thermore, 14-3-3 protein binds to a member of the
family of forkhead transcription factors named fork-
head box protein (FOXO), blocking its translocation
to the nucleus and later activation of death genes [26].
These functions of 14-3-3 proteins have been reported
to be dependent on their dimeric structure. The
dimeric status of 14-3-3 proteins is regulated by site-
specific serine (Ser58) phosphorylation by sphingosine-
dependent kinase 1. This serine is located within the
dimer interface of 14-3-3 proteins, and its phosphoryla-
tion promotes the formation of a monomeric form of

14-3-3. Thus, phosphorylation of Ser58 on 14-3-3f
controls its ability to modulate target protein activity,
and this may have significant implications for the regu-
lation of many cellular processes, including apoptosis,
by preventing dimer-dependent inactivation of proa-
poptotic BAD or BAX [27]. Ceramide, a bioactive
lipid mediator, was found to be an apoptosis inducer
that activates sphingosine-dependent kinase 1, regu-
lates Bcl-2 expression, blocks survival signals, and acti-
vates phosphatases (protein phosphatase 1 and protein
phosphatase 2A) [28–31]. Several studies have pro-
posed that ceramide and its metabolic derivatives be
therapeutically applied in cancer-suppressing strategies
[32–36].
Inhibition of apoptosis by 14-3-3, through known
processes such as association with BAD, FOXO, and
ASK1, and other unknown processes that involve
mitogen-activated protein kinase (MAPK) and phos-
phoinositide 3-kinase cascades, suggests that 14-3-3
has an important antiapoptotic function. Expression of
a polypeptide that prevents 14-3-3 proteins from bind-
ing to targets in mammalian cells triggers apoptosis
and decreases viability in prostate, lung and cervix
cancer cell lines [37,38]. Furthermore, treatment with
2-methoxyestradiol resulted in decreased 14-3-3 expres-
sion that, in parallel with apoptosis induction,
decreased cell growth [39], and the use of 14-3-3f anti-
sense in cancer cell lines increased the sensitivity of the
cells to stress-induced apoptosis, such as that induced
by UV light, IR light, and doxorubicin [40–42]. On the

other hand, several studies found increased expression
of 14-3-3f in lung, stomach and breast cancers [42–47].
These data suggest that 14-3-3 proteins have a role in
regulating cancer cell proliferation and, as such, could
be targeted by cancer therapies.
Several proteomics studies have been performed to
find new 14-3-3-interactor proteins under physiological
conditions or even during mitosis [12–16,18–20]. Never-
theless, the work reported here is the first study
to include a comprehensive proteomics analysis of
14-3-3-binding proteins under physiological conditions
as compared with apoptosis stimulation, with the aim of
increasing our knowledge of the role of 14-3-3 proteins
in the apoptotic pathway. Because antineoplastic thera-
pies ultimately eliminate tumor cells by the induction of
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3322 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
apoptosis, a comprehensive understanding of how
14-3-3-mediated survival pathways inhibit apoptosis
may allow the use of 14-3-3 antagonists to sensitize
tumor cells for effective therapy.
Thus, to identify novel cellular survival functions of
14-3-3 proteins, global proteomics and biochemical
analyses were carried out to identify proteins that
bind 14-3-3 proteins during apoptotic and survival
conditions. These 14-3-3-interacting proteins were
purified from extracts of both control and C2-cera-
mide-stimulated HeLa cells, using tandem affinity
purification (TAP) methodology. The proteins, identi-
fied by liquid chromatography–tandem MS (LC-

MS ⁄ MS) analysis, were involved in multiple cellular
biological processes, but a pool of these proteins
had important functions in apoptosis through regula-
tion of intermediate filament integrity, cell blebbing,
formation of apoptotic bodies, DNA repair, and
regulation of oncogenic or death promoters dur-
ing apoptosis. Using the small interfering RNA
(siRNA) technique, the survival role of 14-3-3f during
C2-ceramide-induced apoptosis was characterized.
The involvement of identified C2-ceramide-regulated
14-3-3-binding proteins with several processes that
control apoptosis suggests possible survival roles of
14-3-3 proteins in addition to others that have been
previously characterized.
Results
Identification of 14-3-3-binding proteins related
to apoptosis
A few years ago, in a proteomics study of 14-3-3-affin-
ity purification of over 200 human phosphoproteins,
new links of 14-3-3 proteins with the regulation of
cellular metabolism, proliferation and trafficking were
shown [12]. Related to the functions of 14-3-3 proteins
as regulators of cell survival with central roles in inhib-
iting apoptosis, several apoptotic-related 14-3-3-bind-
ing proteins were identified in our study. Thus, we
found further 14-3-3-interactor proteins that are regu-
lators of apoptosis, such as receptor-interacting protein
kinase 1 (RIP1), programmed cell death protein
6 ⁄ ALG2 (apoptosis-linked gene 2), second mitochon-
drial-derived activator of caspase (Smac), signal trans-

ducer and activator of transcription 3 (STAT3), and
hematopoietic progenitor kinase 1-interacting protein
of 55 kDa (HIP-55). Using both MS and MALDI-
TOF ⁄ TOF MS tryptic mass fingerprinting, those
proteins were identified as 14-3-3-interactor proteins;
however, studies of their presence in the eluted fraction
from the 14-3-3-affinity chromatography column to
confirm these data were not performed at the time.
Here, western blotting analysis showed the presence of
the corresponding protein with the appropriate molec-
ular mass in the ARAApSAPA elution pool from the
14-3-3-affinity chromatography column (Fig. 1). These
data show that proteins such as RIP1, Smac, STAT3
and HIP-55 were eluted from the affinity column, con-
firming these proteins as 14-3-3-interactor proteins
under physiological conditions. Note that none of
these proteins was eluted from the column by either
extensive washing under high-salt conditions or mock
elution with control phosphopeptides that do not bind
to 14-3-3 proteins. These results indicate that isolated
proteins bind to the phosphopeptide-binding sites on
the 14-3-3 proteins, either directly or as components of
protein complexes.
As mentioned above, 14-3-3 interacts with apopto-
sis-related proteins such as BAD, FOXO or ASK1 to
perform its apoptosis-suppressing role in cells. Here,
Crude
Flow through
1st Wash
2nd Wash

3rd Wash
Control
14-3-3BP
BAX
BID
Caspase-8
Caspase-9
FADD
HIP-55
BAK
BAD
RIP1
Smac
STAT3
RIP3
Fig. 1. 14-3-3-affinity chromatography of human HeLa cell extracts.
Clarified HeLa cell extract was subjected to chromatography on
14-3-3–Sepharose, as described in Experimental procedures. Column
fractions were subjected to SDS ⁄ PAGE, using 10% Tris ⁄ glycine
gels, and transferred to nitrocellulose membranes. The amounts of
protein subjected to SDS ⁄ PAGE were as follows: extract, flow
through and beginning of salt wash (1st Wash), 40 lg of each;
middle and end of salt wash (2nd Wash and 3rd Wash, respectively),
protein undetectable; control (phospho)peptide pool, < 1 lg; and
ARAApSAPA elution pool, 2 lg. Western blots were probed with
antibodies against the indicated proteins related to apoptosis.
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3323
14-3-3 interaction with other apoptosis-related proteins
was analyzed by its presence in the 14-3-3-affinity

chromatography elution pool. Thus, proapoptotic pro-
teins such as receptor-interacting protein kinase 3
(RIP3) and Bcl-2-antagonist ⁄ killer (BAK) were eluted
from the 14-3-3-affinity chromatography column, sug-
gesting a broad role of 14-3-3 proteins in apoptosis
regulation. Note that the well-known apoptosis-related
14-3-3-binding protein BAD [23] was eluted from the
affinity chromatography column, giving confidence in
this technique. Additionally, the proapoptotic protein
BAX [24], which is known to be a 14-3-3-interactor
protein, did not appear to be eluted from the column,
probably because its defined interaction with 14-3-3
proteins is independent of phosphorylation (which is a
requirement for elution from the column). On the
other hand, members of extrinsic apoptosis pathways,
such as caspase-8, Fas-associated death domain
(FADD), and Bcl-2-interacting domain, did not bind
to 14-3-3 proteins under the conditions tested.
C2-ceramide promotes changes in 14-3-3-binding
patterns in HeLa cells during C2-ceramide-
induced apoptosis
With the aim of further analyzing the role of 14-3-3
proteins in apoptosis, an evaluation of the ability of
proteins to bind and to be regulated by 14-3-3 proteins
during C2-ceramide-induced apoptotis was carried out.
Previous results have established C2-ceramide as an
inducer of programmed cell death [28]. Thus, C2-cera-
mide-induced cell death in HeLa cells was analyzed,
and the time when this death occurred was established.
HeLa cells were left untreated or exposed to C2-cera-

mide (50 lm) for the indicated times (Fig. 2A). Sample
extracts were processed, and cell death was determined
as a percentage of the sub-G
1
population. The results
in Fig. 2A show that 50 lm C2-ceramide promoted cell
death in HeLa cells in a time-dependent manner.
In order to evaluate the 14-3-3-binding status of
proteins from HeLa cell extracts during C2-ceramide-
induced cell death, cells were treated in the presence or
absence of C2-ceramide (50 lm), and clarified extracts
were run into a gel and electrotransferred to a nitrocel-
lulose membrane. Ponceau dyes showed differential
protein expression, probably because ceramide is
linked to nuclear factor-jB (NFjB) and SAPK ⁄ JNK
cascades, which control protein expression in cells
[48,49], or perhaps because death initiation requires
caspase-dependent cleavage of specific targets [50–54].
Nevertheless, a digoxigenin (DIG)–14-3-3 overlay assay
showed protein bands with a significantly decreased
14-3-3-binding signal during C2-ceramide-induced cell
death (Fig. 2B). These data are intriguing, and may
suggest deregulation of the association of 14-3-3 pro-
teins with their targets during C2-ceramide treatment.
To further investigate the role of 14-3-3 proteins dur-
ing C2-ceramide treatment, downregulation of 14-3-3
proteins was performed and its effects on C2-ceramide
cell death were analyzed in HeLa cells.
First, levels of expression of seven human 14-3-3 iso-
forms were analyzed in a cervical cancer cell line

