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MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase
BBRC Biochemical and Biophysical Research Communications 321 (2004) 448–454 www.elsevier.com/locate/ybbrc
MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase q Honghe Wang a, Erik P. Lillehoj a, K. Chul Kim
a,b,*
a
b
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA Division of Pulmonary and Critical Care Medicine, School of Medicine, University of Maryland, Baltimore, MD 21201, USA Received 15 June 2004
Abstract MUC1 is a transmembrane glycoprotein expressed on the apical surface of epithelial cells and exhibiting structural features characteristic of receptors for cytokines and growth factors. Its intracellular cytoplasmic tail (CT) contains multiple amino acid sequence motifs that, once phosphorylated, serve as docking sites for SH2 domain-containing proteins mediating signal transduction. Most studies examining MUC1 signaling have focused on cancer cells where MUC1 is overexpressed, aberrantly glycosylated, and constitutively phosphorylated. No studies have determined the signaling pathways activated in response to stimulation of its ectodomain. To better understand the signaling mechanisms of MUC1, we stably transfected HEK293 cells with an expression plasmid encoding a chimeric protein consisting of the extracellular and transmembrane domains of CD8 and the MUC1 CT (CD8/MUC1). Extracellular treatment of HEK293-CD8/MUC1 cells with CD8 antibody induced intracellular Tyr phosphorylation of the MUC1 CT and activated ERK1/2, but not the p38, SAPK/JNK, or ERK5 MAP kinases. Moreover, phosphorylation of ERK1/2 was completely blocked using a CT deletion mutant or a mutant construct in which all Tyr residues in the CT were changed to Phe. These results establish that Tyr phosphorylation of the MUC1 CT is required for activation of a downstream ERK1/2 pathway. 2004 Elsevier Inc. All rights reserved. Keywords: CD8/MUC1 chimera; Mucin; Mitogen-activated protein kinase
MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of glandular epithelia that is believed to play a key role in immune protection and anti-adhesion [1–3]. MUC1 is overexpressed in human carcinomas, including more than 90% of breast tumors and metastases, and has been identified as a human breast tumor antigen and prognostic marker of the disease [4,5]. In tumor cells, MUC1 expression loses its polarity, becoming uniformly expressed on the entire cell surface [5]. Further evidence implicating a role for
q Abbreviations: Ab, antibody; CT, cytoplasmic tail; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; pTyr, phosphotyrosine; SAPK/JNK, stress-activated protein kinase/ Jun N-terminal kinase; WT, wild type. * Corresponding author. Fax: 1-410-706-1702. E-mail address: [email protected] (K.C. Kim).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.167
MUC1 in malignant transformation is suggested by the observation that MUC1-transfected 3Y1 fibroblasts, unlike non-transfected cells, are oncogenic in nude mice [6]. MUC1 consists of two polypeptide subunits of dissimilar size as a result of proteolytic cleavage of the initially translated precursor protein. The larger extracellular subunit is extensively O-glycosylated with terminal sialic acid giving the molecule a net negative charge. The smaller subunit consists of a juxtamembrane region of the extracellular portion, a transmembrane domain, and a cytoplasmic tail (CT) [1,7]. The amino acid sequence of the CT is highly conserved among different metazoan species and contains multiple Tyr, Ser, and Thr residues as potential phosphorylation sites. At least six residues in the CT have been shown to be phosphorylated in vitro [8–11]. In addition, recent studies have shown that the MUC1 phosphorylated
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intracellular region, like those of cell surface receptors for cytokines and growth factors, interacts with SH2 domain-containing adaptor proteins and kinases involved in signal transduction [12–17]. To further elucidate the signaling mechanisms initiated from the MUC1 CT, a chimeric plasmid (pCD8/ MUC1) comprising the extracellular and transmembrane regions of CD8 and the CT of MUC1 was constructed [18] and expressed in HEK293 cells. CD8 antibody (Ab) treatment of HEK293-CD8/MUC1 cells stimulated Tyr phosphorylation of the MUC1 CT [10]. In the current studies, we investigated the downstream signaling events activated following CD8 Ab-stimulated CT phosphorylation. Based on prior studies that demonstrated a mitogen-activated protein (MAP) kinase pathway was activated by MUC1 expression [14], we assessed phosphorylation of the extracellular signal-regulated kinases (ERK), p38 kinase, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) in the CD8/ MUC1 system. Treatment of HEK293-CD8/MUC1 cells with CD8 Ab resulted in phosphorylation of ERK1/2, but not p38, SAPK/JNK, or ERK5. In addition, using CD8/MUC1 mutants containing a deletion of the CT (DCT) or a CT in which all seven Tyr residues were changed to Phe (7YF), we established Tyr phosphorylation as a necessary event leading to ERK1/2 activation. The functional significance of MUC1 signaling in the context of its potential role as a cell surface receptor is discussed.
