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of June 18, 2017.
Nuclear Shuttling of Mitogen-Activated
Protein (MAP) Kinase (Extracellular
Signal-Regulated Kinase (ERK) 2) Was
Dynamically Controlled by MAP/ERK
Kinase After Antigen Stimulation in
RBL-2H3 Cells
Tadahide Furuno, Naohide Hirashima, Shinobu Onizawa,
Noriko Sagiya and Mamoru Nakanishi
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 166:4416-4421; ;
doi: 10.4049/jimmunol.166.7.4416
http://www.jimmunol.org/content/166/7/4416
Nuclear Shuttling of Mitogen-Activated Protein (MAP) Kinase
(Extracellular Signal-Regulated Kinase (ERK) 2) Was
Dynamically Controlled by MAP/ERK Kinase After Antigen
Stimulation in RBL-2H3 Cells
Tadahide Furuno, Naohide Hirashima, Shinobu Onizawa, Noriko Sagiya, and
Mamoru Nakanishi1
T
he mitogen-activated protein kinase (MAPK)2 cascade
consisting of MAPK and its activator, MAPK kinase
(MAPKK), is essential for signaling of various extracellular stimuli to the nucleus. Stimulation of T cells, B cells,
and mast cells through aggregation of multimeric immune receptors and other stimulants results in activation of MAPK (1–
5). The enzymes regulate genes that are responsible for cell
growth and differentiation (6). However, the mechanisms for
activation of MAPK have not been defined yet in cells.
Rat basophilic leukemia (RBL-2H3) cells provide a useful
model for studying signaling mechanisms in mast cells and basophils. RBL-2H3 cells are activated by Ags via Fc⑀RI to induce release of secretory granules and generation of cytokines
(7). MAPK is thought to mediate divergent signals for proliferation, differentiation, and other cellular responses when RBL2H3 cells are stimulated via the surface receptors. Stimulation
of RBL-2H3 cells, whether by Ags or other stimulants, has been
reported to lead to the transient activation of MAPK (8). The
mechanisms for activation of MAPK have not been defined in
Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
Received for publication January 28, 2000. Accepted for publication January
17, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Mamoru Nakanishi, Faculty of
Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya
467-8603, Japan. E-mail address: [email protected]
2
Abbreviations used in this paper: MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase; ERK, extracellular signal-regulated kinase; MEK, MAP/
ERK kinase; RBL-2H3, rat basophilic leukemia; GFP, green fluorescent protein; YFP,
yellow fluorescent protein; CFP, cyan fluorescent protein; CLSM, confocal laser scanning microscopy; fura 2-AM, fura 2-acetyl-methyl ester; [Ca2⫹]i, intracellular free
calcium ion concentration; NES, nuclear export signal.
Copyright © 2001 by The American Association of Immunologists
mast cells, except that Syk-dependent tyrosine phosphorylation
of Vav and Shc might provide potential links between Fc⑀RI
and the MAPK pathway (9). The transient activation of MAPK
through Fc⑀RI contrasts with the relatively weak and shortlived activation through the G protein-coupled muscarinic and
adenosine receptors. The transient activation of MAPK is consisted with the functional responses of mast cells to Ag. These
include not only a rapid discharge of secretory granules, but
also the synthesis of various cytokines over the course of several hours through gene transcription, for which sustained signals may be needed (7).
The recent characterization of a chromophore, the green, yellow, or cyan fluorescent protein (GFP, YFP, or CFP), provides
a general method to label proteins in living cells. Chimeras
formed with a highly efficient variant of GFP (YFP or CFP)
afford a unique opportunity to examine the organization of proteins of interest within various cellular compartments (10 –12).
