Báo cáo khoa học: PKCd-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate pdf

PKCd-dependent cleavage and nuclear translocation of annexin A1
by phorbol 12-myristate 13-acetate
Yoon S. Kim
1,
*, Jesang Ko
2,
*, In S. Kim
1
, Sung-Wuk Jang
2
, Ho J. Sung
2
, Hye J. Lee
1
, Si Y. Lee
1
, Youngho Kim
3
and Doe S. Na
1
1
1
Department of Biochemistry and Molecular Biology;
2
Asan Institute for Life Sciences, and
3
Genome Research Center for Birth
Defects and Genetic Diseases, University of Ulsan College of Medicine, Seoul, Korea
Annexin A1 (ANX-1), a calcium-dependent, phospholipid
binding protein, is known to be involved in diverse cellular
processes, including regulation of cell growth and differen-

tiation, apoptosis, and inflammation. The mitogen phorbol
12-myristate 13-acetate (PMA) induces expression and
phosphorylation of ANX-1. However, the roles of ANX-1
in PMA-induced signal transduction is unknown. Here,
we study the cellular localization of ANX-1 in the
PMA-induced signal transduction process. We have found
that PMA induces the cleavage of ANX-1 in human
embryonic kidney (HEK) 293 cells, and that the cleaved
form of ANX-1 translocates to the nucleus. The PMA-
induced nuclear translocation of ANX-1 was inhibited by
the protein kinase C (PKC)d-specific inhibitor rottlerin,
indicating that PKCd plays a role in nuclear translocation of
the cleaved ANX-1. We propose a novel mechanism of
PMA-induced translocation of ANX-1 to the nucleus that
may participate in the regulation of cell proliferation and
differentiation.
Keywords: annexin A1; PMA; cleavage; nuclear transloca-
tion; PKCd.
Annexins (ANXs) are a family of calcium-dependent,
phospholipid-binding proteins. Several members of the
ANX family are known to be involved in various physio-
logical functions including anti-inflammatory processes, cell
signaling, regulation of cell growth and differentiation,
apoptosis, membrane fusion, exocytosis, and interaction
with cytoskeletal proteins [1–3]. Although there have been
recent advances in understanding the molecular mechanisms
by which ANXs play a role in these cellular processes, the
regulatory mechanism in cell proliferation and differenti-
ation remains to be characterized. It has been reported that
ANX-1 is involved in regulation of the mitogenic signal

transduction pathways including the mitogen-activated
protein (MAP) kinase-, the epidermal growth factor receptor
(EGFR)-, and the hepatocyte growth factor receptor
(HGFR) kinase-mediated signaling pathways [4–6]. The
expression level of ANX-1 increases in response to phorbol
12-myristate 13-acetate (PMA) and interleukin (IL)-6 [7,8],
and dys-regulation of ANX-1 results in development of
various cancers [9,10]. Mitogens such as EGF and PMA
induce phosphorylation, cleavage, and translocation of
ANX-1 to the membrane [11–13] and this phosphorylation
event is mediated by protein kinase C (PKC) [14].
ANX-1 mainly exists in the cytosol, but also exists in the
membrane or the nucleus [15]. Recent reports suggest that
subcellular localization of ANX-1 can be redistributed by
treatment with specific stimuli. ANX-1 translocates to the
membrane and is secreted to the extracellular surface of the
cell membrane in response to glucocorticoid and PMA
[16,17]. Although we have previously proposed that EGF,
oxidative stress and heat shock induce translocation of
ANX-1 to the nucleus [18,19], the mechanism for nuclear
translocation of ANX-1 is still unknown.
PKCs are serine-threonine kinases that are activated by
diverse stimuli including mitogens and participate in a
variety of cellular processes such as cell proliferaction and
differentiation, and apoptosis [20,21]. The PKC family
consists of 12 isoforms that are grouped into three
subfamilies: the classical PKCs (a, b1, b2, v), the novel
PKCs (d, e, g, h), and the atypical PKCs (n, k/i). PKCd,a
member of the novel PKC subfamily, is activated by
diacylglycerol (DAG) and phorbol esters in a calcium-