Materials and methods Materials. All reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. The sources of Abs were as follows: CD8 Ab A (used for treatment of HEK293-CD8/MUC1 cells, #MCA1226XZ, Serotech, Raleigh, NC), CD8 Ab B (used for immunoprecipitation, immunoblot, and immunofluorescence analyses, #sc7188, Santa Cruz Biotechnology, Santa Cruz, CA), phosphotyrosine (pTyr) Ab (Cell Signaling Technology, Beverly, MA), and ERK2 Ab (Santa Cruz Biotechnology); phospho-ERK1/2 (pERK1/2) Ab (Sigma), phospho-p38 Ab (Cell Signaling Technology), phospho-SAPK/ JNK Ab (Cell Signaling Technology), c-tubulin Ab (Sigma), horseradish peroxidase-conjugated mouse IgG and rabbit IgG Abs (KPL, Gaithersburg, MD), and Alexa Fluor 488-conjugated mouse IgG and Alexa Fluor 546-conjugated rabbit IgG Abs (Molecular Probes, Eugene, OR). Normal mouse IgG was from Serotech. Generation of the pCD8/MUC1-DCT and -7YF mutant plasmids. Construction of the pCD8/MUC1 plasmid has been described [18]. The pCD8/MUC1-DCT plasmid containing a deletion of the CT was constructed by BamHI/KpnI digestion of pCD8/MUC1, purification of the 7.2 kb fragment containing the CD8 coding region and pcDNA3.1 vector (Invitrogen, Carlsbad, CA) on a 1.5% agarose gel, and re-ligation. The pCD8/MUC1-7YF plasmid was constructed as previously described [10] by sequential steps of nested PCR using pCD8/MUC1 as template and primers encoding Tyr-to-Phe substitutions at the seven Tyr residues of the CT. All mutations were verified by nucleotide sequence analysis at the University of Maryland Biopolymer Core Facility (Baltimore, MD) on an Applied Biosystems 373A automated sequencer by dye terminator cycle sequencing with Taq polymerase (Perkin–Elmer, Wellesley, MA) as specified by the manufacturer.
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Transfection and establishment of HEK293 cells expressing the CD8/ MUC1-WT, -DCT, and -7YF proteins. HEK293 cells (American Type Culture Collection, Manassas, VA) were maintained in DulbeccoÕs modified EagleÕs (DME) medium supplemented with penicillin (100 U/ ml), streptomycin (100 lg/ml), and 10% fetal bovine serum (GibcoBRL, Gaithersburg, MD). HEK293 cells were transfected with pCD8/ MUC1 wild type (WT) pCD8/MUC1-DCT, or pCD8/MUC1-7YF plasmids using PolyFect (Qiagen, Santa Clara, CA) according to the manufacturerÕs procedure. Stable cell clones were established by continuous passage in the presence of 800 lg/ml G418 (Gibco-BRL). At least 10 passages were carried out for each clone. CD8 Ab treatment and immunoprecipitation/immunoblot analyses. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were stimulated with CD8 Ab A and cell lysates were subjected to immunoprecipitation and/ or immunoblotting as previously described [10]. Briefly, cells were seeded at 4 · 105 cells/well in six-well plates, cultured to 90% confluence, incubated in serum-free DME medium at 37 C in 5% CO2 for 4 h, and treated at 37 C for 15 min with 7.5 lg/well CD8 Ab A or isotype-matched normal mouse IgG. For direct immunoblot analysis, the cells were immediately lysed in SDS–PAGE sample buffer. For immunoprecipitation, the cells were lysed on ice for 20 min in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM benzamidine, 6.25 mM phenylmethylsulfonyl fluoride, 10 lg/ml leupeptin, 200 lM sodium orthovanadate, and 1% NP-40, the lysates were centrifuged at 12,000g for 10 min at 4 C, and protein concentrations were measured by the procedure of Bradford [19] using bovine serum albumin as standard (Bio-Rad, Richmond, CA). Lysates containing equal amounts of protein were immunoprecipitated with CD8 Ab B and protein A– agarose (Invitrogen) at 4 C for 16 h with continuous agitation, the immunoprecipitates were separated by SDS–PAGE [20] and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Positive control lysates were from HEK293 cells treated with 0.6 mM Na3VO4 and 0.6 mM H2O2 for 15 min. Membranes were blocked in 10 mM Tris–HCl, pH 7.5, 150 mM NaCl (TBS) containing 0.05% Tween 20, and 5% nonfat dried milk and incubated for 2 h at room temperature with Abs to pERK1/2, total ERK2, phospho-ERK5, phospho-p38, or phospho-SAPK/JNK (diluted 1:1000). c-Tubulin Ab (1:5000) was used as a control to confirm equal gel loading of the samples. Immunoblots were washed and incubated with appropriate secondary Ab (1:10,000) and visualized using SuperSignal West Pico chemiluminescence reagents (Pierce, Rockford, IL). Immunofluorescence analysis. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were cultured on round 18-mm glass coverslips (VWR Scientific Products, Media, PA) in six-well plates and subjected to immunofluorescence analysis. Briefly, after treatment with CD8 Ab A as described above, the cells were rapidly rinsed in cold PBS, 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, the cells were sequentially incubated for 1 h in TBS containing 0.1% Tween 20 and 3% BSA, 1 h with CD8 Ab B and pERK1/2 Ab (1:150 each), and 1 h with Alexa Fluor 546- and Alexa Fluor 488-conjugated Abs to rabbit and mouse IgGs, respectively (0.8 lg/ml each). Cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI) in PBS. Images were photographed using a confocal fluorescence microscope (Nikon, Eclipse E800, Melville, NY) and processed using Nikon software (EZ-C1 version 2.10).
Results CD8/MUC1-DCT and -7YF are not Tyr-phosphorylated in response to CD8 Ab treatment We previously reported that HEK293 cells expressing a recombinant CD8/MUC1 protein and treated with
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CD8 Ab exhibited increased Tyr phosphorylation on the MUC CT [10]. To investigate the relationship between CT phosphorylation and MAP kinase signaling, HEK293 stable clones expressing either the CD8/ MUC1-DCT or CD8/MUC1-7YF mutations were produced and compared with HEK293-CD8/MUC1-WT cells. Initially, we verified that the WT and mutant-expressing cells expressed the expected gene products. As shown in Fig. 1A, CD8 Ab immunoblot analysis demonstrated that HEK293-CD8/MUC1-WT and -7YF cells expressed 32.0 kDa proteins while the apparent molecular mass of that expressed in HEK293-CD8/MUC1DCT cells was 28.0 kDa (i.e., truncated CD8). Identical results were observed using 3–5 independent clones of each cell type. Furthermore, membrane localization of all three recombinant proteins was verified by immunofluorescence microscopy (see below, Fig. 4). Next, to assess stimulation of Tyr phosphorylation, HEK293-CD8/ MUC1-WT, -DCT, and 7YF cells were treated with normal IgG or CD8 Ab A and lysates were analyzed by immunoprecipitation with CD8 Ab B and immunoblotting with pTyr Ab. As shown in Fig. 1B, treatment of HEK293-CD8/MUC1-WT cells with CD8 Ab resulted
Fig. 1. CD8/MUC1-DCT and -7YF are not Tyr-phosphorylated in response to CD8 Ab treatment. (A) Equal protein aliquots of cell lysates from HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were immunoblotted with CD8 Ab. (B) HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A as described in Materials and methods and equal protein aliquots of lysates were immunoprecipitated with CD8 Ab B followed by immunoblotting with pTyr Ab. (C) HEK293-CD8/MUC1-WT, DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and lysates were immunoblotted with c-tubulin Ab. These results are representative of 3–5 experiments.
in a dramatic increase in Tyr phosphorylation compared with IgG-treated cells. In contrast, CD8 Ab treatment of HEK293-CD8/MUC1-DCT and -7YF cells revealed no evidence of Tyr phosphorylation. To control for equal gel loadings and immunoblot transfers, all transfected cells had equivalent amounts of c-tubulin (Fig. 1C). HEK293-CD8/MUC1-WT cells exhibit activation of ERK1/2, but not p38, SAPK/JNK, or ERK5, in response to CD8 Ab treatment MAP kinases are a conserved family of enzymes involved in a variety of cellular activities such as proliferation, differentiation, and apoptosis. To investigate which MAP kinase pathway(s) are involved in MUC1 signaling, we activated HEK293-CD8/MUC1-WT cells with normal IgG or CD8 Ab and probed cell lysates for phosphorylated forms of ERK1/2, p38, SAPK/ JNK, and ERK5. As shown in Fig. 2A, increased levels of pERK1/2 were detected following treatment with CD8 Ab compared with IgG. Levels of total ERK2 protein were equal in the two treatment conditions indicating that CD8 Ab did not increase overall ERK expression (Fig. 2B). In contrast, phosphorylation of p38 (Fig. 2C), SAPK/JNK (Fig. 2D), and ERK5 (data not shown) was not observed. Positive controls obtained by treatment with pervanadate (0.6 mM Na3VO4 and 0.6 mM H2O2) confirmed the ability of HEK293-CD8/ MUC1-WT cells to activate p38 and SAPK/JNK.