(HeLa) and in several breast cancer cell lines (Fig. 3).
The data showed that four different 14-3-3 isoforms
were expressed in HeLa cells, 14-3-3f and 14-3-3h
being the best expressed. Note that similar results were
A
0
10
20
30
40
50
0h 8h 24h
% of apoptosis
Time
B
94 kDa
67 kDa
43 kDa
30 kDa
DIG–14-3-3 Ponceau
C
C2
C
C2
Fig. 2. C2-ceramide induces changes in the pattern of 14-3-3 bind-
ing in HeLa cell protein extracts. (A) HeLa cells were incubated
with 50 l
M C2-ceramide for the indicated times. Apoptosis was
measured as percentage of cells with sub-G
1

DNA content, as
described in Experimental procedures. Columns represent the aver-
age of three different experiments. (B) Clarified extract from control
HeLa cells or HeLa cells treated with 50 l
M C2-ceramide overnight
were subjected to SDS ⁄ PAGE, using 10% Tris ⁄ glycine gels, and
transferred to a nitrocellulose membrane. Line (C) corresponds to a
nontreated control sample, and (C2) corresponds to an extract from
C2-ceramide-treated cells. The membrane was stained for protein
(Ponceau) and analyzed by DIG–14-3-3 overlay assay.
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3324 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
found in different breast cancer cell lines, where
14-3-3f and 14-3-3h were expressed well and uni-
formly. Meanwhile, other human 14-3-3 isoforms
showed low expression levels in HeLa cells, and were
also differently expressed in several types of breast
cancer cell line. Previous reports suggested that 14-3-3f
overexpression occurs in a high percentage of breast
tumors in the early stage of the disease, contributing
to the transformation of cells and also to the further
progression of breast cancer [42]. On the other hand,
downregulation of 14-3-3f reduced anchorage-indepen-
dent growth and sensitized cells to stress-induced
apoptosis [42]. These data suggest an important role of
14-3-3f overexpression in cancer; it is considered to be
a molecular marker for disease recurrence in breast
cancer patients, and may serve as an effective thera-
peutic target in patients whose tumors overexpress
14-3-3f. On the other hand, many reports suggest

important regulatory functions of this isoform in the
apoptotic pathway, through interactions with specific
components of the apoptotic process [55,56].
Downregulation of 14-3-3f with siRNA
oligonucleotide enhances C2-ceramide-induced
apoptosis in HeLa cells
To investigate the role of 14-3-3f downregulation
during C2-ceramide-induced apoptosis, sensitization
effects on cell death were analyzed in Hela cells in
which 14-3-3 binding was blocked by decreasing the
levels of 14-3-3f expression, using 14-3-3f siRNA.
Clarified extracts from HeLa cells, transfected with
14-3-3f siRNA or scrambled siRNA, were immunob-
lotted with antibodies against all human 14-3-3 iso-
forms (note that all mammal isoforms were tested, but
only four of them were visible in HeLa cells). Fig-
ure 4A shows specific downregulation of 14-3-3f iso-
forms by 14-3-3f siRNA oligonucleotide, but no
difference was observed in other human isoforms.
Cell death was determined as the percentage of the
sub-G
1
population, in order to evaluate the effects of
14-3-3f downregulation on C2-ceramide induced
apoptosis in transfected HeLa cells with 14-3-3f or
scrambled siRNA. The results in Fig. 4B show that
14-3-3f siRNA did not promote cell death on its own
after 48 h of transfection (or after an additional 24 h;
data not shown). Otherwise, downregulation of
endogenous 14-3-3f sensitized HeLa cells to cell death

promoted by C2-ceramide at 50 lm. Previously, it
was reported that downregulation of 14-3-3 proteins
sensitized cells to stress-induced apoptosis, such as
that induced by UV light and doxorubicin [41,42]. To
my knowledge, this is the first study to analyze
in detail the effects of 14-3-3 downregulation on
C2-ceramide-induced apoptosis. These results suggest
an important role of 14-3-3f in C2-ceramide-induced
cell death, probably by binding to and regulation of
specific targets that play important roles in C2-cera-
mide-induced cell death.
Knockdown of 14-3-3f promotes C2-ceramide-
induced activation of caspase-8 and regulation
of the mitochondrial apoptotic pathway
Mitochondrial dysfunction appears to be important
in C2-ceramide signaling of apoptosis. In vitro studies
have shown that C2-ceramide itself is not an efficient
inducer of nuclear apoptosis, unless mitochondria are
present [57]. It is still a matter of debate whether
C2-ceramide acts directly or indirectly on mitochon-
dria, but some data suggest that C2-ceramide could
signal mitochondrial apoptosis by inhibiting the pro-
tein kinase Akt, which is responsible for BAD phos-
phorylation, hence leading to inhibition of the
antiapoptotic protein Bcl-2 by BAD [58–60]. More-
over, C2-ceramide induces cytochrome c release from
mitochondria in a caspase-independent fashion,
leading to the activation of executioner caspases and
also activation of the initiator caspase-8 [61], effects
that are completely abolished by Bcl-2 and Bcl-xL

[62,63].
MDA-MB- 435
MCF-7/C4
MDA-MB- 231
MCF-7/E6
EvsaT
BT 474
HeLa
SKBR3
14-3-3γ
14-3-3ζ
14-3-3ε
14-3-3σ
14-3-3θ
Tubulin
Fig. 3. Analysis of expression levels of several 14-3-3 isoforms in
cervical and breast cancer cell lines. Extracts from cervical cancer
cells (HeLa) and several breast cancer cell lines (EvsaT, MDA-MB-
435, MDA-MB-231, MCF-7 ⁄ E6, MCF-7 ⁄ C4, BT-474, and SKBR3)
(30 lg), grown under physiological conditions, were subjected to
SDS ⁄ PAGE, using 10% Tris ⁄ glycine gels, and transferred to a nitro-
cellulose membrane. Western blots were probed with antibodies
against several isoforms of 14-3-3 proteins.
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3325
As downregulation of 14-3-3f has been seen to
enhance C2-ceramide-induced cell death, the aim was
to obtain further insights into the mechanism of sensi-
tization to C2-ceramide with 14-3-3f siRNA by investi-
gating the C2-ceramide-induced mitochondrial

apoptotic pathway. Therefore, western blot analysis
was performed to examine the presence of cyto-
chrome c in cytosolic and membrane fractions from
extracts of HeLa cells transfected with 14-3-3f siRNA
and treated with C2-ceramide. The results in Fig. 4C
show lowered cytochrome c levels in the mitochondria-
containing membrane fraction and the release of cyto-
chrome c to the cytosolic fraction on C2-ceramide
treatment when 14-3-3f was downregulated.
To confirm that the apoptosis cascade was fully
active in 14-3-3f siRNA-transfected HeLa cells treated
with C2-ceramide, the proteolytic degradation of the
nuclear protein poly(ADP-ribose) polymerase (PARP),
a substrate of effector caspases, and of the effector cas-
pase-8 were analyzed. As shown in Fig. 4D, PARP
cleavage was clearly induced in C2-ceramide-treated
HeLa cells previously transfected with 14-3-3f siRNA,
but no PARP cleavage was observed in untreated
HeLa cells. Cell extracts of indicated samples were
analyzed by western blot to determine caspase-8 acti-
vation. Procaspase-8 is first cleaved to the p43 ⁄ p41
intermediate fragments, releasing the small subunit
p12, and then subsequently processed to generate the
large, catalytically active p18 subunit [64]. On the
other hand, procaspase-8 has been reported to be
cleaved in the presence of C2-ceramide, both native
and exogenous, releasing active caspase-8, showing
that caspase-8 plays a role downstream of C2-ceramide
in the cell death process [65,66]. As shown in Fig. 4D,
neither the downregulation of 14-3-3f nor C2-ceramide

