Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
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ABSTRACT |
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MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.
CD8; chimera; transfection; Ras; mitogen-activated protein kinase kinase; extracellular signal-regulated kinase
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INTRODUCTION |
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MUC1 MUCIN IS A TRANSMEMBRANE glycoprotein initially detected in various cancer cell lines and later in secretory epithelial cells lining the respiratory, reproductive, and gastrointestinal tracts (5, 7-9). Primary hamster tracheal surface epithelial cells grown on a thick collagen gel matrix undergo goblet cell differentiation at confluence (25). These cells express Muc1 mucin on the cell surface (11, 18) and the expression of Muc1 mRNA correlates with goblet cell differentiation (17). However, the exact role of MUC1 mucin in the airway remains unknown.
From a structural point of view, MUC1 mucin resembles the type II cytokine receptor, which has a number of tyrosine phosphorylation sites on its cytoplasmic (CT) domain yet does not autophosphorylate (27). In a breast cancer cell line (MCF7), MUC1 mucin is constitutively tyrosine phosphorylated but rapidly dephosphorylated (27). Using the same cell line, Pandy et al. (16) showed that MUC1 mucin can directly interact with the SH2 domain of the adaptor protein Grb2. They also demonstrated that the MUC1-Grb2 complex associates with the guanine nucleotide exchange protein Sos. Because Sos binds to the SH3 domains of Grb2 and thereby associates with Ras at the plasma membrane, their results seem to support a role for MUC1 mucin in intracellular signaling. An extracellular binding protein responsible for the tyrosine phosphorylation of MUC1 mucin in MCF7 cells has been identified as the extracellular domain of MUC1 mucin containing a tandem repeat array (1). This is the first potential ligand for MUC1 mucin that has been reported. The downstream signaling pathways of MUC1 mucin, however, remain to be uncovered.
In an attempt to understand the signaling pathways of MUC1 mucins without identifying "true" ligand(s), we constructed a chimeric plasmid (pCD8/MUC1), which contained the extracellular and transmembrane domains of CD8 and the CT domain of MUC1 mucin (14). COS-7 cells transiently transfected with the chimeric plasmid expressed a chimeric protein (CD8/MUC1), which was tyrosine phosphorylated after treatment with anti-CD8 antibody (14). Using this simple, reproducible system, we were interested in exploring whether the mitogen-activated protein (MAP) kinase pathway is activated after activation of MUC1 mucin. Here we report that treatment with the anti-CD8 antibody of COS-7 cells stably expressing CD8/MUC1 protein results in activation of extracellular signal-regulated kinase (ERK) 2, a major MAP kinase located downstream of the Grb2-Sos-Ras signaling pathway.
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METHODS |
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Materials. All the reagents used in this experiment were purchased from Sigma (St. Louis, MO) unless otherwise stated. The sources of antibodies used for the present experiment are as follows: anti-ERK2 antibody (rabbit polyclonal, C-14, Santa Cruz Biotechnology, Santa Cruz, CA); anti-diphosphorylated ERK1/2 (ppERK1/2) antibody (mouse monoclonal); and anti-CD8 antibody A (mouse monoclonal, Serotec, Raleigh, NC); and anti-CD8 antibody B (rabbit polyclonal, Santa Cruz Biotechnology). Anti-CD8 antibody A was used for activation of the chimera, whereas anti-CD8 antibody B was used for both immunofluorescence staining and immunoblotting. Anti-mouse IgG and anti-rabbit IgG conjugated with horseradish peroxidase (Pierce, Rockford, IL) were used for immunoblotting. Fluorescein isothiocyanate or Texas Red-conjugated anti-rabbit IgG or anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used for immunofluorescence staining.
Transfection and establishment of CD8/MUC1 cells. COS-7 cells (CRL 1651; American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD). Transient transfection was carried out using SuperFect (QIAGEN, Santa Clarita, CA) as previously described (14). CD8/MUC1 cells, a stable COS-7 cell line stably expressing the chimera, was established by continuous passage of positive clones after transient transfection in the presence of 800 µg/ml G418 (GIBCO BRL). At least 10 passages were carried out.