Fig. 2. HEK293-CD8/MUC1-WT cells exhibit activation of ERK1/2, but not p38 or SAPK/JNK, in response to CD8 Ab treatment. HEK293-CD8/MUC1-WT cells were treated with normal IgG or CD8 Ab and equal protein aliquots of lysates were analyzed by immunoblotting with Abs to pERK1/2 (A), total ERK2 (B), phospho-p38 (C), phospho-SAPK/JNK (D), or c-tubulin (E). Positive control lysates were prepared from cells treated with 0.6 mM Na3VO4 and 0.6 mM H2O2. These results are representative of three experiments.
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Identical results were seen in three independent clones of HEK293-CD8/MUC1-WT cells. HEK293-CD8/MUC1-DCT and -7YF cells do not exhibit ERK1/2 activation in response to CD8 Ab treatment Next, we used biochemical and visual approaches to determine whether CD8 Ab-stimulated Tyr phosphorylation of the MUC1 CT was required for ERK1/2 activation. HEK293-CD8/MUC1-WT, -DCT, or -7YF cells were treated with IgG or CD8 Ab and pERK1/2 were detected by immunoblotting. As seen previously, CD8 Ab treatment of HEK293-CD8/MUC1-WT cells increased pERK1/2 levels compared with IgG-treated cells (Fig. 3A). In contrast, pERK1/2 were not detected in HEK293-CD8/MUC1-DCT- or -7YF cells. As controls, total ERK2 (Fig. 3B) and c-tubulin (Fig. 3C) levels were equivalent in all samples. To confirm that Tyr phosphorylation of the MUC1 CT was necessary for ERK1/2 activation, we performed immunofluorescence analysis of IgG- or CD8 Ab-treated HEK293-CD8/ MUC1-WT, -DCT, and -7YF cells (Fig. 4). CD8 Ab treatment of HEK293-CD8/MUC1-WT cells induced pERK1/2 expression and nuclear localization of the MAP kinase. In contrast, pERK1/2 were not seen in IgG-treated HEK293-CD8/MUC1-WT cells or CD8 Ab-treated CD8/MUC1-DCT or -7YF cells. Identical immunoblot and immunofluorescence results were obtained using three independent clones of HEK293CD8/MUC1-WT, -DCT, and -7YF cells.
Fig. 4. Immunofluorescence analysis of ERK1/2 activation in HEK293-CD8/MUC1-WT, -DCT, and -7YF cells in response to CD8 Ab treatment. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and immunostained with CD8 Ab B plus Alexa Fluor 546-labeled goat anti-rabbit IgG Ab (green) and pERK1/2 Ab plus Alexa Fluor 488-labeled goat antimouse IgG Ab (red). Nuclei were visualized by DAPI staining (blue). These results are representative of three experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Discussion
Fig. 3. HEK293-CD8/MUC1-DCT and -7YF cells do not exhibit ERK1/2 activation in response to CD8 Ab treatment. HEK293-CD8/ MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and equal protein aliquots of lysates were analyzed by immunoblotting with Abs against pERK1/2 (A), total ERK2 (B), or ctubulin (C). These results are representative of three experiments.