Here, we have prepared fusion proteins of MAPK (extracellular
signal-regulated kinase (ERK) 2) with GFP or YFP and
MAPKK (MAP/ERK kinase; MEK) with CFP. ERK2 chimera
proteins functioned normally in the cytoplasmic/nuclear translocation and gene activation. Using these chimera proteins, we
have monitored subcellular translocation of ERK2 and regulation of MEK in RBL-2H3 cells. It was clear that the GFP-ERK2
or YFP-ERK2 was invaluable in exploring the dynamic movements of ERK2 between the cytoplasm and the nucleus depending on the sustained increase of calcium ions in the cytoplasm
after Ag stimulation. The results showed that ERK2 rapidly
shuttled between the cytoplasm and the nucleus in RBL-2H3
cells, but MEK-CFP remained mainly in the cytoplasm to regulate the nuclear shuttling of ERK2 in RBL-2H3 cells. From the
present results, we discussed the effect of MEK on the nuclear
shuttling of ERK2 between the cytoplasm and the nucleus.
0022-1767/01/$02.00
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The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and
its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined
yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat
basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after
the ligation of IgE receptors. The import of ERK2 reached the maximum at 6 –7 min, and then the imported ERK2 was exported
from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after
ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle
between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in
the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of
ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear
shuttling of RBL-2H3 cells after the ligation of IgE receptors. The Journal of Immunology, 2001, 166: 4416 – 4421.
The Journal of Immunology
Materials and Methods
Materials
GFP expression vector (pCMX-SAM/Y145F) was given by Professor K.
Umesono (Kyoto University, Kyoto, Japan) (13). pEYFP-C1 and
pECFP-N1 were obtained from Clontech Laboratories (Palo Alto, CA).
Mouse ERK2 and human MEK (MKK1) genes were obtained from the
American Type Culture Collection (Manassas, VA). Anti-ERK1/2, antiphospho-ERK1/2, anti-MEK1/2, and anti-phospho-MEK1/2 Abs were obtained from New England Biolabs (Beverly, MA). Fura 2-acetoxymethyl
ester (fura 2-AM) was obtained from Molecular Probes (Eugene, OR).
PD98059 was obtained from Calbiochem (La Jolla, CA), and wortmannin
was obtained from Wako Pure Chemicals (Osaka, Japan).
Construction of GFP-ERK2
Cell culture and electroporation
ature of the observation chambers was maintained at 37°C during experiments. Conventional fluorescence microscopy was done using Argus
50/CA (Hamamatsu Photonics, Hamamatsu City, Japan). The excitation
wavelengths were 340 nm for fura 2 and 490 nm for GFP. A long-path filter
(above 530 nm) was used to observe their fluorescence emission.
Immunocytochemical experiments were conducted as described previously (16). RBL-2H3 cells were harvested from culture dishes and transferred to an observation chamber. After Ag stimulation RBL-2H3 cells
were fixed by 3% formaldehyde in PBS for 15 min and were permeabilized
by 0.2% Triton X-100 in PBS for 15 min. Subsequently, anti-MEK1/2 Ab
and FITC-labeled anti-rabbit IgGs were added to the cell.
Results
Characterization of the fluorescent protein-tagged ERK2 and
MEK
First, we examined whether the fluorescent protein-tagged ERK2
and MEK could be activated as well as endogenous kinases after
ligation of IgE receptors. Results by Western blot analysis are
shown in Fig. 1. Here, we used Abs against kinases and phosphorylated kinases. Fig. 1 shows that the induction pattern of the kinase
activation for YFP-tagged ERK2 (YFP-ERK2) and CFP-tagged
MEK (MEK-CFP) was consistent with that for the endogeneous
ERK2 and MEK. GFP-tagged ERK2 (GFP-ERK2) gave the pattern of the kinase activation similar to that shown in Fig. 1a.
Subcellular localization of ERK2 and MEK
We studied the subcellular localization of the GFP-ERK2 chimera
proteins by CLSM (Fig. 2, a– c). GFP-ERK2 resided mainly in the
RBL-2H3 cells were cultured in MEM supplemented with 10% FCS from
Boehringer Mannheim (Indianapolis, IN). The cells were electroporated in
cold K⫹-PBS buffer with 20 ␮g of plasmid DNA at 300 V and 950 ␮F
using Gene Pulser (Bio-Rad, Richmond, CA) (14). RBL-2H3 cells transfected with plasmid DNAs were cultured in the observation chamber (Elekon, Chiba, Japan) for a few days and were used for the experiments.
Stable transfectant cells were obtained by the selection with antibiotic
G418 (Life Technologies, Rockville, MD).