ERK2 activation and immunoblotting.
CD8/MUC1 cells were seeded at 4 × 105 cells/60-mm
tissue culture dish. After a 48-h incubation, the cells were starved in
serum-free medium for 8 h and then activated with 15 µg of
anti-CD8 antibody A for 15 min with or without pretreatment with 0.1, 0.3, or 1 µM MEK inhibitor U-0126 (3) (Promega, Madison,
WI) for 30 min as previously described (23). After
activation, cells were immediately lysed using either SDS-PAGE sample
buffer or lysis buffer (1% Triton X-100, 10% glycerol, 20 mM HEPES,
pH 7.2, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 1 mM
Na3VO4). The cell lysate was centrifuged to remove the detergent-insoluble material. Equal amounts of the supernatant were mixed with SDS-PAGE sample buffer, separated on 15%
SDS-polyacrylamide gels, and then transferred to polyvinylidene difluoride membrane. The membrane was blocked for 1 h at room temperature in Tris-buffered saline (TBS) [10 mM Tris-Cl, pH 7.5, 150 mM NaCl and 0.05% (vol/vol) Tween 20] containing 5% nonfat dry milk.
The membrane was incubated for 1 h at room temperature with either
anti-ERK2 or anti-ppERK1/2 antibody (diluted 1:1,000). Anti--tubulin
antibody (diluted 1:5,000) was used as a control to confirm equal
protein loading of the samples. Immunoblots were washed with TBS (50 mM
Tris, pH 7.6, and 0.15 M NaCl) containing 0.1% (vol/vol) Tween 20 for
45 min, incubated with the secondary antibody conjugated with
horseradish peroxidase (diluted 1:10,000), and visualized by
chemiluminescence using enhanced chemiluminescence reagents (Pierce).
In vitro kinase assay.
Total cell lysates from both activated and unactivated CD8/MUC1 cells
were subjected to in vitro kinase assay as described previously
(21). Briefly, cell lysates were incubated with 2 µg of
anti-ERK2 antibody for 2 h on ice, followed by addition of 20 µl
of protein A Sepharose (Pierce) and further incubation for 2 h at
4°C with constant mixing. On completion of incubation, the protein A
Sepharose precipitate was washed twice with 0.5 ml of a buffer
containing 25 mM HEPES, pH 7.4, 25 mM MgCl2, and 1 mM
dithiothreitol (DTT). Activity of immunoprecipitated ERK2 was measured
by incubating the washed, immobilized immune complexes for 10 min at
30°C in a final volume of 15 µl of kinase buffer, which consisted
of 25 mM HEPES, pH 7.4, 15 mM MgCl2, 1 mM
Na3VO4, 1 mM DTT, 10 µCi of
[-32P]ATP (3,000 Ci/mmol; DuPont-NEN Research
Products, Boston, MA), 10 µM ATP, and 2.5 µg of myelin basic
protein (MBP). Reactions were quenched with SDS-PAGE sample buffer,
resolved on 20% SDS-polyacrylamide gels, and 32P
incorporation was quantified by phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA).
Immunofluorescence analysis. Cells grown on round 18-mm glass coverslips (VWR Scientific Products, Media, PA) inside 60-mm tissue culture dishes were transiently transfected with pCD8/MUC1 as described above and subjected to immunofluorescence analysis (23). Briefly, on activation, the coverslips were rapidly rinsed in cold PBS and then immediately fixed using 4% formaldehyde in PBS for 5 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min. After fixation, cells were incubated for 1 h in TBS containing 0.1% Tween 20 and 3% BSA. The coverslips were incubated with primary antibodies (1:150 dilutions of anti-ppERK1/2 antibody and anti-CD8 antibody B) for 1 h, washed four times with TBS containing 0.1% Tween 20, incubated for 1 h with fluorescein isothiocyanate- or Texas Red-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (0.8 µg/ml each). After incubation, the coverslips were washed four times with TBS containing 0.1% Tween 20 and counterstained with 0.4 µg/ml 4',6-diamidino-2-phenylindole in PBS. Fluorescent images were viewed and photographed using a fluorescence microscope (Nikon, E-800, Melville, NY). Images were processed using IP-LAB Spectrum software (Scanalytics, Fairfax, VA).