Previously, we reported that HEK293 cells expressing the chimeric membrane protein CD8/MUC1 and stimulated with CD8 Ab exhibited increased Tyr phosphorylation of the MUC1 CT [10]. Additionally, Schroeder et al. [14] observed elevated ERK1/2 activation in transgenic mice expressing human MUC1, although phosphorylation of the CT was not reported by these investigators. Therefore, we performed the current studies to address the following questions: (1) Is ERK1/2 activation dependent upon MUC1 CT phosphorylation? (2) Are other MAP kinases activated through MUC1 phosphorylation? (3) What is the physiological role for CT phosphorylation? The information presented in this report clearly establish that Tyr phosphorylation of the MUC1 CT is required for activation of the downstream ERK1/2 pathway and this pathway, as opposed to those involving the p38, SAPK/JNK, or ERK5 kinases, appears to be preferentially activated. While discovering the function of MUC1 remains an intense effort of several laboratories, some studies have implicated roles for this membrane-tethered glycoprotein in cell–cell and cell–matrix adhesion, proliferation, oncogenesis, immunity, and apoptosis mediated through
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CT phosphorylation [6,9,14–17,21–24]. Zrihan-Licht et al. [12] were the first to observe Tyr phosphorylation of MUC1 although the specific sites of modification were not reported. Pandey et al. [13] subsequently identified phosphorylation at Tyr60 leading to MUC1 association with the adaptor proteins Grb2 and Sos. Studies by Li et al. [9,15,17] identified a Tyr46–Glu–Lys–Val tetrapeptide motif in the MUC1 CT that was phosphorylated by the epidermal growth factor (EGF) receptor (ErbB1), c-Src, and Lyn kinases. Using the CD8/MUC1 chimera, we recently confirmed phosphorylation at Tyr46 and Tyr60 and identified Tyr20 and Tyr29 as phosphorylation sites previously unreported. Much interest has focused on identifying the downstream manifestations of MUC1 CT phosphorylation. Schroeder et al. [14] reported that the CT of human MUC1 expressed in transgenic mice was phosphorylated and interacted with all four members of the ErbB receptor family. Activation of MUC1 transgenic mouse mammary glands also led to elevated pERK1/2 activity compared with Muc1 null and wild type animals suggesting a functional link between the introduced membrane protein and MAP kinase signaling. In other studies, Tyr46 phosphorylation by EGF receptor, c-Src, or Lyn increased the interaction between MUC1 and b-catenin, a component of the adherens junctions of mammalian epithelial cells [9,15]. b-Catenin translocates to the nucleus and forms a transcriptional activator in association with LEF-1/TCF [25] and nuclear localization of the MUC1 CT and b-catenin, as well as the catenin-like protein p120ctn, was reported by several investigators [8,25,26]. These studies suggest that overexpression of MUC1 by cancer cells could potentially contribute to oncogenesis by disruption of b-catenin/ p120ctn function during cell–cell interactions and/or transcriptional regulation. The MUC1 CT also has been shown to be constitutively Tyr-phosphorylated in activated T lymphocytes and dendritic cells [16,27]. Li et al. [27] showed that MUC1 was a potential downstream effector of Lck and ZAP-70, tyrosine kinases essential for T cell activation. Whereas Lck phosphorylated Tyr46, ZAP-70 phosphorylated Tyr20. In both cases, however, Lck- or ZAP-70-mediated phosphorylation stimulated binding of MUC1 to b-catenin. The Tyr20-His-Pro-Met site also fits the canonical sequence motif for binding of the SH2 domain of phosphatidylinositol 3-kinase (PI3K) and over-expression of MUC1 activated the anti-apoptotic PI3K/Akt pathway [24]. Collectively, these studies suggest not only that MUC1 contributes to normal processes of intercellular adhesion, proliferation, and apoptosis, but also inappropriate MUC1 expression may induce a transformed phenotype by dysregulation of these, or other, cellular activities. Based on our prior report that intracellular Tyr phosphorylation of the MUC1 CT was stimulated by