independent manner and plays a critical role in the control
of cell growth and apoptosis [22].
In this study, we aimed to elucidate whether PMA
induces the translocation of ANX-1 to the nucleus in
human embryonic kidney (HEK) cells and the roles of
PKCd in the nuclear translocation of ANX-1. We propose
a mechanism for the nuclear translocation of ANX-1 in
response to PMA that may be involved in cellular processes
such as cell proliferation and differentiation.
Correspondence to D. S. Na, Department of Biochemistry and
Molecular Biology, University of Ulsan College of Medicine,
388–1 Poongnap-dong, Songpa-gu, Seoul 138–736, Korea.
Fax: + 82 2 477 9715, Tel.: + 82 2 3010 4275,
E-mail:
Abbreviations: ANX, annexin; PMA, phorbol 12-myristate 13-acetate;
HEK, human embryonic kidney; PKC, protein kinase C; MAP kinase,
mitogen-activated protein kinase; EGFR, epidermal growth factor
receptor; HGFR, hepatocyte growth factor receptor; DAG,
diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium.
*Note: the first two authors contributed equally to this work.
(Received 15 July 2003, revised 17 August 2003,
accepted 21 August 2003)
Eur. J. Biochem. 270, 4089–4094 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03800.x
Materials and methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM), and fetal
bovine serum (FBS) were purchased from Life Technolo-
gies, Inc. (Gaithersburg, MD, USA). Rottlerin, Ro-31-8425,
PD98059, Ly294002, SB202190 were from Calbiochem
(San Diego, CA, USA). PMA and goat anti-(mouse IgG) Ig

conjugated with tetramethylrhodamine isothiocyanate
(TRITC) were products of Sigma (St Louis, MO, USA).
Anti-ANX-1 monoclonal antibody was purchased from
Transduction Laboratories (Lexington, KY, USA).
Cell culture
HEK 293 cells were maintained in DMEM supplemented
with 10% heat-inactivated FBS, penicillin (100 UÆmL
)1
),
and streptomycin (100 lgÆmL
)1
)at37°Cunder5%CO
2
atmosphere. For Western blot analysis, cells were
seeded into 60 mm dishes at 1 · 10
6
cells per dish. After
18–24 h, cells were further grown in the same medium
supplemented without FBS for 24 h. Serum-starved cells
were treated with PMA for the indicated times. For
immunostaining, 2 · 10
5
cells grown on cover slides
(22 · 22 mm) were starved for 24 h before stimulation
with PMA.
Immunocytochemistry
HEK 293 cells grown on cover slides were fixed with
3.7% paraformaldehyde for 15 min and permeabilized
with 0.2% Triton X-100 in NaCl/KCl/P
i

(NaCl/P
i
,
137 m
M
NaCl, 2.7 m
M
KCl, 8 m
M
Na
2
HPO
4
,1.5m
M
KH
2
PO
4
) for 5 min. After washing the cells with NaCl/
KCl/P
i
three times, the cells were blocked for 30 min in
NaCl/P
i
containing 1% bovine serum albumin. Immuno-
staining was performed by incubation with anti-ANX-1
monoclonal antibody (0.05 lgÆmL
)1
)for2h.After

washing the cells with NaCl/P
i
three times, the cells
were incubated with TRITC-conjugated goat anti-(mouse
IgG) Ig for 1 h. Cover slides were washed with NaCl/P
i
,
mounted, and examined using a Leica TCS SP2 Confo-
cal microscope (Leica Microsystems, Wetzlar GmBH,
Germany).
Cell fractionation
HEK 293 cells were seeded into 60 mm dishes at 1 · 10
6
cells/dish and cultured in DMEM for 18–24 h. The cells
were starved for 24 h in serum-free media. After
treatment with PMA for a given time, the cells were
harvested and washed with ice-cold NaCl/P
i
.Thecells
were then resuspended in 100 lL of lysis buffer (10 m
M
Hepes, 10 m
M
NaCl, 0.1 m
M
EDTA, 0.1 m
M
EGTA, 1%
NP-40, 0.5 m
M

phenylmethylsulfonyl fluoride, 0.1 m
M
dithiothreitol, 0.1 m
M
sodium orthovanadate, and prote-
ase inhibitors) and incubated on ice for 10 min. The
nuclei were collected by centrifugation at 2000 g for
5min at 4°C. The supernatant was collected as a
cytosolic fraction. Protein concentration of each sample
was determined.
Western blot analysis
For preparing whole cell lysates, 1 · 10
6
cells were lysed in
1 · SDS gel-loading buffer (50 m
M
Tris/HCl, pH 6.8,
100 m
M
dithiothreitol, 2% SDS, 0.1% bromophenol blue,
10% glycerol). Protein samples were separated on 12%
SDS/polyacrylamide gels and transferred to nitrocellulose
filters. The blots were incubated with anti-ANX-1 mono-
clonal antibody for 1 h. After washing three times with
NaCl/P
i
containing 0.05% Tween 20, the blots were
incubated with goat anti-(mouse IgG) Ig conjugated with
horseradish peroxidase (HRP) for 1 h. The blots were
washed three times with NaCl/P