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RESULTS |
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After activation of CD8/MUC1 cells with anti-CD8 antibody A,
immunoblot analysis revealed a significant increase in the intensity of
anti-ppERK1/2 immunoreactivity compared with unactivated control cells
(Fig. 1A), indicating
activation of ERK1/2. Immunoblotting of the same samples with anti-ERK2
antibody, which recognizes both inactive (unphosphorylated) and active
(phosphorylated) ERK2, revealed an additional band just above the
inactive ERK2 (Fig. 1B), which represents phosphorylation.
Treatment of untransfected control cells with anti-CD8 antibody A
neither affected the level of ppERK1/2 nor revealed the ERK2 band shift
(data not shown). Thus Fig. 1 indicates that treatment of CD8/MUC1
cells resulted in an increase in the amount of activated ERK2.
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To measure the kinase activity of ERK2 after chimera activation,
we purified total ERK2 by immunoprecipitation and measured its ability
to phosphorylate MBP, a substrate of activated ERK2. Figure
2 shows that activation of the chimera
resulted in a drastic increase (over 200%) in 32P
incorporation into MBP compared with control, indicating an increased
kinase activity in the activated sample compared with the unactivated
sample despite the presence of equal amounts of total ERK2.
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The subcellular distribution of activated ERK2 in response to
activation of the chimera was assessed by immunofluorescence staining
of active ERK2 in COS-7 cells expressing the chimera either stably
(Fig. 3A) or transiently (Fig.
3B, shown in green). Figure 3 shows that treatment of cells
with anti-CD8 antibody A resulted in the appearance of activated ERK2
(shown in red; right) and the presence of active ERK2 was
limited only to cells expressing the chimera (bottom
right). Active ERK2 was found in both the cytoplasm and the
nucleus.
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Because it is well established in receptor tyrosine kinase signaling
systems that ERK2 activation requires activation of Ras and MEK
(reviewed in Ref. 12), we determined whether activation of
these two components are necessary for activation of ERK2 in our
chimera system. Figure 4 shows that
CD8/MUC1 cells transiently transfected with a Ras dominant negative
mutant (4) completely lost the ability to activate ERK2 on
activation of the chimera, suggesting the involvement of Ras in this
system. Likewise, CD8/MUC1 cells pretreated with the MEK inhibitor
U-0126 (3) also failed to activate ERK2 after activation
of the chimera based on both immunoblot (Fig.
5A) and in vitro kinase assay
(Fig. 5B). In the presence of U-0126, ERK2 activity was
significantly reduced even without the activation of the chimera (Fig.
5). Thus these results suggest that activation of both Ras and MEK is
required for ERK2 activation after activation of the chimera.
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DISCUSSION |
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Recently, we have shown that treatment with anti-CD8 antibody of COS-7 cells transiently expressing a chimeric membrane protein (CD8/MUC1 protein) results in tyrosine phosphorylation of the CT domain of MUC1 mucin (14). Based on the presence of the consensus motif in the CT domain for binding to the adaptor protein Grb2, it was demonstrated that the CT domain of MUC1 mucin can physically associate with Grb2 and then with Sos, a guanine nucleotide exchange factor (16). Given the fact that Grb2 often initiates sequential activation of a signaling pathway involving Ras, Raf, MEK and MAP kinase (12), we were interested in determining whether phosphorylation of the CT domain of MUC1 mucin also initiates a similar signaling pathway involving ERK, a major MAP kinase.