extracellular stimulation [10], we were interested in determining whether this process initiated MAP kinase signaling. In particular, we focused on the major MAP kinase families, including ERK1/2, p38, SAPK/ JNK, and the big mitogen kinase1 (BMK1/ERK5), mediators of a diverse array of cell functions [28,29]. MAP kinase pathways are not mutually exclusive and, in general, an initially specific extracellular signal may be amplified or attenuated by intracellular crosstalk between different pathways. In the present study, we observed activation solely of ERK1/2 following treatment of HEK293-CD8/MUC1-WT cells with CD8 Ab and pERK1/2 were detected only if Tyr phosphorylation of the MUC1 CT was permissible. Moreover, by immunocytochemistry, pERK1/2 were localized in the nucleus. Nuclear translocation of ERK1/2 is critical for activation of other protein kinases (e.g., RSK1, RSK2, and RSK3) involved in transcriptional activation of the cAMP response element binding protein (CREB), the co-activator CBP, and c-Fos [30]. pERK1/2 also phosphorylates c-Jun, activating transcription factor-2 (ATF-2), and Elk-1, an ETS family domain protein that mediates transcription from serum response elements [31–33]. Interestingly, some of these factors induce expression of proinflammatory cytokine genes including interleukin-1b and tumor necrosis factor-a [34,35]. What could be the relationship between MUC1 Tyr phosphorylation and inflammation? Recent studies in animal models suggest that MUC1 is a cell surface receptor mediating innate immunity at epithelial surfaces. For example, hamster Muc1 expressed by epithelial cells was shown to be an adhesion site for Pseudomonas aeruginosa, an opportunistic pathogen primarily responsible for morbidity and mortality associated with cystic fibrosis [36–38]. P. aeruginosa binding to hamster Muc1 resulted in phosphorylation of its CT and activation of ERK1/2 [39]. Furthermore, Muc1 null mice exhibited increased bacterial conjunctivitis due to Staphylococcus, Streptococcus, and Corynebacterium spp. compared with wild type mice [40]. DeSouza et al. [22] observed that female Muc1 null mice housed under normal conditions displayed chronic infection and inflammation of the uterus as a result of infection by the normal bacterial flora of the reproductive tract. This phenotype was not observed in mice raised in a pathogen-free environment. In vitro studies also have suggested that MUC1 plays a role in protecting the endometrium from microbial attack [23]. Our current working model hypothesizes that binding of microorganisms to MUC1 expressed on mucosal surface stimulates phosphorylation of its CT, activation of a MAP kinase signaling pathway, elaboration of proinflammatory cytokines, and induction of innate immune responses ultimately leading to clearance of the offending microbes. Research to
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further validate this theory is currently ongoing in our laboratory. [16]
Acknowledgments This work was supported by grants from NIH (RO1 HL-47125) and the Cystic Fibrosis Foundation.
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MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase q Honghe Wang a, Erik P. Lillehoj a, K. Chul Kim
a,b,*
a
b
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA Division of Pulmonary and Critical Care Medicine, School of Medicine, University of Maryland, Baltimore, MD 21201, USA Received 15 June 2004
Abstract MUC1 is a transmembrane glycoprotein expressed on the apical surface of epithelial cells and exhibiting structural features characteristic of receptors for cytokines and growth factors. Its intracellular cytoplasmic tail (CT) contains multiple amino acid sequence motifs that, once phosphorylated, serve as docking sites for SH2 domain-containing proteins mediating signal transduction. Most studies examining MUC1 signaling have focused on cancer cells where MUC1 is overexpressed, aberrantly glycosylated, and constitutively phosphorylated. No studies have determined the signaling pathways activated in response to stimulation of its ectodomain. To better understand the signaling mechanisms of MUC1, we stably transfected HEK293 cells with an expression plasmid encoding a chimeric protein consisting of the extracellular and transmembrane domains of CD8 and the MUC1 CT (CD8/MUC1). Extracellular treatment of HEK293-CD8/MUC1 cells with CD8 antibody induced intracellular Tyr phosphorylation of the MUC1 CT and activated ERK1/2, but not the p38, SAPK/JNK, or ERK5 MAP kinases. Moreover, phosphorylation of ERK1/2 was completely blocked using a CT deletion mutant or a mutant construct in which all Tyr residues in the CT were changed to Phe. These results establish that Tyr phosphorylation of the MUC1 CT is required for activation of a downstream ERK1/2 pathway. 2004 Elsevier Inc. All rights reserved. Keywords: CD8/MUC1 chimera; Mucin; Mitogen-activated protein kinase
MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of glandular epithelia that is believed to play a key role in immune protection and anti-adhesion [1–3]. MUC1 is overexpressed in human carcinomas, including more than 90% of breast tumors and metastases, and has been identified as a human breast tumor antigen and prognostic marker of the disease [4,5]. In tumor cells, MUC1 expression loses its polarity, becoming uniformly expressed on the entire cell surface [5]. Further evidence implicating a role for