Western blot analysis
To prepare whole-cell lysate, collected RBL-2H3 cells were suspended in
lysis buffer (20 mM HEPES (pH 7.3), 1% Triton X-100, 1 mM EDTA, 50
mM NaF, 2.5 mM p-nitrophenyl phosphate, 1 mM Na3VO4, 10 ␮g/ml
PMSF, 10 ␮g/ml leupeptin, and 10% glycerol) and allowed to stand on ice
for 30 min (15). The suspension was clarified by centrifugation (150,000 ⫻
g for 20 min). After centrifugation, the resulting supernatants were solubilized by treatment with Laemmli buffer at 100°C for 3 min and separated
by electrophoresis in 8% SDS-polyacrylamide gel. The electrophoresed
proteins were transferred to polyvinylidene difluoride membrane with an
electroblotter. After blocking with 0.5% casein, the membranes were
probed with rabbit anti-ERK1/2 Ab (1:1000 dilution), antiphospho-ERK1/2 Ab (1:1000 dilution), anti-MEK1/2 Ab (1:1000 dilution),
or anti-phospho-MEK1/2 Ab (1:1000 dilution) and treated with 1:1000
dilution HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa
Cruz, CA; 0.4 ␮g/ml) (14). The amount of HRP-conjugated IgG bound to
each protein band was determined by LAS-1000 (Fuji Film, Tokyo, Japan)
and was analyzed by Image Gauge (Fuji Film).
Microscopic measurements
Fluorescence microscopic measurements were performed by the previous
procedure (14, 16, 17). The transfected RBL-2H3 cells were harvested
from culture dishes and transferred to an observation chamber. After that,
the cells were treated with HEPES buffer (10 mM HEPES, 140 mM NaCl,
5 mM KCl, 0.6 mM MgCl2, and 1 mM CaCl2 (pH 7.2)) with mouse antiDNP IgE. In our present experiments, seven 2,4-DNP groups, on average,
were conjugated with BSA (DNP7-BSA). Fluorescence microscopic images were observed with confocal laser scanning microscopes (CLSM)
(LSM-410 and LSM-510; Zeiss, Oberkochen, Germany) with argon ion
lasers (458 and 488 nm). GFP and YFP fluorescence was excited at 488
nm, and its emission was observed through a long-path filter (above 515
nm). EYFP and fura red fluorescence were excited at 488 nm, and their
emissions were observed through a band filter (505–530 nm) and a longpath filter (above 585 nm), respectively. ECFP and EYFP fluorescence was
excited at 458 nm, and their emissions were observed through one band
filter (475– 490 nm) and another (560 – 615 nm), respectively. The temper-
FIGURE 1. Western blot analysis making a comparison of the kinase
activation between fluorescent protein-tagged kinases and endogenous kinases in RBL-2H3 cells after Ag stimulation at 37°C. The transfected RBL2H3 cells were stimulated with Ag (500 ng/ml DNP7 -BSA), and their
kinase activation was analyzed by Western blotting using Abs against
ERK1/2, phospho-ERK1/2, MEK1/2, and phospho-MEK1/2. a, Lower
panels, Western blots of YFP-tagged ERK2 and endogenous ERK1/2 before (lane 1), at 6 min after (lane 2), and at 15 min after (lane 3) Ag
stimulation. Upper panels, Western blots of phosphorylated YFP-tagged
ERK2 and endogenous ERK1/2 before (lane 1), at 6 min after (lane 2), and
at 15 min after (lane 3) Ag stimulation. b, Lower panels, Western blots of
CFP-tagged MEK and endogenous MEK before (lane 1), at 6 min after
(lane 2), and at 15 min after (lane 3) Ag stimulation. Upper panels, Western blots of phosphorylated CFP-tagged MEK and endogenous MEK before (lane 1), at 6 min after (lane 2), and at 15 min after (lane 3) Ag
stimulation.