treatment alone promoted caspase-8 activation at the
indicated times, but a combination of both led to
the processing of procaspase-8 to its 43 and 41 kDa
Fig. 4. Downregulation of endogenous 14-3-3f sensitizes cells to
C2-ceramide-dependent apoptosis. (A) HeLa cells were transfected
either with siRNA oligonucleotide targeting 14-3-3f or with a scram-
bled RNA oligonucleotide, as described in Experimental procedures.
After 48 h, extracts from untransfected cells (C) or cells transfected
either with siRNA 14-3-3f (14-3-3) or with scrambled siRNA (SC)
were harvested for immunoblot analysis to verify knockdown of
endogenous 14-3-3f but not other isoforms (14-3-3r, 14-3-3e, and
14-3-3h). Tubulin was used as a protein loading control. (B) HeLa
cells transfected either with 14-3-3f or scrambled siRNA oligonu-
cleotide, or without siRNA (control), were treated with 50 l
M
C2-ceramide for the indicated times. Apoptosis was measured as
percentage of cells with sub-G
1
DNA content, as described in
Experimental procedures. Columns represent the average of three
different experiments. (C) HeLa cells were transfected as in (A) and
treated with 50 l
M C2-ceramide for an additional 4 or 8 h. Follow-
ing treatment, cells were lysed, and cytosolic proteins were sepa-
rated from mitochondria as described in Experimental procedures.
Levels of cytochrome c in cytosolic and membrane fractions were
determined by western blot. COX IV was used as a mitochondrial
loading control, and tubulin was used as a cytosolic protein loading
control. (D) HeLa cells untransfected (C) or transfected either with
siRNA oligonucleotide targeting 14-3-3f (14-3-3) or with a scram-

bled RNA oligonucleotide (SC) were treated in the presence or
absence of 50 l
M C2-ceramide for an additional 4 h. HeLa cells
were harvested for immunoblotting to analyze caspase-8 process-
ing with mouse monoclonal antibody against human caspase-8.
Both the 55 ⁄ 53 kDa native forms and the 43 ⁄ 41 kDa intermediate
cleavage products are indicated by arrows. PARP cleavage was
detected by immunoblotting with antibody against PARP; intermedi-
ate cleavage products are indicated by arrows. 14-3-3f antibodies
were used to verify knockdown of this isoform, and tubulin was
used as a protein loading control.
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3326 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
intermediate fragments. In conclusion, downregulation
of endogenous 14-3-3f sensitizes HeLa cells to the
C2-ceramide-induced mitochondrial apoptotic pathway
and activation of caspase-8 and PARP cleavage. These
data suggest an extensive and important role of 14-3-3
proteins in C2-ceramide-induced apoptosis, probably
through regulation of already known apoptosis-related
14-3-3-binding proteins, some of them most likely still
to be identified.
Purification of 14-3-3-binding proteins from HeLa
cells stably expressing green fluorescent protein
(GFP)–TAP–14-3-3f by TAP method
The data shown above suggest an interesting role of
14-3-3 proteins in C2-ceramide-induced apoptosis,
taking into consideration that 14-3-3 downregulation
sensitizes cells to C2-ceramide-induced apoptosis.
Thus, it was considered that 14-3-3 proteins modulated

C2-ceramide-induced apoptosis by binding to well-
known apoptosis-related proteins, but possibly also by
association with other targets with central roles in the
apoptotic process that remain to be identified. There-
fore, the aim was to identify new targets of 14-3-3
proteins involved in C2-ceramide-induced apoptosis.
To identify proteins associated with 14-3-3 in vivo,
a TAP tag approach was used, which allows the isola-
tion of native protein complexes from cells ectopically
expressing the tagged protein of interest [67]. The TAP
tag was fused to 14-3-3f as previously described [68].
This construct, generously provided by D. Alessi
(MRC, Dundee, UK), was successfully used to analyze
LKB1 phosphorylation-dependent 14-3-3 binding of
protein kinases closely related to AMP-activated pro-
tein kinase, such as QSK and SIK, in 293 cells [68].
Here, HeLa cells stably expressing GFP–TAP–14-3-
3f were generated and analyzed to determine the size,
level of expression and distribution of stably transfect-
ed fusion protein (Fig. 5A,B). Western blot analysis
with polyclonal antibody against 14-3-3f showed GFP–
TAP–14-3-3f of the expected size with a similar level of
expression to that of endogenous protein (Fig. 5A).
Moreover, the fusion protein showed a cytoplasmic
localization identical to the previously described locali-
zation for endogenous 14-3-3f [4,69] (Fig. 5B).
With regard to the goal of purifying and identifying
new 14-3-3-binding proteins involved in C2-ceramide-
induced apoptosis, HeLa cells stably expressing
GFP–TAP–14-3-3f were used for subsequent protein

purification and identification by the TAP method.
Thus, stably transfected HeLa cells were either exponen-
tially proliferating (untreated) or treated with C2-cera-
mide to induce apoptosis (see Experimental procedures).
Eluted pools from control and C2-ceramide-treated
GFP–TAP–14-3-3f-expressing HeLa cells, purified by
TAP, were further analyzed by LC-MS ⁄ MS.
Identification of 14-3-3-affinity purified proteins
by LC-MS ⁄ MS analysis
Analysis by LC-MS ⁄ MS of purified 14-3-3-binding
proteins from cells undergoing control and C2-cera-
mide-induced apoptosis showed different potential
ligands of 14-3-3f in both conditions. The 14-3-3 inter-
actors were grouped according to the processes in
which they had previously been involved (Tables 1 and
S1). The identified 14-3-3-binding proteins included
proteins involved in cell signaling, metabolic pathways,
A
115 kDa
82 kDa
49 kDa
64 kDa
37 kDa
26 kDa
GFP-TAP-14-3-3ζ
Endogenous 14-3-3ζ
GAPDH
HeLa
HeLa
14-3-3ζ

Wb: Anti-GFP Wb: Anti-14-3-3
19 kDa
HeLa
HeLa
14-3-3ζ
GAPDH
B
GFP-TAP-14-3-3
ζ
Fig. 5. Stable expression of GFP–TAP–14-3-3f in HeLa cells. (A)
Cell extracts from HeLa cells stably expressing GFP–TAP–14-3-3f
or control HeLa cells were harvested for immunoblotting to verify
expression of 14-3-3f fusion or endogenous protein with GFP (left
panel) and 14-3-3f (right panel) antibodies from Santa Cruz. Molecu-
lar masses of the transfected (GFP–TAP–14-3-3f ) and endogenous
protein indicated by western blotting were in agreement with
expected masses. GAPDH was used as a protein loading control.
(B) HeLa cells stably expressing GFP–TAP–14-3-3f were fixed in
3% (v ⁄ v) paraformaldehyde, and GFP localization was visualized
directly by observing GFP fluorescence. The cells were viewed
with a Leica CTR 6000 confocal microscope. A full color version of
this figure can be found in FEBS Journal online edition.
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3327
cytoskeletal dynamics, RNA binding, DNA binding
and chromatin structure, cellular trafficking, and pro-
tein folding. Some of them were previously shown to
be associated with 14-3-3 isoforms (indicated in
Table S1). Detection of those 14-3-3 ligands already
Table 1. Comparative analysis of 14-3-3-binding proteins identified

by TAP–MS from control or C2-ceramide-treated GFP–TAP–14-3-3f-
expressing HeLa cells. This is an abbreviated version of Table S1;
proteins identified by TAP and LC-MS ⁄ MS analysis were grouped
into functional classes, and data were searched against the Euro-
pean Bioinformatics Institute ⁄ International Protein Index human
database, using the
MASCOT search algorithm (see Experimental
procedures). The data were obtained by LC-MS ⁄ MS analysis of
tandem affinity-purified 14-3-3f-associated proteins from GFP–TAP–
14-3-3f-expressing HeLa cells left untreated (control) or stimulated
with C2-ceramide to induce apoptosis. Each protein identification
was manually confirmed to ensure that no other human proteins
matched the peptide sequences obtained. Interactions validated by
biochemical methods are indicated in bold.
Control Ceramide
Chromatin structure, DNA binding
Histone H1.0 Histone H1.0
Histone H1.3
Histone H1t
Histone H2A.x
Histone H2A type 1
Histone H2B type 1 Histone H2B type 1
Histone H4 Histone H4
B23
Ttransforming growth factor-b
-induced transcription factor
2-like protein
RNA binding
Heterogeneous nuclear
ribonucleoproteins C1 ⁄ C2

Heterogeneous nuclear
ribonucleoproteins A2 ⁄ B1
RNA-binding protein Raly
Translation
40S Ribosomal protein S3 Elongation factor 1 a1
Protein folding and processing
E3 ubiquitin-protein ligase CBL
Cellular trafficking
Voltage-dependent L-type
calcium channel subunit a1S
Metabolism
ATP synthase subunit a,
mitochondrial precursor
ATP synthase subunit a,
mitochondrial precursor
Ubiquitin C-terminal
hydrolase 42
ATP synthase subunit b
Carbamoyl-phosphate
synthase, mitochondrial
precursor
Hydroxymethylglutaryl-
CoA synthase
U6 snRNA-specific
terminal
uridylyltransferase 1
Cellular signaling
Histone H1.2
Myosin regulatory light chain 2 Myosin light chain
kinase 2