In the present study, we first identified ERK2 activation by three different methods. First, we determined the level of activated ERK1/2 (ppERK1/2) by immunoblotting with specific antibodies. Figure 1A clearly shows the appearance of activated ERK2 after activation of the chimera. Second, we determined the enzymatic activity of total ERK2. As seen in Fig. 2, the kinase activity of total ERK2 increased drastically after activation of the chimera. Third, our immunocytochemistry results also showed the presence of activated ERK2 in CD8/MUC1 cells treated with anti-CD8 antibody A (Fig. 3), again confirming activation of ERK2 as a result of activation of the chimera. Localization of active ERK2 in the nucleus is due to the translocation of activated ERK2 from the cytoplasm (10). Based on these biochemical, functional, and morphological experiments, we conclude that activation of ERK2 is a result of chimera activation, most likely a consequence of tyrosine phosphorylation of the CT domain of MUC1 mucin.
Having identified ERK2 activation after activation of the chimera, we next examined whether ERK2 activation involves activation of Ras as well as of MEK as predicted from the ERK signaling pathway. In determining the involvement of Ras, we employed a Ras dominant negative mutant generated by replacing serine at position 17 with asparagine (N17) (4), which is a powerful tool widely used to assess the function of Ras (15, 22). Normally, inactive Ras is in GDP-bound form. Sos activated by Grb2 binds to the inactive Ras, which then mediates the exchange of GDP for GTP, thus resulting in GTP-bound form of Ras, an active form. The Ras mutant also binds to GDP, but its affinity for GTP, even in the presence of Sos, is too low to produce a significant amount of active Ras (4). Therefore, the excess mutant will interfere with "normal" activation of endogenous Ras, resulting in a blockade of Ras activation in the pathway. As can be seen in Fig. 4, transient transfection of the mutant in CD8/MUC1 cells completely abolished activation of ERK2 induced by chimera activation, suggesting the involvement of Ras in the activation of ERK2 following chimera activation. To examine the involvement of MEK in ERK2 activation induced by chimera activation, we employed the MEK inhibitor U-0126 (3), which has been used extensively to block MEK activity in intact cells (22, 23, 26). As can be seen in Fig. 5, U-0126 blocked ERK2 activation induced by chimera activation, suggesting the involvement of MEK in ERK2 activation following chimera activation. Suppression of ERK2 activity without activation of the chimera in the presence of U-0126 (Fig. 5) compared with cells transiently transfected with the dominant negative Ras mutant (Fig. 4B) is most likely due to the efficiency of the transfection (~50%). The inhibition of ERK2 activation in the presence of U-0126 was confirmed using an in vitro kinase assay (Fig. 5B). These results are consistent with previous studies demonstrating that MEK activation is essential for ERK2 activation (2) and, collectively, strongly suggest that activation of both Ras and MEK is necessary for ERK2 activation induced by activation of the chimera. Thus it is very likely that the classical Ras-Raf-MEK-ERK signaling pathway is involved following activation of the chimera in CD8/MUC1 cells. In summary, activation of the CT domain of MUC1 mucin in this chimera system leads to activation of ERK2 via a pathway involving Ras and MEK. To the best of our knowledge, this is the first demonstration of the involvement of a MAP kinase in the MUC1 signaling pathway.
How is this finding related to the "real world," especially in the
airway? Recently we showed that Muc1 mucins are an adhesion site for
Pseudomonas aeruginosa in a Chinese hamster ovary cell line
stably expressing hamster Muc1 mucins (13). Adhesion of P. aeruginosa to these Muc1 mucins induced
tyrosine phosphorylation of their CT domain based on both metabolic
radiolabeling with inorganic phosphate and immunoblot analysis with
anti-phosphotyrosine antibody. Because ERK2 activation has been shown
to transcriptionally stimulate and release proinflammatory cytokines
such as interleukin-1 (19, 20) and tumor necrosis
factor-
(6, 19, 24), we speculate that MUC1 mucin might
have an important role in P. aeruginosa infection and
inflammation in the airway. Research on this hypothesis is currently
under way.
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ACKNOWLEDGEMENTS |
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We thank Dr. Erik Lillehoj in our department for editing this manuscript.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants RO1-HL-47125 and HL-10289 (F32, D. Meerzaman) and a Designated Research Incentive Fund from the University of Maryland School of Pharmacy.
Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Rm 446, Baltimore, MD 21201 (E-mail: kkim{at}umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 10 August 2000; accepted in final form 9 February 2001.
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