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Preparation of fluorescent protein-tagged ERK2 and MEK was as follows:
ERK2 cDNA served as a template in PCR amplification using appropriate
oligonucleotide primers, and GFP was conjugated to the N terminal of
ERK2. For the generation of a GFP-ERK2 chimera protein, ERK2 cDNA
was amplified with oligonucleotides such that SalI and NheI restriction
sites were introduced at the 5⬘ and 3⬘ ends, respectively. SalI and NheI
restriction sites, as described above, achieved fusion between GFP and N
terminal of ERK2. Fusion between enhanced (E)YFP and N terminal of
ERK2 was achieved at HindIII and BamHI restriction sites. HindIII and
BamHI also did fusion between ECFP and C terminal of MEK restriction
sites. Western blot analysis showed that the molecular mass of GFP-ERK2
(YFP-ERK2) and MEK-CFP were 68 and 72 kDa, respectively. They were
well consistent with the calculated molecular mass of GFP-ERK2 (YFPERK2) and MEK-CFP.
4417
4418
NUCLEAR SHUTTLING OF ERK2
FIGURE 2. Fluorescence microscopic images of GFPERK2 in a single RBL-2H3 cell after Ag stimulation. GFPERK2 was expressed in RBL-2H3 cells. Fluorescence
pseudo-color images of GFP-ERK2 were observed before
and after Ag stimulation (500 ng/ml DNP7-BSA). a– c, Fluorescence images of GFP-tagged ERK2 before (a), at 6 min
after (b), and at 11 min after (c) Ag stimulation in an RBL2H3 cell. d–f, Fluorescence images of GFP-tagged ERK2
before (d), at 6 min after (e), and at 11 min after (f) Ag
stimulation in an RBL-2H3 cell pretreated with an inhibitor
of MEK (PD98059, 20 ␮M) for 30 min.
pletely by the pretreatment of RBL-2H3 cells with PD98059 and
that, subsequently, the phosphorylation of endogenous ERK2 and
YFP-tagged ERK2 was blocked. However, the nuclear shuttling of
ERK2 was not blocked by the pretreatment with wortmannin (an
inhibitor of phosphatidylinositol 3-kinase, 100 nM), although degranulation in RBL-2H3 cells was blocked after Ag stimulation.
To study the movements of ERK2 and MEK at the same time,
we cotransfected plasmids of YFP-ERK2 and MEK-CFP into a
single RBL-2H3 cell. Fluorescence intensities of ERK2 and MEK
were observed mainly in the cytoplasm before Ag stimulation (Fig.
4, a and c). The fluorescence intensity of YFP-ERK2 increased in
the nucleus of the cotransfected RBL-2H3 cell after Ag stimulation, but that of MEK-CFP did not increase (Fig. 4, b and d). Time
courses of the fluorescence intensity changes of YFP-ERK2 and
MEK-CFP in the nucleus of the RBL-2H3 cell after Ag stimulation
are shown in Fig. 3c. The import of ERK2 reached the maximum
at 6 –7 min, and then the imported ERK2 was exported from the
nucleus. MEK mainly resided in the cytoplasm, and no significant
MEK translocation was detected after the ligation of IgE receptors.
Furthermore, we checked the translocation of endogenous MEK
proteins by immunocytochemistry. We confirmed that endogenous
MEK as well as MEK-CFP remained mainly in the cytoplasm after
Ag stimulation (data not shown).
Effects of calcium ions on the nuclear shuttling of ERK2
Next, we studied the effects of calcium ions on the nuclear shuttling of ERK2 in RBL-2H3 cells. Here, we measured both the
intracellular free calcium ion concentration ([Ca2⫹]i) and the distribution of GFP-ERK2 in RBL-2H3 cells by a conventional fluorescence microscope. In the presence of the external calcium ions
(1 mM), the [Ca2⫹]i increased after stimulation with Ag (500
ng/ml DNP7-BSA), and it was sustained at a higher level in the
cytoplasm for ⬎20 min (Fig. 3d; E). Simultaneously, GFP-ERK2
translocated from the cytoplasm to the nucleus after the removal of
the stored calcium ions (Fig. 3e; E). In the absence of the external
calcium ions, the transient increase of the [Ca2⫹]i was observed;
however, the sustained increase of the [Ca2⫹]i disappeared, as
shown in Fig. 3d (F). The CLSM images in Fig. 5 also showed the
effect of calcium ions on the translocation of ERK2 into the nucleus. These results indicated that the sustained calcium increase
was required for optimal ERK2 translocation. We further checked
by Western blot analysis whether the calcium increase affects
MEK activation and subsequently affects ERK2 translocation or
whether it has direct effect on ERK2 translocation. Results of
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cytoplasm before Ag stimulation, and, in some cases, the expression of excess amounts of ERK2 resulted in its nuclear accumulation. After Ag (DNP7-BSA) stimulation, GFP-ERK2 translocated to the nucleus with a dose-dependent manner (5–500 ng/ml
DNP7-BSA) similar to the endogenous ERK2. Thus, the GFP
functioned as a chromophore in essentially all of the expressing
ERK2 in RBL-2H3 cells, as described above.
