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Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 RBL-2H3 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 mast cells, except that Syk-dependent tyrosine
phosphorylation of Vav and Shc might provide potential links
between FcRI and the MAPK pathway (9). The transient
activation of MAPK through FcRI contrasts with the relatively weak
and short-lived 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, 11, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, anti-phospho-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
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 (YFP-ERK2) and MEK-CFP.
Cell culture and electroporation
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 µg of plasmid DNA at 300 V and 950 µF 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 µg/ml PMSF, 10 µg/ml leupeptin, and 10% glycerol) and allowed to stand on ice for 30 min (15). The suspension was clarified by centrifugation (150,000 x 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), anti-phospho-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 µg/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 anti-DNP 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 long-path 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 temperature 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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We studied the subcellular localization of the GFP-ERK2 chimera
proteins by CLSM (Fig. 2, a–c). GFP-ERK2 resided mainly in the 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.
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a. 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 (
). 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 (•). 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 GFP-ERK2 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 (X). This
indicated that photobleaching did not affect on the time courses shown
in Fig. 3a. The results indicated that GFP-ERK2 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
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b (also see CLSM images shown in Fig. 2, d–f). Western blot analysis showed that the phosphorylation
of endogenous MEK was blocked completely 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).
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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; ).
Simultaneously, GFP-ERK2 translocated from the cytoplasm to the nucleus
after the removal of the stored calcium ions (Fig. 3e; ).
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 (•). 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 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present results indicated that there was no significant appearance
of MEK-CFP in the nucleus of the cotransfected RBL-2H3 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 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.
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c.
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Acknowledgments |
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Footnotes |
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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.
Received for publication January 28, 2000. Accepted for publication January 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI: rapamycin inhibits activation of pp70–S6 kinase and proliferation in mouse mast cells. Eur. J. Immunol. 23:3286.[Medline]
B with a distinct lag time in living cells. FEBS Lett. 444:1.[Medline]
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) 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. 