Titin
Table 1. (Continued).
Control Ceramide
CaM
Centrosomal Nek2-associated
protein 1
TSC2
Myosin light chain kinase 2
14-3-3f ⁄ d 14-3-3f ⁄ d
14-3-3e 14-3-3e
14-3-3c
14-3-3h 14-3-3h
14-3-3g
B-cell scaffold protein with
ankyrin repeats
14-3-3r
14-3-3b ⁄ a
DNA-PK catalytic subunit
Serine ⁄ threonine protein
kinase WNK4
Cellular organization
Vimentin
Lamin-A ⁄ C
a-Actinin-2
a-Actinin-3
Desmin
VASP
Myosin-2
Myosin-3
Myosin-7 (myosin heavy

chain 7)
Myosin-7
Myosin-9 Myosin-9
Myosin-11
Myosin-13 Myosin-13
Myosin light polypeptide 3 Myosin light
polypeptide 3
a-Actin-2 Actin, cytoplasmic 1
c-Actin
Actin, cytoplasmic 1
Tubulin b Tubulin a
Ankyrin repeat
domain-containing protein 18A
Ankyrin repeat
domain-containing
protein 18A
Heat-shock protein b1
a-Actin-2
Unclassified
Keratin, type II cytoskeletal 8 Keratin, type II
cytoskeletal 8
Keratin, type I cytoskeletal 17 Keratin, type I
cytoskeletal 17
Keratin, type I cytoskeletal 18
Tropomyosin-1 a chain
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3328 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
known implies that the conditions used here for the
TAP tag purification allowed the identification of
genuine 14-3-3 ligands. Detailed analysis of the

14-3-3-asociated proteins found showed that 46 of them
were exclusively present in one of the conditions ana-
lyzed and 15 of them were involved, to a greater or les-
ser extent, in the apoptotic process, according to
previous reports (detailed in Table 2). It is interesting to
note that 14-3-3f copurified with other 14-3-3 isoforms,
which is in accordance with previous reports showing
heterodimerization among different 14-3-3 isoforms [1].
Detection of 14-3-3-binding motifs on purified
and identified 14-3-3-binding proteins
The TAP tag approach allows the isolation of native
protein complexes from cells ectopically expressing the
tagged protein of interest, so proteins associated with
14-3-3 proteins were purified and identified in this
study (Tables 1 and 2). Frequently, 14-3-3 proteins
regulate cellular processes by binding to phosphory-
lated motifs (phosphoserine and phosphothreonine)
within target proteins [6], but, because of the methodo-
logical characteristics of the TAP tag approach,
this phosphorylation-dependent binding of identified
proteins is not evident.
Two optimal 14-3-3 phosphopeptide ligands with the
consensus sequences [RSX(pS ⁄ T)XP and RX(Y ⁄ F)X
(pS ⁄ T)XP] have been defined [7], although some 14-3-3
proteins bind to phosphorylated motifs that are com-
pletely different to the consensus sites, or even bind to
unphosphorylated motifs [9]. To investigate the phos-
phorylation-dependent binding to 14-3-3 proteins of
the identified proteins, the presence of putative 14-3-3
consensus binding sites was determined for identified

14-3-3f-associated proteins, using the software scan-
site [70] (Table 2) (detailed in Table S3). Low-strin-
gency settings of the scansite algorithm were applied
to analyze 14-3-3-binding consensus motif mode I
[RSX(pS ⁄ T)XP] on identified proteins. Note that most
proteins studied were identified in normal cell growth
conditions, and lost association with 14-3-3 in the treat-
ments with ceramide. To determine whether this associ-
ation was phosphorylation-dependent, extracts from
GFP–TAP–14-3-3f HeLa cells were loaded onto an
IgG–agarose chromatography column. Phosphoryla-
tion-dependent 14-3-3-binding proteins were eluted
using a phosphopeptide (ARAApSAPA) that competes
with proteins for 14-3-3 binding in a phosphorylation-
dependent manner. The data in Fig. 6A show desmin
to be a protein eluted from the affinity column. To my
knowledge, desmin, a protein that has been shown to
actively participate in the execution of apoptosis [51],
was clearly identified here for the first time as a
phosphorylation-dependent 14-3-3-associated protein
under normal growth conditions, using LC-MS ⁄ MS
(Table 2) and biochemical validation (Fig. 6A). Fur-
thermore, the data shown here confirm vasodilator-
stimulated phosphoprotein (VASP), nucleophosmin
(B23) and calmodulin (CaM), whose 14-3-3 binding
was suggested in previous studies, as phosphorylation-
dependent 14-3-3-associated proteins (Fig. 6A).
The data in Fig. 6A show vimentin to be a phos-
phorylation-dependent 14-3-3-binding protein in con-
trol conditions. Analysis using the highest-stringency

settings in the scansite algorithm showed Ser39 in
vimentin to be the most probable 14-3-3-binding site
(Table S3). These data support previous findings sug-
gesting that 14-3-3 binding of vimentin is a phosphory-
lation-dependent mechanism [71]. Tuberin [tuberous
sclerosis protein 2 (TSC2)], a tumor suppressor protein
that antagonizes the mTOR signaling pathway,
was also found to be a phosphorylation-dependent
14-3-3-binding protein. These data support previous
results showing that Akt phosphorylation of Ser939 in
TSC2 is required for its association with 14-3-3 [72].
Both results gave confidence in this technique.
On the other hand, the TAP tag approach and phos-
phopeptide-specific elution from IgG–agarose chroma-
tography columns allows the isolation of native proteins
from cells either directly or as components of protein
complexes. To determine whether isolated proteins
undergo direct interactions with 14-3-3, immunoprecipi-
tation assays for several isolated apoptotis-related
14-3-3-binding proteins were performed. Figure 6B
shows VASP and B23 to be phosphorylation-dependent
14-3-3-associated proteins that undergo direct interac-
tions with 14-3-3 proteins. TSC2 also showed a direct
interaction with 14-3-3 proteins, supporting previous
results [72], and giving confidence in this technique.
Biochemical validation of identified
14-3-3-associated proteins related to apoptosis
The combination of TAP and LC-MS ⁄ MS allowed iden-
tification of 14-3-3-binding proteins from both control
cells and those subjected to C2-ceramide treatments.

These data showed a pool of 14-3-3-interactor proteins
involved in apoptosis, the 14-3-3-binding pattern being
regulated during C2-ceramide-induced apoptosis
(Table 2). Silver staining of a gel loaded with the eluted
fractions from TAP purification showed different bands
of 14-3-3-binding proteins between control and C2-cera-
mide-induced apoptosis conditions (Fig. 7). These data
also support the idea that C2-ceramide-induced apop-
tosis promoted changes in the 14-3-3-binding pattern
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3329
Table 2. Selected apoptosis-related 14-3-3-associated proteins identified by TAP–MS and immunoblotting analysis. Apoptosis-related 14-3-3-
binding proteins identified in this study by LC-MS ⁄ MS and ⁄ or western blot analysis are listed and grouped by their functions. The role of
every protein in the apoptotic process is reported. References are cited for consensus binding sites (CBSs) for every protein; for the details
of every site found, see Table S3. Underlining indicates proteins that undergo 14-3-3-binding under control conditions but lose this associa-
tion after C2-ceramide treatment. Nonunderlined proteins bind to 14-3-3 proteins under conditions of C2-ceramide-induced apoptosis.
Function GI accession no. Name CBS Notes
Chromatin
structure, DNA
binding
121992
Histone H2A.x
a
[2] Histone H2AX induction occurs only in apoptotic nuclei in cells, and
is implicated in the restoration of genomic integrity in response to
DNA double-strand breaks [80]
114762
B23
b,c
[4] B23 negatively regulates p53 and antagonizes stress-induced

apoptosis in human normal and malignant hematopoietic cells [76]
RNA binding 108935845
Heterogeneous nuclear
ribonucleoproteins C1 ⁄ C2
a
[4] Upregulation of hnRNP C1 ⁄ C2 during ischemia or
staurosporine-induced apoptosis in mice may foster the
synthesis of XIAP as a protective pathway against apoptotic
effects [95]
Protein folding
and processing
115855
E3 ubiquitin-protein
ligase CBL
a
[1] Caspase-3 negatively regulates Bim expression by stimulating its
degradation through E3-ubiquitin ligases Cbl, thus creating a
negative feedback loop in the Bim caspase axis [88]
Metabolism 4033707 Carbamoyl-phosphate
synthase,
mitochondrial
precursor
a
[2] Carbamoyl-phosphate synthase (CPS) is part of a multienzymatic
protein (CAD) required for the de novo synthesis of pyrimidine
nucleotides and cell growth. CAD is a target for caspase-dependent
regulation during apoptosis, in this case a fast inactivation of
CPS occurs [89]
Cellular
signaling

417101
Histone H1.2
a
[1] Histone H1.2 is translocated to mitochondria and associates with
BAK in cells undergoing bleomycin-induced apoptosis. Upon DNA
damage, histone H1.2 acts as a positive regulator of apoptosome
formation, triggering activation of caspase-3 and caspase-7 via
APAF-1 and caspase-9 [96–98]
127169
Myosin regulatory
light chain 2
a
[1] Myosin regulatory light chain phosphorylation is critical for
apoptotic membrane blebbing and the active morphological
changes during apoptosis [90]
108861911
Titin
a
[9] Titin expression is induced by cyclosporin A via activation of MAPK
pathways, and this may promote proliferation, promote invasion
and inhibit apoptosis of human first trimester trophoblasts [91]
49037474
CaM
a,b
[0] CaM has been shown to regulate apoptosis in tumor models.
CaM-specific inhibitor increased apoptotic cell death with
morphological changes characterized by cell shrinkage and nuclear
condensation [92]
1717799
TSC2

b,c
[17] TSC2 is a tumor suppressor that antagonizes the mTOR signaling
pathway, thus regulating cell growth and proliferation. TSC2
activates BAD to promote apoptosis and negatively regulate
Bcl-2’s antiapoptotic effects on low serum deprivation-induced
apoptosis [99–101]
74751216
B-cell scaffold
protein with ankyrin
repeats (BANK1)
a
[2] It has been reported that the BANK1 gene is one of the most
downregulated genes in colorectal cancer patients, and this
suggests that it can be used as a novel blood marker for
colorectal cancer [93]
4506539
RIP1
b
[4] RIP1 is a specific mediator of the p38 MAPK response to TNF-a
[94]
205371831
RIP3
b
[4] Overexpression studies revealed RIP3 to be a potent inducer of
apoptosis, being capable of selectively binding to large prodomain
initiator caspases and attenuating both RIP1 and TNF-a
receptor-1-induced NF-jB activation [102–104]
38258929 DNA-PK catalytic
subunit
b,c