Upon the stimulation with Ag, translocation of GFP-ERK2 into
the nucleus occurred mostly in the fluorescing RBL-2H3 cells. A
typical example of time courses of GFP-ERK2 fluorescence in the
nucleus and cytoplasm of an RBL-2H3 cell is shown in Fig. 3a.
After a few minutes of a lag time, ERK2 translocated from the
cytoplasm to the nucleus in the RBL-2H3 cell, as shown in Fig. 3a
(E). The fluorescence intensity of GFP-ERK2 increased first, and
it decreased again in the nucleus of the RBL-2H3 cell. On the
contrary, the fluorescence of GFP-ERK2 decreased first in the cytoplasm with a similar extent of the lag time and increased again,
as shown in Fig. 3a (F). The translocation of ERK2 was dependent
on the concentration of Ag (5–500 ng/ml). Five nanograms per
milliliter DNP7-BSA was able to induce the import of ERK2 to the
nucleus; however, it took a longer lag time for translocation of
ERK2 to the nucleus. Five hundred nanograms per milliliter
DNP7-BSA was saturated enough to induce the import of GFPERK2 from the cytoplasm to the nucleus. The import of ERK2
reached the maximum at ⬃6 –7 min, and the export to the cytoplasm almost finished at ⬃15 min after Ag stimulation (500 ng/ml
DNP7-BSA). The average of the fluorescence intensities in the
nucleus and cytoplasm was not changed during 20 min, as shown
in Fig. 3a (✕). This indicated that photobleaching did not affect on
the time courses shown in Fig. 3a. The results indicated that GFPERK2 was shuttling between the cytoplasm and nucleus after stimulation with Ag. Quite similar results were also observed in the
nuclear shuttling of YFP-ERK2. These kinetic studies on ERK2
translocation were consistent with the phosphorylation patterns of
transfected and endogenous ERK, as shown in Fig. 1.
It is demonstrated that ERK2 is activated both by MEK and by
Raf-1. ERK2 is translocated from the cytoplasm to the nucleus,
where the activated ERK2 phosphorylates and regulates nuclear
proteins including transcriptional factors. When RBL-2H3 cells
were pretreated with the inhibitor of MEK (PD98059, 20 ␮M), the
translocation of fluorescent protein-tagged ERK2 (GFP-ERK2,
YFP-ERK2) was completely blocked, as shown in Fig. 3b (also see
CLSM images shown in Fig. 2, d–f). Western blot analysis showed
that the phosphorylation of endogenous MEK was blocked com-
The Journal of Immunology
4419
FIGURE 3. Time courses of the fluorescence intensity changes of fluorescent protein-tagged ERK2 and MEK and those of calcium ion concentration in RBL-2H3 cells after Ag stimulation (500 ng/ml DNP7-BSA). a,
Fluorescence intensity changes of GFP-ERK2 in the nucleus (E), in the
cytoplasm (F), and the average in the nucleus and the cytoplasm (✕). b,
The effect of PD98059 on ERK2 translocation after Ag stimulation. The
figure shows that MEK regulates the nuclear shuttling of ERK2. Here, Ag
(500 ng/ml DNP7-BSA) was added to RBL-2H3 cells pretreated with
PD98059 (20 ␮M). c, Fluorescence intensity changes of YFP-ERK2 (E)
and MEK-CFP (‚) in the nucleus of the RBL-2H3 cell after Ag stimulation. The import of ERK2 reached the maximum at 6 –7 min, although
MEK mainly resided in the cytoplasm. d, Time courses of calcium signals
in the RBL-2H3 cell after Ag stimulation. Fura 2 fluorescence intensities
were observed by a conventional fluorescence microscope. E, With the
external calcium ions (1 mM); F, without the external calcium ions. e,
Fluorescence intensities of GFP-ERK2 in the nucleus with the external
calcium ions (1 mM) (E) and without the external calcium ions (F).