i
containing 0.05% Tween
20 and developed with the enhanced chemiluminescence
detection system (Amersham Pharmacia Biotech., Piscata-
way, NJ, USA).
Results
PMA induces the cleavage of ANX-1
As ANX-1 is cleaved by treatment with PMA in epithelial
A549 cells [7], we first examined whether PMA induces the
cleavage of ANX-1 in HEK 293 cells. Cells were treated
with 10 n
M
PMA for 30 min and cell lysates were subjected
to Western blot analysis using ANX-1 antibody. As shown
in Fig. 1, the cleaved form of ANX-1 was detected in PMA-
stimulated HEK 293 cells, whereas only the intact form of
ANX-1 was found in control cells. ANX-1 was partially
cleaved by the treatment of PMA and most of ANX-1
remained in the intact form. This result indicates that PMA
induces the cleavage of ANX-1 in HEK 293 cells.
ANX-1 translocates to the nucleus by PMA stimulation
There have been reports that the cleaved form of ANX-1 is
secreted and presents mainly on the outer surface of cell
membrane [17]. To examine the subcellular localization of
ANX-1 in response to PMA in HEK 293 cells, cells were
incubated in the absence or presence of 10 n
M
PMA for
30 min and immunostained with anti-ANX-1 monoclonal
antibody. In unstimulated cells, ANX-1 was evenly detected

throughout the cells including cytosol and nucleus (Fig. 2).
However, the level of ANX-1 significantly increased in the
nucleus in about 20–30% of PMA-treated cells observed
(Fig. 2), indicating that ANX-1 translocates to the nucleus
by PMA stimulation.
Fig. 1. PMA induces the cleavage of ANX-1. Serum-starved HEK 293
cells were incubated in the absence or presence of 10 n
M
PMA for
30minandlysedwith1· SDS-loading dye. Whole cell lysates were
separated in a 12% SDS/polyacrylamide gel and transferred to nitro-
cellulose membrane. Cleavage of ANX-1 was detected by Western
blotting with anti-ANX-1 monoclonal antibody.
4090 Y. S. Kim et al.(Eur. J. Biochem. 270) Ó FEBS 2003
PMA induces the nuclear translocation of the cleaved
form of ANX-1
As PMA induced the cleavage of ANX-1 and accumulation
of ANX-1 in the nucleus, we wondered if the cleaved
ANX-1 is responsible for the increase of ANX-1 in the
nucleus. To determine this possibility, HEK 293 cells were
treated with 10 n
M
PMA for 30 min, and fractionated into
cytosolic and nuclear fractions. As shown in Fig. 3A, the
cleaved ANX-1 was detected in the nuclear fraction of
PMA-treated cells and the amount of the intact ANX-1
level was not changed between the nuclear fractions of
treated and untreated cells. These results indicate that
accumulation of ANX-1 in the nucleus is a result of
translocation of the cleaved ANX-1 to the nucleus.

We next examined dose- and time-dependency of the
nuclear translocation of the cleaved ANX-1. HEK 293 cells
were treated with the indicated concentrations of PMA for
30 min and subjected to Western blot analysis. The cleavage
and translocation of ANX-1 was induced at a concentration
of more than 1 n
M
(Fig. 3B). Figure 3C shows that the
cleaved ANX-1 in the nuclear fraction began to be found
after 15 min of exposure to PMA. These results indicate
that the cleavage and translocation of ANX-1 to the nucleus
in response to PMA are immediate early events.
PMA-induced nuclear translocation of ANX-1
is mediated via PKCd
It has been reported that PKC induces phosphorylation and
cleavage of ANX-1 [12]. Therefore, we examined whether
PKC is involved in the cleavage and nuclear translocation of
ANX-1. HEK 293 cells were preincubated in the presence
of rottlerin or Ro-31-8425, and were stimulated with PMA.
The cleavage and nuclear translocation of ANX-1 were
inhibited by a PKCd-specific inhibitor rottlerin, but not by
Ro-31-8425 (Fig. 4A). To investigate whether other signa-
ling molecules are involved in the cleavage and nuclear
translocation of ANX-1, Western blot analysis was con-
ducted in the absence or presence of inhibitors of ERK
(PD98059), p38 (SB202190), or PI-3 kinase (LY294002).
Figure 4A shows that the PMA-induced cleavage and
Fig. 2. ANX-1 accumulates in the nucleus by PMA stimulation. Serum-
starved HEK 293 cells were incubated in the absence or presence of
10 n