[6] The crucial survival role of DNA-PK in the repair of DNA double
strand breaks is highlighted by the hypersensitivity of
DNA-PK(– ⁄ –) mice to IR light. DNA-PK is robustly activated in
apoptotic cells during C2-ceramide treatment [82]
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3330 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
of proteins from cell extracts. Additionally, to validate
the LC-MS ⁄ MS data, antibodies against some of the
identified proteins were used to analyze their 14-3-3
interaction in survival or apoptotic conditions (Fig. 7).
Thus, a combination of LC-MS ⁄ MS and western blot
analysis showed that proteins related to the apoptotic
process, such as RIP1, VASP, and RIP3, were 14-3-3-
binding proteins whose association was lost during
C2-ceramide-induced apoptosis. Meanwhile, the cata-
lytic unit of DNA-dependent protein kinase (DNA-
PK), a protein with an essential role in DNA double-
strand break repair in the early stages of apoptosis,
raised their 14-3-3-binding status after treatment with
C2-ceramide (Fig. 7). Thus, LC-MS ⁄ MS and biochem-
ical validation analysis confirms a pool of apoptosis-
related proteins whose 14-3-3-binding status changes
during apoptosis, suggesting an extensive role for
14-3-3 proteins during apoptosis initiation.
Stable expression of GFP–TAP–14-3-3f in HeLa
cells delays C2-ceramide-induced cell death
Previous studies have clearly shown that 14-3-3 pro-
teins are survival proteins with antiapoptotic effects in
cells, by binding to well-known antiapoptotic proteins
and probably also to the apoptosis-related proteins

reported in the present work [37]. According to the
effect of 14-3-3f knockdown in sensitizing cells to
C2-ceramide-induced apoptosis, overexpression of
14-3-3f is able to delay cell death promoted by cera-
mide at longest time analysis (36 h) (Fig. 8). Thus,
evaluation of mitochondrial membrane potential
changes, using 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethyl-
benzimidazolylcarbocyanine iodide (JC-1), clearly
shows a delay in apoptosis induction in those cells that
overexpress 14-3-3f. These data support the important
role that 14-3-3 proteins have in C2-ceramide-induced
apoptosis, by binding to well-known apoptosis-related
proteins, and probably also to other apoptosis-related
proteins that have been suggested here.
Discussion
The aim of this study was to gain further understand-
ing of the role of 14-3-3 proteins in cellular fate,
promoting cell survival or inhibiting proapoptotic pro-
cesses in cells. New aspects are evident from this work:
(a) apoptosis-related proteins such as RIP3, BAK and
desmin were identified as new phosphorylation-depen-
dent 14-3-3-binding proteins under normal growth
conditions; (b) apoptosis-related proteins previously
identified by others, using MS ⁄ MS analysis, such as
RIP1, Smac, STAT3, B23, and CaM, were confirmed
here by immunoblot analysis to be phosphorylation-
dependent 14-3-3-associated proteins; (c) C2-ceramide-
induced apoptosis promoted decay of the 14-3-3-binding
signal of proteins in cell extracts; (d) depletion of
14-3-3f sensitized cells to C2-ceramide-induced cell

death, whereas overexpression of this isoform delayed
cell death; (e) a combination of TAP purification and
LC-MS ⁄ MS identified 15 proteins involved in cell
survival processes, their 14-3-3-binding status being
changed when apoptosis was promoted; and (f) immu-
noblot analysis showed that the 14-3-3-binding status
of VASP, RIP1 and RIP3 decayed during induced
apoptosis, whereas the association of DNA-PK with
14-3-3 increased during cell death.
Several regulators of apoptosis, such as RIP1, Smac,
STAT3, and HIP-55, were previously identified as
14-3-3-interactor proteins by a combination of 14-3-3-
affinity chromatography purification and MALDI-TOF
MS ⁄ MS techniques [12]. In this study, we identified
these proteins as 14-3-3-interactor proteins basically by
MS. However, a proper study confirming these data
Table 2. (Continued).
Function GI accession no. Name CBS Notes
Cellular
organization
55977767
Vimentin
a,b
[3] Mutations in vimentin disrupt the cytoskeleton in fibroblasts and
delay the execution of apoptosis. Cleavage of the p53–vimentin
complex enhances TNF-a-related apoptosis-inducing
ligand-mediated apoptosis in fibroblasts [50,52,53]
6686280
Desmin
a,b

[4] Caspase proteolysis of desmin at Asp263 produces a
dominant-negative inhibitor of intermediate filaments, and actively
participates in the execution of apoptosis [51]
1718079
VASP
b,c
[1] VASP binds to aII-spectrin and this association attenuates aII-spectrin
cleavage during apoptotic cells. Cleavage of the plasma membrane-associated
spectrins leads to cell shrinkage, membrane blebbing, the
formation of apoptotic bodies, and irreversible cell death [79]
a
Identification by LC-MS ⁄ MS.
b
Identification by biochemical approaches.
c
Proteins with low scores identified by LC-MS ⁄ MS.
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3331
by immunoblot analysis, showing their presence in the
eluted fraction from 14-3-3-affinity chromatography
columns, was not performed at the time. Here, western
blot analysis confirms that RIP1, Smac and STAT3
are 14-3-3-interactor proteins through a phosphoryla-
tion-dependent mechanism. Additionally, HIP-55 was
found in the eluted pool from the column, supporting
previous studies showing HIP-55 to be an Akt phos-
phorylation-dependent 14-3-3-binding protein [73].
Furthermore, other well-known proapoptotic 14-3-3-
binding proteins, such as BAD [23], were found in the
eluted fraction from the affinity column. Both of these

findings provide confidence in immunoblot analysis of
phosphorylation-dependent 14-3-3-binding proteins
eluted from the column. Thus, other proapoptotic
B23
VASP
CaM
A
WEL
Input
Purification
Desmin
Vimentin
TSC2
C
B
VASP
DIG–14-3-3
IP: VASP
DIG–14-3-3
B23
DIG–14-3-3
TSC2
C IP
IP: B23
C IP
IP: TSC2
C IP
Fig. 6. Analysis of phosphorylation-dependent 14-3-3 interaction of
identified targets. (A) Clarified extracts from untreated GFP–TAP–
14-3-3f-expressing HeLa cells were chromatographed on IgG–aga-

rose, as described in Experimental procedures. Eluted fractions
were obtained by using ARAApSAPA phosphopeptide, which pro-
motes dissociation of phosphorylation-dependent interactions of
14-3-3 proteins. Extract (C), wash (W) and eluted (EL) fractions from
the column were subjected to SDS ⁄ PAGE. Western blots were
probed with antibodies against the indicated proteins. The molecular
masses of the proteins indicated by western blotting were in agree-
ment with the masses predicted from the gene sequences
(Table S1). (B) Clarified extracts from untreated HeLa cells were
used for immunoprecipitation assay, as described in Experimental
procedures. Antibodies against VASP, B23 and TSC2 were used for
immunoprecipitation assays. Control (C) and immunoprecipitated
(IP) fractions were subjected to SDS ⁄ PAGE, transferred to a nitro-
cellulose membrane, and analyzed by DIG–14-3-3 overlay assay and
with antibodies against the indicated proteins. The molecular masses
of the proteins indicated by western blotting were in agreement
with the masses predicted from the gene sequences (Table S1).
Fig. 7. Western blot analysis confirms association of apoptosis-
related proteins with 14-3-3 proteins in HeLa cells. HeLa cells sta-
bly expressing GFP–TAP–14-3-3f were left untreated or stimulated
with C2-ceramide to induce apoptosis. Clarified extracts from both
conditions were processed by TAP methodology. Extract (EX),
wash (W) and eluted (EL) fractions were subjected to 10%
SDS ⁄ PAGE, and silver stained. Arrows indicate bands in both
eluted fractions that showed differences in control and C2-cera-
mide-stimulated GFP–TAP–14-3-3f-expressing HeLa cells. A similar
band in both eluted fractions (*) is shown (top panel). Fractions
from tandem affinity purifications were processed for immunoblot
analysis. Western blots were probed with antibodies against the
indicated proteins. The molecular masses of proteins indicated by

western blotting were in agreement with the masses predicted
from the gene sequences (Table S1). Levels of identified proteins
were quantified in eluted fractions from the column relative to the
amounts in the extracts, and the ratio is presented as arbitrary
units (n =3,P £ 0.0007, Student’s t-test).
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3332 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
proteins, such as RIP3 and BAK, were found to be
14-3-3-interactor proteins through a phosphorylation-
dependent mechanism. BAK is a ‘multidomain’ proa-
poptotic member that, together with BAX, appears to
be an essential gateway to the mitochondrial dysfunc-
tion required for cell death in response to diverse
stimuli [74].
These data suggest that 14-3-3 proteins may have an
extensive role in controlling cell survival beyond that
known to date. Comprehensive proteomics and bio-
chemical analyses were used to identify polypeptides
that are associated with 14-3-3 proteins in vivo in con-
trol or C2-ceramide-stimulated HeLa cells. Ceramide,
a bioactive lipid mediator, was found to be an apopto-
sis inducer [28–31]. Here, the important role of 14-3-3
proteins during C2-ceramide-induced apoptosis was
shown, because downregulation of 14-3-3f, using
siRNA, sensitized cells to C2-ceramide-induced cell
death, whereas overexpression of 14-3-3f delayed cell
death. These data are consistent with previous reports
suggesting that 14-3-3f downregulation sensitizes cells
to UV light or doxorubicin [41,42]. Taking into consid-
eration the important role of 14-3-3 proteins during