retain it in the cytoplasm. There are at least three explanations for
the translocation of MAPKs from the cytoplasm to the nucleus.
One explanation proposed by Seger’s group is that the MEKMAPK complex translocates upon serum stimulation to the nucleus, where only MEK is rapidly excluded via its active NES
sequence following dissociation (19). Another explanation proposed by Fukuda et al. (20) and Lenormand et al. (21) is a rapid
dissociation of MAPK in the cytoplasm after retrophosphorylation
Western blot analysis using Abs against phosphorylated kinase are
shown in Fig. 6. The data shows that the difference in calcium
levels did not affect the expression of ERK and MEK. However,
the difference in calcium levels affected the expression of the phosphorylated ERK and the phosphorylated MEK. These results suggested that the calcium increase affects MEK activation and subsequently affects ERK2 translocation.
Discussion
We showed here that GFP-ERK2 (YFP-ERK2) in living RBL-2H3
cells was able to shuttle rapidly between the cytoplasm and the
nucleus after Ag stimulation depending on the sustained increase
of [Ca2⫹]i. As described in the previous paper, ERK2 has neither
nuclear localization signal nor nuclear export signal (NES) sequence, although MEK has an NES sequence in the N-terminal
region (residues 33– 44) (18). Thus, it seemed that the NES sequence of MEK was used to export ERK2 from the nucleus and
FIGURE 5. CLSM images of nuclear translocation of fluorescent protein-tagged ERK2 in the presence or in the absence of external calcium
ions (1 mM). CLSM images of YFP-ERK2 before (a) and at 6 min after (b)
Ag stimulation in the presence of external calcium ions (1 mM), and those
before (c) and at 6 min after (d) Ag stimulation in the absence of external
calcium ions.
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FIGURE 4. CLSM images of fluorescent protein-tagged ERK2 and
MEK coexpressed in a single RBL-2H3 cell. Fluorescence pseudo-color
images of YFP-ERK2 (green) and MEK-CFP (red) were observed before
and at 6 min after Ag stimulation (500 ng/ml DNP7-BSA) at 37°C. a,
CLSM image of ERK2 before Ag stimulation; b, that of ERK2 at 6 min
after Ag stimulation. A plain fluorescence of ERK2 was observed in the
nucleus. c, CLSM image of MEK before Ag stimulation; d, that of MEK
at 6 min after Ag stimulation. In this case, a plain fluorescence of MEK was
not observed in the nucleus.
4420
of the anchoring complex via MAPKs (ERK1 and ERK2). Subsequent to this dissociation, MAPKs could easily translocate to the
nucleus via simple diffusion (18), although a cotransport involving
association of MAPKs with their nuclear localization signal-containing substrates cannot be excluded. This explanation is not in
contradiction with the previous results, in which nonphosphorylatable mutants of MAPKs translocated to the nucleus upon mitogen stimulation (22, 23). A third explanation proposed by
Khokhlatchev et al. is that phosphorylation of ERK2 promotes its
homodimerization and nuclear translocation. The explanation was
based on microinjection studies in fibroblasts (24). In contrast, it
was suggested that the export of MAPK from the nucleus to the
cytoplasm was mediated by the dephosphorylation of MAPK
and/or by the NES sequence of MAPKK (20, 24).