M
PMA for 30 min and processed for immunocytochemical
detection of endogenous ANX-1 using anti-ANX-1 monoclonal anti-
body as described in Materials and methods. Confocal images of
untreated (A) and PMA-treated (B) cells are representatives of the cells
observed in three independent experiments.
2
Fig. 3. PMA induces the nuclear translocation of the cleaved ANX-1.
(A) Serum-starved HEK 293 cells were incubated in the absence or
presence of 10 n
M
PMA for 30 min. Cells were then fractionated into
cytosol and nucleus, and separated in a 12% SDS/polyacrylamide gel
and subjected to immunoblotting using anti-ANX-1 monoclonal
antibody. (B) HEK 293 cells were incubated in serum-free DMEM for
24 h and stimulated with PMA at indicated concentrations for 30 min.
Cells were harvested and separated into cytosolic and nuclear frac-
tions, then samples were resolved by 12% SDS/polyacrylamide gels
and transferred to nitrocellulose membrane. Translocation of ANX-1
was detected by Western blotting. (C) Serum-starved HEK 293 cells
were treated with 10 n
M
PMA for indicated times, then harvested and
fractionated into cytosol and nucleus. Samples were separated in 12%
SDS/polyacrylamide gels. Time-dependent translocation of ANX-1 to
the nucleus was detected by Western blotting with anti-ANX-1
monoclonal antibody.
Ó FEBS 2003 PMA-induced nuclear translocation of annexin A1 (Eur. J. Biochem. 270) 4091
nuclear translocation of ANX-1 were not inhibited by the
addition of these inhibitors suggesting that these molecules

are not involved in the PMA-induced cleavage and nuclear
translocation of ANX-1.
We next confirmed the inhibitory effect of rottlerin on
the cleavage and nuclear translocation of ANX-1 using
immunocytochemical analysis (Fig. 4B). In cells preincu-
bated with rotterin, PMA was not able to induce the
increase of ANX-1 in the nucleus. These results indicate that
PKCd plays a role in the PMA-induced cleavage and
nuclear translocation of ANX-1.
Discussion
Despite recent advances in our understanding of the roles
of ANX-1 in mitogenic signal transduction, the exact
mechanism through which ANX-1 functions in response
to mitogenic stimuli remains unclear. In this work, we
attempted to elucidate the regulatory mechanism of ANX-1
in the PMA signaling pathway in HEK 293 cells. We have
demonstrated that (a) PMA induces the cleavage of ANX-1.
(b) the cleaved ANX-1 translocates to the nucleus in a time-
dependent manner, and (c) the cleavage and nuclear
translocation of ANX-1 is mediated by a PKCd-dependent
mechanism.
Several lines of evidences suggest that ANX-1 is cleaved
in response to IL-6 and PMA in different cell lines [7,12] and
that the cleaved ANX-1 is translocated to the extracellular
surface of the cell membrane [16,17]. To investigate the role
of ANX-1 in the PMA-induced signal transduction path-
way, we first examined whether PMA induces the cleavage
of ANX-1 in HEK 293 cells. Our data showed that PMA
induced the cleavage of ANX-1 and the cleavage is
comparable to that reported previously in other cell lines

[7,12]. It has been reported that truncated ANX-1 that is
missing the first 29 N-terminal amino acids is secreted from
the prostate cancer cells [23]. Whether the same type of
cleavage occurs in PMA treated cells is not clear. Never-
theless it is reasonable to assume that the cleavage site of
ANX-1 in response to PMA is at the N-terminal region of
ANX-1. The cleavage site of ANX-1 and the enzyme that is
responsible for the cleavage is under investigation in this
laboratory.
Interestingly, data from confocal microscopy show that
ANX-1 is accumulated in the nucleus. We have previously
reported that ANX-1 translocates to the nucleus by EGF,
oxidative stress, and heat shock [18,19]. Our results suggest
that PMA also induces the translocation of ANX-1 to the
nucleus. However, the nuclear translocation of ANX-1 was
observed in about 20–30% of PMA-treated cells indicating
that this may be cell cycle-dependent event. When PMA-
treated HEK 293 cells were fractionated into cytosolic and
nuclear fractions, the cleaved form of ANX-1 was found in
the nuclear fraction, indicating that the cleaved form but not
the intact form of ANX-1 translocates to the nucleus. The
cleaved ANX-1 was detected in the cytosolic fraction with a
longer exposure, indicating that ANX-1 is cleaved in the
cytosol, then the cleaved ANX-1 translocates to the nucleus.
However, results from the Western blotting with a longer
exposure and confocal microscopy demonstrate that the
minimal level of the cleaved ANX-1 was also found in the
nuclear fraction of control cells (data not shown). Therefore,
it cannot be ruled out the possibility that the cleavage of
ANX-1 occurs both in the cytosol and the nucleus.