C2-ceramide-induced apoptosis polypeptides that are
associated with 14-3-3 proteins in vivo in control or
C2-ceramide-stimulated HeLa cells were identified using
LC-MS ⁄ MS. The data presented here show a broad
range of 14-3-3-associated proteins involved in diverse
potential functions, such as many different aspects of
metabolism, cytoskeletal organization, RNA binding,
DNA binding and chromatin structure, protein fold-
ing, ubiquitination and proteolysis, translation, and
cell signaling. These results reveal that 14-3-3 proteins
are involved in a wide range of cellular processes in
survival or apoptotic conditions. Previous proteomics
studies used techniques similar to those used here to
identify 14-3-3-binding proteins; FLAG-tagged 14-3-
3c, TAP-tagged 14-3-3r, TAP-tagged 14-3-3f, His-
tagged 14-3-3e or FLAG-tagged 14-3-3e were
expressed in HEK293 cells or in transgenic mice [14–
16,18,20], whereas our previous data were obtained
with the in vitro binding of human HeLa cell proteins
to immobilized homologous 14-3-3 proteins from yeast
14-3-3 homolog (BMH1 ⁄ BMH2) [12]. Also, Meek
et al. and Ruri et al. used mammalian GST–14-3-3f
and GST–14-3-3 [13,19]. The TAP technology, when
coupled to MS and confirmed by western blot analysis,
has proven to be efficient in allowing the characteriza-
tion of protein complexes from Escherichia coli, yeast,
and mammalian cells in culture, as well in transgenic
mice [75]. All of these previous proteomics studies
on 14-3-3-binding proteins using TAP purification
increased the possibility of finding genuine 14-3-3-asso-

ciated proteins. In fact, half of the 14-3-3-binding pro-
teins found here were previously described as 14-3-3
targets, such as vimentin and TSC2. However, several
of the identified proteins have not previously been
shown to bind 14-3-3, such as desmin and RIP3. The
fact that several known 14-3-3 targets were identified
in this screen suggests that many of the proteins identi-
fied here are likely to form complexes with 14-3-3,
either directly or indirectly. Analysis in detail of identi-
fied 14-3-3-binding proteins showed that more than
half of the proteins lost their ability to bind in
C2-ceramide-induced HeLa cells, and just 8% of the
total identified proteins increased their binding after
C2-ceramide treatment. A detailed study of identified
proteins showed 15 proteins to be involved in survival
processes in cells in which 14-3-3 binding was regu-
lated by C2-ceramide.
Among those proteins identified as 14-3-3-interactor
proteins under physiological conditions by LC-MS ⁄ MS,
some that have been already identified as 14-3-3-asso-
ciated proteins, B23, TSC2, and CaM, were found.
B23 is a nuclear protein that has previously been iden-
tified as a 14-3-3 interactor under normal growth con-
ditions in HeLa cells, by MS analysis [13]. Here, MS
data and biochemical validation showed, for the first
time, B23 to be a direct phosphorylation-dependent
14-3-3-interactor protein under physiological condi-
tions. B23 is a tumor marker, and exerts its oncogenic
effect through binding and by suppressing numerous
tumor suppressors. B23 is an antiapoptotic protein

that negatively regulates p53 in human normal and
malignant hematopoietic cells [76]. Moreover, proteo-
lytic cleavage of B23 during the apoptotic process has
been identified, whereas nuclear Akt interacts with B23
0
20
40
60
80
100
120
0 h 16 h 36 h
% JC-1 red/green
GFP–TAP–14-3-3
ζ
C2-ceramide 50 µM
*
Fig. 8. HeLa cells stably expressing GFP–TAP–14-3-3f delay
C2-ceramide-induced cell death. Control cells (white bars) or HeLa
cells stably expressing GFP–TAP–14-3-3f (black bars) were left
untreated or stimulated with C2-ceramide (50 l
M) to induce apopto-
sis. At the indicated times, apoptosis was assessed by mitochon-
drial membrane potential changes, using JC-1 (see Experimental
procedures). The percentage JC-1 red ⁄ green of C2-ceramide-trea-
ted cells was quantified as mean ± standard deviation (n =3,
*P = 0.0008, Student’s t-test).
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3333
and protects it from proteolytic cleavage, enhancing cell

survival [54]. Speculating on B23–14-3-3 interaction
under survival conditions by an Akt-dependent mechan-
ism and taking into consideration the survival role of
14-3-3 proteins, it could be suggested a role of 14-3-3
proteins supporting the oncogenic character of B23.
Novel 14-3-3-binding partners identified here include
desmin, for which 14-3-3 binding is a phosphorylation-
dependent mechanism, as has been well established by
LC-MS ⁄ MS and western blot analysis. scansite analy-
sis of the desmin sequence showed several 14-3-3-bind-
ing sites with relatively low stringency. It is interesting
to note that well-known 14-3-3-associated proteins
showed medium or low stringency by scansite
analysis [77]. Desmin has an important role during
apoptosis, because it is cleaved selectively by caspase-
6, producing a dominant-negative inhibitor of interme-
diate filaments that promotes apoptosis. Moreover,
stable expression of a caspase cleavage-resistant desmin
mutant partially protects cells from tumor necrosis
factor-a (TNF-a)-induced apoptosis [51].
This selective cleavage of desmin and B23 as part of
the apoptotic process could also be observed during
C2-ceramide-dependent apoptosis (data not shown). It
is interesting to note that Ponceau staining of proteins
in cell extracts under control conditions and C2-cera-
mide stimulation show differential protein expression,
probably because ceramide is linked to NF-jB and
SAPK ⁄ JNK cascades, which control protein expression
[48,49], but probably also because of activation of
apoptotic processes that selectively cleave some target

in a necessary mechanism during apoptosis activation,
such as desmin or B23.
A few years ago, an interesting study on Arabidopsis
cells provided a new function for 14-3-3 proteins: con-
trolling the cleavage of many targets in sugar-starved
cells [78]. These data provided the first evidence that
14-3-3 proteins could protect their own targets from
proteolysis. Here, analysis of Ponceau staining of pro-
teins in cell extracts suggests that some proteins could
be cleaved during apoptosis initiation; this fact sug-
gests an interesting hypothesis concerning the role of
14-3-3-binding in protecting 14-3-3 targets from prote-
olysis in the early stages of apoptosis.
Here, LC-MS ⁄ MS analysis and biochemical valida-
tion suggest that apoptotic-related proteins such as
VASP, RIP1 and RIP3 bind to 14-3-3 proteins under
physiological conditions, losing this association when
apoptosis is induced with C2-ceramide. DNA-PK
increases its 14-3-3 binding during C2-ceramide treat-
ment. Previously, we identified VASP as a 14-3-3-bind-
ing protein by using a combination of 14-3-3-affinity
chromatography and MALDI-TOF MS analysis under
normal growth conditions in HeLa cells, although con-
firmation by immunoblot analysis was not performed
at the time [12]. Here, a combination of LC-MS ⁄ MS
and western blot analysis showed VASP to be a 14-3-
3-binding protein that loses its association with 14-3-3
during C2-ceramide-induced apoptosis. It has been
reported that VASP binds to aII-spectrin and this
complex has a role in the regulation of cell shrinkage,

membrane blebbing and the formation of apoptotic
bodies during cell death [79]. The data presented here
show that 14-3-3 associates with VASP in a C2-cera-
mide-dependent manner. This association may suggest
the involvement of 14-3-3 proteins in the regulation of
membrane transformation processes during apoptosis.
Biochemical validation identified DNA-PK as a 14-3-
3-binding protein during C2-ceramide-induced apopto-
sis. Previous proteomics studies, using a different
approach, suggest that DNA-PK could bind 14-3-3
during interphase [13]. Here, LC-MS ⁄ MS analysis sug-
gests that this interaction occurs under apoptotic con-
ditions; moreover, the biochemical approach clearly
showed DNA-PK to be a 14-3-3-binding protein dur-
ing C2-ceramide-promoted apoptosis. DNA-PK is a
nuclear serine ⁄ threonine protein kinase working as a
molecular sensor for DNA double-strand breaks under
conditions of DNA damage and apoptosis. DNA-PK
promotes DNA repair, enhancing the signal via phos-
phorylation of many downstream targets, such as p53
and histone H2AX [80,81]. Furthermore, DNA-PK is
robustly activated in apoptotic cells, as shown by auto-
phosphorylation at Ser2056, and is able to initiate an
early step in the DNA damage response, before it is
inactivated by cleavage in order to forestall DNA
repair. Moreover, it has been reported that C2-cera-
mide induces DNA-PK activity before the appearance
of apoptotic markers, whereas the loss in activity was
coincident with cell death [82]. The role of 14-3-3 pro-
teins in promoting cell survival and data showing