q Abbreviations: Ab, antibody; CT, cytoplasmic tail; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; pTyr, phosphotyrosine; SAPK/JNK, stress-activated protein kinase/ Jun N-terminal kinase; WT, wild type. * Corresponding author. Fax: 1-410-706-1702. E-mail address: [email protected] (K.C. Kim).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.167
MUC1 in malignant transformation is suggested by the observation that MUC1-transfected 3Y1 fibroblasts, unlike non-transfected cells, are oncogenic in nude mice [6]. MUC1 consists of two polypeptide subunits of dissimilar size as a result of proteolytic cleavage of the initially translated precursor protein. The larger extracellular subunit is extensively O-glycosylated with terminal sialic acid giving the molecule a net negative charge. The smaller subunit consists of a juxtamembrane region of the extracellular portion, a transmembrane domain, and a cytoplasmic tail (CT) [1,7]. The amino acid sequence of the CT is highly conserved among different metazoan species and contains multiple Tyr, Ser, and Thr residues as potential phosphorylation sites. At least six residues in the CT have been shown to be phosphorylated in vitro [8–11]. In addition, recent studies have shown that the MUC1 phosphorylated
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intracellular region, like those of cell surface receptors for cytokines and growth factors, interacts with SH2 domain-containing adaptor proteins and kinases involved in signal transduction [12–17]. To further elucidate the signaling mechanisms initiated from the MUC1 CT, a chimeric plasmid (pCD8/ MUC1) comprising the extracellular and transmembrane regions of CD8 and the CT of MUC1 was constructed [18] and expressed in HEK293 cells. CD8 antibody (Ab) treatment of HEK293-CD8/MUC1 cells stimulated Tyr phosphorylation of the MUC1 CT [10]. In the current studies, we investigated the downstream signaling events activated following CD8 Ab-stimulated CT phosphorylation. Based on prior studies that demonstrated a mitogen-activated protein (MAP) kinase pathway was activated by MUC1 expression [14], we assessed phosphorylation of the extracellular signal-regulated kinases (ERK), p38 kinase, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) in the CD8/ MUC1 system. Treatment of HEK293-CD8/MUC1 cells with CD8 Ab resulted in phosphorylation of ERK1/2, but not p38, SAPK/JNK, or ERK5. In addition, using CD8/MUC1 mutants containing a deletion of the CT (DCT) or a CT in which all seven Tyr residues were changed to Phe (7YF), we established Tyr phosphorylation as a necessary event leading to ERK1/2 activation. The functional significance of MUC1 signaling in the context of its potential role as a cell surface receptor is discussed.
Materials and methods Materials. All reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated. The sources of Abs were as follows: CD8 Ab A (used for treatment of HEK293-CD8/MUC1 cells, #MCA1226XZ, Serotech, Raleigh, NC), CD8 Ab B (used for immunoprecipitation, immunoblot, and immunofluorescence analyses, #sc7188, Santa Cruz Biotechnology, Santa Cruz, CA), phosphotyrosine (pTyr) Ab (Cell Signaling Technology, Beverly, MA), and ERK2 Ab (Santa Cruz Biotechnology); phospho-ERK1/2 (pERK1/2) Ab (Sigma), phospho-p38 Ab (Cell Signaling Technology), phospho-SAPK/ JNK Ab (Cell Signaling Technology), c-tubulin Ab (Sigma), horseradish peroxidase-conjugated mouse IgG and rabbit IgG Abs (KPL, Gaithersburg, MD), and Alexa Fluor 488-conjugated mouse IgG and Alexa Fluor 546-conjugated rabbit IgG Abs (Molecular Probes, Eugene, OR). Normal mouse IgG was from Serotech. Generation of the pCD8/MUC1-DCT and -7YF mutant plasmids. Construction of the pCD8/MUC1 plasmid has been described [18]. The pCD8/MUC1-DCT plasmid containing a deletion of the CT was constructed by BamHI/KpnI digestion of pCD8/MUC1, purification of the 7.2 kb fragment containing the CD8 coding region and pcDNA3.1 vector (Invitrogen, Carlsbad, CA) on a 1.5% agarose gel, and re-ligation. The pCD8/MUC1-7YF plasmid was constructed as previously described [10] by sequential steps of nested PCR using pCD8/MUC1 as template and primers encoding Tyr-to-Phe substitutions at the seven Tyr residues of the CT. All mutations were verified by nucleotide sequence analysis at the University of Maryland Biopolymer Core Facility (Baltimore, MD) on an Applied Biosystems 373A automated sequencer by dye terminator cycle sequencing with Taq polymerase (Perkin–Elmer, Wellesley, MA) as specified by the manufacturer.