Fig. 4. PMA-induced nuclear translocation of
ANX-1 is mediated via PKCd. (A) Serum-
starved HEK 293 cells were preincubated in
the absence or presence of rottlerin (5 l
M
),
Ro-31-8425 (50 l
M
), PD98059 (50 l
M
),
LY294002 (10 l
M
), and SB202190 (20 l
M
)for
30 min and stimulated with 10 n
M
PMA for
30 min. Cells were fractionated into cytosolic
and nuclear fractions, and analyzed by 12%
SDS/polyacrylamide gels and transferred to
nitrocellulose membrane. Translocation of
ANX-1 was probed with anti-ANX-1 mono-
clonal antibody. (B) HEK 293 cells were
incubatedinserum-freeDMEMfor24h.
Serum-starved cells were preincubated in the
absence or presence of PKCd-specific inhibitor
rottlerin (5 l
M

) for 30 min and treated with
10 n
M
PMA for 30 min. Cells were then
immunostained with anti-ANX-1 monoclonal
antibody as described in Materials and
methods. The micrographs are representatives
of the cells observed in three independent
experiments.
3
4092 Y. S. Kim et al.(Eur. J. Biochem. 270) Ó FEBS 2003
PMA induced the cleavage and nuclear translocation of
ANX-1 in a time-dependent manner. The cleaved ANX-1
began to be detected in the cytosolic fraction at 10 min of
PMA treatment. After 15 min of exposure to PMA, the
cleaved ANX-1 began to translocate to the nucleus. This
result confirms the data that ANX-1 is cleaved first in the
cytosol, then translocates to the nucleus. The nuclear
translocation of ANX-1 seems to be an immediate early
process and is comparable to that of other signaling
molecules such as MAP kinase and NF-jB [24,25]. As
ANX-1 binds both DNA and RNA [26], there is a
possibility that the cleaved ANX-1 translocates to the
nucleus and participates in cell proliferation and differen-
tiation processes by regulating transcription. Taken
together, the cleavage and translocation of ANX-1 to the
nucleus by PMA may be a physiological process involved in
cell proliferation and differentiation.
The PKC family, which comprises several isoforms, plays
an important role in cell proliferation and differentiation

[20,21]. PKC is known to be involved in the cleavage and
secretion of ANX-1 [12]. However, it is not clear which
isoform of PKC mediates the cleavage of ANX-1. PKCd is a
member of the novel PKC subfamily and is known to play
a critical role in the regulation of cell proliferation and
apoptosis [22]. Our data indicate that PKCd is responsible
for the cleavage and nuclear translocation of ANX-1. It has
been known that the major phosphorylation sites of ANX-1
by PKC are Ser-27 and Thr-41, and the phosphorylation of
ANX-1 at Ser-27, Ser-28, and Thr-24 has also been
identified [27]. PKCd probably phosphorylates ANX-1 at
one or several of these sites directly or indirectly and induces
the cleavage and nuclear translocation of ANX-1. We also
investigated the involvement of other signaling molecules
such as MEK, PI-3 kinase, and p38 in PMA-induced
translocation of ANX-1. Inhibition of these molecules did
not affect the cleavage and nuclear translocation of ANX-1
in response to PMA, indicating that these molecules are not
required for the PMA-induced cleavage and nuclear trans-
location of ANX-1.
ANX-1 is involved in the regulation of cell proliferation
and differentiation [28,29]. Recently, ANX-1
–/–
mice were
generated and partially characterized [30] and it has been
confirmed that ANX-1 is a mediator of glucocorticoid-
induced growth inhibition [31]. However, the exact mech-
anism by which ANX-1 plays a role in cell growth is not
known. In the present study, we propose the mechanism by
which PMA induces the translocation of ANX-1 to the

nucleus. We have demonstrated that PMA induces the
cleavage of ANX-1 leading to the nuclear translocation of
ANX-1, and that PKCd plays a critical role in this process.
While further studies are required to characterize the exact
functions of ANX-1 in the nucleus, from this study we can
begin to understand the role of ANX-1 in the PMA-induced
signal transduction, which may provide an important
clue for understanding the molecular mechanism of cell
proliferation and differentiation.
Acknowledgements
This work was supported by a Korea Research Foundation Grant
(KRF-2002-042-C00076) (to D. S. N and J. K).
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