DNA-PK–14-3-3 interaction during apoptosis initia-
tion raise the hypothesis that 14-3-3 binds DNA-PK to
contribute to the survival function of DNA-PK, pro-
moting DNA repair in the early stages of the apoptotic
process.
The data shown here suggest that 14-3-3 proteins pro-
mote cell survival not only by regulation of well-known
proapoptotic proteins, but also by binding proteins
involved in several cellular processes involved in survival
and death mechanisms in cells. Thus, 14-3-3 proteins
could promote cell survival through regulation of inter-
mediate filament integrity and cell shrinkage, such as
desmin and VASP, and oncogenic or death promoters,
such as B23 and RIP3. 14-3-3 proteins may also contrib-
ute to the survival role of DNA-PK, repairing DNA
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3334 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
during early stages of apoptotic. Finally, this biochemi-
cal and functional analysis proposes 14-3-3 as a survival
factor during C2-ceramide-induced apoptosis, and iden-
tifies novel 14-3-3 interactor proteins under survival or
death conditions in HeLa cells.
Future research can now focus on dissecting the
details of the signaling pathways involved in phosphory-
lation and 14-3-3-binding of identified targets; moreover,
the effects of 14-3-3 interactions on target functions will
be investigated. These data will help provide a com-
prehensive picture of the survival role of 14-3-3 proteins,
which may improve cancer therapy treatments.
Experimental procedures

Reagents
C2-ceramide was from Calbiochem (Merck Chemicals, Not-
tingham, UK); JC-1, active tobacco etch virus protease,
Optimen, Geneticin (G418) and protease inhibitor cocktail
tablets were from Invitrogen (Paisley, UK); protein IgG–
agarose and propidium iodide were from Sigma (St Louis,
MO, USA); CaM and CaM–Sepharose 4B were from GE
Heathcare (Waukesha, WI, USA). Peptide (ARAApSAPA)
was synthesized by Graham Bloomberg at the University of
Bristol, and generously provided by C. MacKintosh (MRC,
Dundee, UK).
Antibodies
Antibodies against 14-3-3 (types f, r, e, h, and c), Bcl-2-
interacting proteins (BAK, Bcl-2-interacting domain, BAD,
and BAX), RIP3, DNA-PK, CaM, TSC2 and B23 were
from Santa Cruz (Santa Cruz, CA, USA). Monoclonal
antibody against cytochrome c was from BD Pharmingen
(San Jose, CA, USA). Monoclonal antibody against PARP
was from Roche Molecular Biochemicals (San Francisco,
CA, USA). Rabbit antibody against cytochrome c oxidase
subunit IV (COX IV) and antibody against RIP1 were from
Abcam (Cambridge, UK). Antibodies against caspase-8,
caspase-9 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) were kindly provided by A. L. Rivas (CABI-
MER, Seville, Spain). Monoclonal antibody against
a-tubulin was from Sigma. Antibody against STAT3 was
from Cell Signalling (Danvers, MA, USA). Antibodies
against VASP, HIP-55 and Smac were kindly provided
by C. MacKintosh (MRC, Dundee, UK). Antibodies
against desmin and vimentin were generously provided by

K. Hmadcha (CABIMER, Seville, Spain).
siRNA
siRNAs against 14-3-3f were from Santa Cruz (sc-29583).
siRNA non-targeting scramble was synthesized by SIGMA
proligo. Cells were transfected with either 14-3-3f or scram-
bled siRNAs (20 nm), using DharmaFECT 1 (Dharmacon,
Denver, CO, USA), as described by the manufacturer.
After 48 h of transfection, cells were used for further
analysis.
Cell lines
HeLa cells were maintained in DMEM supplemented with
10% fetal bovine serum at 37 °C in a humidified 5%
CO
2
⁄ 95% air incubator. HeLa cells stably transfected with
GFP–TAP–14-3-3f were maintained in DMEM supple-
mented with 10% fetal bovine serum and 3 mg ⁄ mL G418.
Buffers
Lysis buffer contained 50 mm Tris ⁄ HCl (pH 7.5), 1 mm
EGTA, 1 mm EDTA, 0.2% (w ⁄ v) NP-40, 1 mm sodium
orthovanadate, 10 mm sodium-b-glycerophosphate, 50 mm
sodium fluoride, 5 mm sodium pyrophosphate, 0.27 m
sucrose, 1 mm dithiothreitol, and complete proteinase
inhibitor cocktail (one tablet per 50 mL). Buffer A con-
tained 50 mm Tris ⁄ HCl (pH 8), 0.15 m NaCl, 0.1% (w ⁄ v)
NP-40, 0.5 mm EDTA, and 1 mm dithiothreitol. Buffer B
contained: 50 mm Tris (pH 8), 0.15 m NaCl, 1 mm MgAc,
1mm imidazole, and 2 m m CaCl
2
. Buffer C contained

10 mm Tris (pH 7.5), 1 mm imidazole, and 20 mm EGTA.
NaCl ⁄ P
i
-Tween buffer contained 50 mm Tris ⁄ HCl (pH 7.5),
0.15 m NaCl, and 0.1% (v ⁄ v) Tween-20. Buffer D con-
tained 50 mm Tris ⁄ HCl (pH 7.5) and 0.1 mm EGTA.
Analysis of apoptosis
Hypodiploid apoptotic cells were assessed by flow cytometry
according to published procedures [83]. Flow cytometry was
performed on a FACScan cytometer using cell quest soft-
ware (Becton Dickinson, Mountain View, CA, USA). Apop-
tosis was determined by analyzing the cleavage of caspase-8
and the caspase substrate PARP by western blot, using anti-
bodies against caspase-8 and PARP. Cytocrome c release to
the cytosolic fraction was also used to assess apoptosis.
Apoptosis was also assessed by mitochondrial membrane
potential changes, using JC-1, a cationic carbocyanine dye
that accumulates in mitochondria during apoptosis, its
transition from low concentrations (green fluorescence) to
higher concentrations (red fluorescence) indicating mito-
chondrial membrane potential changes during apoptosis
[84]. Values were obtained by flow cytometry performed on
a FACScan cytometer, using cell quest software.
Immunoblotting and 14-3-3 overlay assay
Total cell lysates (30)50 lg) were heated at 90 °C for 5 min
in SDS sample buffer, subjected to PAGE, and electro-
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3335
transferred to nitrocellulose membranes. The membrane
was blocked for 1 h in NaCl ⁄ P

i
-Tween buffer containing
5% (w ⁄ v) skimmed milk. The membrane was probed with
1 lg ⁄ mL of indicated antibodies in NaCl⁄ P
i
-Tween and
5% (w ⁄ v) skimmed milk for 16 h at 4 °C. Detection
was performed using horseradish peroxidase-conjugated
secondary antibodies and the ECL system (Amersham
Biosciences-GE Heathcare, Waukesha, WI, USA). For
14-3-3 overlays, DIG-labeled 14-3-3 proteins were used
instead of a primary antibody. The membrane was incu-
bated with 5 lg ⁄ mL total His-BMH1 and His-BMH2 (yeast
14-3-3 isoforms) in NaCl ⁄ P
i
-Tween containing 1 mg ⁄ mL
BSA for 2 h at room temperature. After several washes, the
membrane was incubated with a horseradish peroxidase-
conjugated secondary antibody against DIG and exposed
using the ECL system [77,85].
Immunoprecipitation assay
Cells were lysed in lysis buffer, the lysate was centrifuged at
4 °C for 10 min at 13 000 g, and an aliquot of the superna-
tant (1.5 mg of protein) was incubated with 2 lg of anti-
body against VASP, antibody against B23 and antibody
against TSC2 coupled to 20 lL of protein G–Sepharose for
3 h at 4 °C. The suspension was centrifuged for 1 min at
13 000 g, the protein G–Sepharose–antibody–protein com-
plex was washed twice with 1 mL of lysis buffer containing
150 mm NaCl and twice with buffer D, and the immuno-

precipitate was resolved by SDS ⁄ PAGE for DIG–14-3-3 or
western blot analysis.
Cellular fractionation
Following treatment, cells were lysed and cytosolic proteins
were separated from mitochondria as previously described
[86]. Briefly, cells (7 · 10
5
) were washed with NaCl ⁄ P
i
and
lysed in 30 lL of ice-cold lysis buffer (80 mm KCl, 250 mm
sucrose, 500 mg ⁄ mL digitonin and protease inhibitors in
NaCl ⁄ P
i
). Then, cell lysates were centrifuged for 5 min at
10 000 g. Proteins from the supernatant (cytosolic fraction)
and pellet (membrane fraction) were mixed with Laemmli
buffer, and resolved on 15% SDS ⁄ PAGE minigels.
Generation of a stable GFP–TAP–14-3-3f-express-
ing HeLa cell line
The pGFP–TAP–14-3-3f construct is an expression vector
that enables the expression of 14-3-3f in cells and incorpo-
rates a GFP tag before the TAP tag to facilitate easier
selection of stable cell lines that express TAP–14-3-3f [68].
This expression vector was generously provided by D. Alessi
(MRC, Dundee, UK).
HeLa cells were cultured in 10 cm diameter dishes to 50–
70% confluence, and transfected with 3 lg of pGFP–TAP–
14-3-3f, using Fugene 6 reagent (Roche) according to the
manufacturer’s instructions. After 24 h, G418 was added to