The present results indicated that there was no significant appearance of MEK-CFP in the nucleus of the cotransfected RBL2H3 cells before and after Ag stimulation (Fig. 4). This did not
exclude the model that MEK might shuttle between the cytoplasm
and the nucleus (19, 20, 24). It rather suggested that the translocation of MEK favors the cytoplasm; in other words, the equilibrium constant of the translocation of MEK to the nucleus (KMEK)
is much smaller than that of ERK2 (KERK2) that was detected in
the nucleus. KMEK can be expressed as a ratio of rate constants of
the export and import of MEK from/to the nucleus, kMEK(ex)/
kMEK(im), and kMEK(ex) was greater than kMEK(im), kERK2(ex),
and kERK2(im), because only MEK has NES sequence. However,
it is possible that the addition of CFP may have affected nuclear
transport of MEK-CFP.
Then, our results showed that the import of ERK2 reached the
maximum several minutes after Ag stimulation, and thereafter it
went back to the cytoplasm again within ⬃15 min. The previous
authors supposed that the NES sequence of MEK might play a role
for the export of ERKs from the nucleus to the cytoplasm for
several minutes. Our results showed that both import and export of
ERK2 to/from the nucleus occurred in several minutes after Ag
stimulation. However, the results in the present paper were not
able to explain that the export rate of MEK was as low as the
previous authors supposed. Because, if so, we can observe the
appearance of MEK-CFP in the nucleus, and/or the NES sequence
of MEK does not play any role for the export of ERK2. In short,
the present experiments suggested that the nuclear shuttling, especially export, of MEK be much faster than that of ERK2, shown in
Fig. 3c. A schematic representation of the dynamics in the nuclear
shuttling of MAPK (ERK2) and MAPKK (MEK) is shown in Fig.
7. In a previous paper, Jaaro et al. described that 5% of MEK
FIGURE 7. A schematic representation shows the nuclear shuttling of MAPK (ERK2) and MAPKK (MEK). Phosphorylation of ERK2 with MEK
induces dissociation of ERK2 from MEK, and the phosphorylated ERK2 translocates to the nucleus. In the nucleus, ERK2 is dephosphorylated with MAPK
phosphatases (e.g., CL100) (25), and the dephosphorylated ERK2 is bound with MEK that is able to be exported from the nucleus much faster than ERK2.
Phosphorylation of ERK2 and dephosphorylation of ERK2 occur within 3–7 min in the cytoplasm and within 7–15 min in the nucleus after Ag stimulation,
respectively. It seems that the reactions of phosphorylation and dephosphorylation of ERK2 are the rate-limiting steps of the import and the export of ERK2
to/from the nucleus, respectively. That is, rate constants of kERK2(im) and kMEK(ex) are greater than those of phosphorylation (kR1) and dephosphorylation
(kR2), respectively. In addition, kMEK(ex) is also much greater than kMEK(im), kERK2(ex), and kERK2(im), because only MEK has NES sequence. It is
probable that a rate constant (kERK2/MEK(ex)) of the export of ERK2/MEK complex from the nucleus is similar to that of MEK alone (kMEK(ex)).
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FIGURE 6. Western blot analysis showing effects of the external calcium ions on the phosphorylation of ERK2 and MEK. Here, Abs against
phosphorylated kinases were used, and the experimental conditions were
same as those in Fig. 1. Lane 1, Phosphorylated kinases before Ag stimulation in the presence of external calcium ions (1 mM). Lanes 2 and 3,
Phosphorylated kinases at 6 min after Ag stimulation in the presence and
absence of external calcium ions (1 mM), respectively. a, Western blots of
YFP-tagged ERK2 and endogenous ERK1/2. b, Western blots of endogenous MEK.
NUCLEAR SHUTTLING OF ERK2
The Journal of Immunology
translocated into the nucleus (19). However, they stressed in
con¥clusion that they were not able to determine whether MEK
translocated into the nucleus because the amount of the nuclear
translocation of MEK was very low. Thus, we think the model
shown in Fig. 7 did not contradict the previous experimental
results.
Last, we must explain that the time courses for the import and
the export of ERK2 were due to the rate-limiting steps of the biochemical reactions in the cytoplasm and in the nucleus, respectively. Thus, the nuclear shuttling of MEK itself should be much
faster than the time courses shown in Fig. 3c.
Acknowledgments
We thank Mariko Kohzai for technical assistance.
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