the medium to a final concentration of 3 mg ⁄ mL, and the
medium was changed every 24 h, with maintenance of the
G418 concentration. After 14–20 days, individual surviving
colonies expressing low levels of GFP fluorescence were
selected and expanded. FACS analysis was also performed
to ensure uniform expression of GFP in the selected cell
lines, and anti-14-3-3f immunoblotting analysis of lysed
cells was performed to ensure a band at the expected appar-
ent molecular mass ( 80 kDa) (the isolated GFP–TAP tag
adds 50 kDa to the molecular mass of a protein).
TAP
The purification method was adapted from the previously
described TAP protocol [67]. For each TAP, 70 dishes
(15 cm) of the confluent cell line, untreated or treated with
50 lm C2-ceramide, were cultured. Apoptosis was evaluated
on cells treated with C2-ceramide, using JC-1 and flow
cytometry. When apoptosis was initiated (only 8–10% of
cells showed apoptotic features), dishes were washed with
NaCl ⁄ P
i
to remove the occasional dead cells, and cells were
lysed in 1.0 mL of ice-cold lysis buffer. The lysates were
centrifuged at 26 000 g for 30 min at 4 °C, and the superna-
tant (150 mg of protein extract) was incubated with 0.4 mL
of rabbit IgG–agarose beads for 90 min at 4 °C. The IgG–
agarose was washed extensively with lysis buffer containing
0.15 m NaCl, and then several times in buffer A prior to
incubation with 0.4 mL of buffer B containing 500 U of
TEV protease. After 3 h at 4 °C,  70–90% of the TAP-
tagged 14-3-3-binding proteins had been cleaved from the

IgG–agarose, and the eluted proteins were incubated with
0.3 mL of rabbit CaM–Sepharose equilibrated in buffer B.
After 1 h at 4 °C, the CaM–Sepharose was washed with
buffer B containing 0.1% (v ⁄ v) NP-40, and this was fol-
lowed by a final wash with buffer B without NP-40. The
CaM–Sepharose was then incubated with 0.25 mL of buf-
fer C to elute 14-3-3-binding proteins. Small portions of
both EGTA-eluted fractions were treated with SDS sample
buffer, subjected to SDS ⁄ PAGE, and then silver stained to
analyze the pattern of protein bands obtained under both
conditions. Eluted samples were treated with trypsin, and
the tryptic peptides were analyzed by LC-MS ⁄ MS.
LC-MS ⁄ MS analysis of tryptic peptides
The resulting tryptic peptide mixtures were injected onto a
C-18 RP nanocolumn (Discovery BIO Wide pore, Supelco),
and analyzed in a continuous acetonitrile (ACN) gradient
consisting of 0–50% 95% ACN ⁄ 0.5% acetic acid in
45 min, and 50–90% 95% ACN ⁄ 0.5% acetic acid in 1 min.
A flow rate of  300 nL ⁄ min was used to elute peptides
from the RP nanocolumn to a PicoTip emitter nanospray
needle (New Objective, Woburn, MA, USA) for real-time
ionization and peptide fragmentation on an Esquire HCT
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3336 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS
Ion Trap (Bruker-Daltoniks) mass spectrometer. Every sec-
ond, the instrument cycled through acquisition of a full-
scan mass spectrum and one MS ⁄ MS spectrum. A 3 Da
window (precursor m ⁄ z ± 1.5), an MS ⁄ MS fragmentation
amplitude of 0.90 V and a dynamic exclusion time of
0.30 min were used for peptide fragmentation. Nano-LC

was automatically performed on an advanced nanogradient
generator (Ultimate nano-HPLC; LC Packings, Amster-
dam, The Netherlands) coupled to an autosampler (Famos;
LC Packings). hystar 2.3 was used to control the whole
analytical process and the peak list. MS ⁄ MS data were
searched against the human database of the European
Bioinformatics Institute and the Swiss Institute for Bioin-
formatics [Swiss-Prot 51.6 (257 964 sequences; 93 947 433
residues) (taxonomy: mammal, 49 863 sequences)] was
searched using the mascot search algorithm (http://
www.matrixscience.com [87]) (mascot version 1.8; Matrix
Science), and GI accession numbers were assigned by com-
parisons with the National Center for Biotechnology Infor-
mation databases. Search parameters were as follows: type
of search, MS ⁄ MS ion search; enzyme, trypsin⁄ P; fixed
modification, carbamidomethylation of cysteine; variable
modifications, oxidation (M); mass values, monoisotopic;
protein mass, unrestricted; peptide mass tolerance, ±2 Da;
fragment mass tolerance, ±1 Da; maximum missed cleav-
ages, 1; instrument type, ESI-TRAP. Most proteins were
unambiguously identified by the sequencing of several inde-
pendent peptides. Identifications with mascot scores indi-
cating a higher than 5% probability that the match could
be a random event were categorically rejected [87]. Informa-
tion about all identified proteins and peptides are provided
in Table S1 and S2, and Fig. S1.
14-3-3-affinity chromatography of human HeLa
cell extracts
HeLa cells were thawed and extracted in 20 mL of lysis buf-
fer [50 mm Tris ⁄ HCl (pH 7.5), 1 mm EGTA, 1 mm EDTA,

0.2% (w ⁄ v) NP-40, 1 mm sodium orthovanadate, 10 mm
sodium-b-glycerophosphate, 50 mm sodium fluoride, 5 mm
sodium pyrophosphate, 0.27 m sucrose, 1 mm dithiothreitol
and a complete proteinase inhibitor cocktail]. The broken
cells were centrifuged at 15 000 g for 60 min, and the super-
natant was collected, filtered and processed for affinity chro-
matography as previously described [82]. Briefly, the clarified
solution was mixed end-over-end for 1 h at 4 °C with 5 mL
of Sepharose linked to BMH1 ⁄ BMH2 (the Saccharomy-
ces cerevisiae 14-3-3 isoforms) [85]. The mixture was poured
into a column, the flow-through was collected, and the col-
umn was washed with 600 column volumes of 50 mm
Tris ⁄ HCl (pH 7.5), 500 mm NaCl, and 1 mm dithiothreitol
(buffer A), and then with 12 mL of a control synthetic phos-
phopeptide that does not bind 14-3-3 proteins (1 mm
RSRTRTDpSYSAGQSV in buffer A). Proteins that bind to
the phosphopeptide-binding site of 14-3-3 proteins were
eluted with 12 mL of 1 mm ARAApSAPA phosphopeptide
in buffer A. Samples (12 mL) from the beginning, middle
and end of the 0.5 NaCl wash, the control peptide mock-elu-
tion pool (12 mL) and the ARAApSAPA elution pool
(12 mL) were each concentrated to  400 mL, desalted in Vi-
vaspin concentrators, and analyzed as indicated in Fig. 1.
Affinity purification of 14-3-3-binding proteins
HeLa cells stably expressing GFP–TAP–14-3-3f were left
untreated or exposed to C2-ceramide (50 lm) to induce
apoptosis. Clarified lysates of HeLa cells containing 50 mg
of total protein were incubated at 4 °C for 3 h on a vibrat-
ing platform with 400 lL of IgG agarose. The beads were
washed 10 times with 1 mL of lysis buffer containing

0.15 m NaCl, and five times with 50 mm Tris ⁄ HCl
(pH 7.4). The 14-3-3-binding proteins were eluted using a
phosphopeptide (ARAApSAPA) that competes with pro-
teins for the 14-3-3 binding. Eluted pools were then sub-
jected to electrophoresis and immunoblotting.
Acknowledgements
This work was supported by Ministerio de Educacio
´
n
y Ciencia grant BFU2006-01088 ⁄ BMC and ‘Programa
Ramo
´
n y Cajal’ contract (B.O.E. 17 ⁄ 02 ⁄ 2004 ORDEN
CTE ⁄ 351 ⁄ 2004) to M. P. Rubio. I thank A. L. Rivas
for antibodies, tools, and helpful discussions. I also
thank D. Alessi, K. Hmadcha and C. MacKintosh for
antibodies, constructs, and other helpful tools. I thank
D. Haun for style supervision.
Conflict of interest statement
The author declares that he has no conflict of interest.
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Supporting information
The following supplementary material is available:
Table S1. Comparative analysis of 14-3-3f-associated
proteins identified by TAP-MS from control or C2-
ceramide-treated GFP–TAP–14-3-3f-expressing HeLa
cells.
Table S2. Peptide sequences for each identified
proteins.
Table S3. List of 14-3-3-binding sites (Mode I) of

proteins listed in Table 2.
Fig. S1. MS ⁄ MS spectra labeled with masses detected
as well as fragment assignments for single-peptide-
based protein identifications.
M. Pozuelo-Rubio 14-3-3-binding status during apoptosis
FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS 3341
This supplementary material can be found in the
online version of this article.
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should be addressed to the authors.
14-3-3-binding status during apoptosis M. Pozuelo-Rubio
3342 FEBS Journal 277 (2010) 3321–3342 ª 2010 The Author Journal compilation ª 2010 FEBS