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Transfection and establishment of HEK293 cells expressing the CD8/ MUC1-WT, -DCT, and -7YF proteins. HEK293 cells (American Type Culture Collection, Manassas, VA) were maintained in DulbeccoÕs modified EagleÕs (DME) medium supplemented with penicillin (100 U/ ml), streptomycin (100 lg/ml), and 10% fetal bovine serum (GibcoBRL, Gaithersburg, MD). HEK293 cells were transfected with pCD8/ MUC1 wild type (WT) pCD8/MUC1-DCT, or pCD8/MUC1-7YF plasmids using PolyFect (Qiagen, Santa Clara, CA) according to the manufacturerÕs procedure. Stable cell clones were established by continuous passage in the presence of 800 lg/ml G418 (Gibco-BRL). At least 10 passages were carried out for each clone. CD8 Ab treatment and immunoprecipitation/immunoblot analyses. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were stimulated with CD8 Ab A and cell lysates were subjected to immunoprecipitation and/ or immunoblotting as previously described [10]. Briefly, cells were seeded at 4 · 105 cells/well in six-well plates, cultured to 90% confluence, incubated in serum-free DME medium at 37 C in 5% CO2 for 4 h, and treated at 37 C for 15 min with 7.5 lg/well CD8 Ab A or isotype-matched normal mouse IgG. For direct immunoblot analysis, the cells were immediately lysed in SDS–PAGE sample buffer. For immunoprecipitation, the cells were lysed on ice for 20 min in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 10 mM benzamidine, 6.25 mM phenylmethylsulfonyl fluoride, 10 lg/ml leupeptin, 200 lM sodium orthovanadate, and 1% NP-40, the lysates were centrifuged at 12,000g for 10 min at 4 C, and protein concentrations were measured by the procedure of Bradford [19] using bovine serum albumin as standard (Bio-Rad, Richmond, CA). Lysates containing equal amounts of protein were immunoprecipitated with CD8 Ab B and protein A– agarose (Invitrogen) at 4 C for 16 h with continuous agitation, the immunoprecipitates were separated by SDS–PAGE [20] and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad). Positive control lysates were from HEK293 cells treated with 0.6 mM Na3VO4 and 0.6 mM H2O2 for 15 min. Membranes were blocked in 10 mM Tris–HCl, pH 7.5, 150 mM NaCl (TBS) containing 0.05% Tween 20, and 5% nonfat dried milk and incubated for 2 h at room temperature with Abs to pERK1/2, total ERK2, phospho-ERK5, phospho-p38, or phospho-SAPK/JNK (diluted 1:1000). c-Tubulin Ab (1:5000) was used as a control to confirm equal gel loading of the samples. Immunoblots were washed and incubated with appropriate secondary Ab (1:10,000) and visualized using SuperSignal West Pico chemiluminescence reagents (Pierce, Rockford, IL). Immunofluorescence analysis. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were cultured on round 18-mm glass coverslips (VWR Scientific Products, Media, PA) in six-well plates and subjected to immunofluorescence analysis. Briefly, after treatment with CD8 Ab A as described above, the cells were rapidly rinsed in cold PBS, 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, the cells were sequentially incubated for 1 h in TBS containing 0.1% Tween 20 and 3% BSA, 1 h with CD8 Ab B and pERK1/2 Ab (1:150 each), and 1 h with Alexa Fluor 546- and Alexa Fluor 488-conjugated Abs to rabbit and mouse IgGs, respectively (0.8 lg/ml each). Cells were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI) in PBS. Images were photographed using a confocal fluorescence microscope (Nikon, Eclipse E800, Melville, NY) and processed using Nikon software (EZ-C1 version 2.10).
Results CD8/MUC1-DCT and -7YF are not Tyr-phosphorylated in response to CD8 Ab treatment We previously reported that HEK293 cells expressing a recombinant CD8/MUC1 protein and treated with
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CD8 Ab exhibited increased Tyr phosphorylation on the MUC CT [10]. To investigate the relationship between CT phosphorylation and MAP kinase signaling, HEK293 stable clones expressing either the CD8/ MUC1-DCT or CD8/MUC1-7YF mutations were produced and compared with HEK293-CD8/MUC1-WT cells. Initially, we verified that the WT and mutant-expressing cells expressed the expected gene products. As shown in Fig. 1A, CD8 Ab immunoblot analysis demonstrated that HEK293-CD8/MUC1-WT and -7YF cells expressed 32.0 kDa proteins while the apparent molecular mass of that expressed in HEK293-CD8/MUC1DCT cells was 28.0 kDa (i.e., truncated CD8). Identical results were observed using 3–5 independent clones of each cell type. Furthermore, membrane localization of all three recombinant proteins was verified by immunofluorescence microscopy (see below, Fig. 4). Next, to assess stimulation of Tyr phosphorylation, HEK293-CD8/ MUC1-WT, -DCT, and 7YF cells were treated with normal IgG or CD8 Ab A and lysates were analyzed by immunoprecipitation with CD8 Ab B and immunoblotting with pTyr Ab. As shown in Fig. 1B, treatment of HEK293-CD8/MUC1-WT cells with CD8 Ab resulted
Fig. 1. CD8/MUC1-DCT and -7YF are not Tyr-phosphorylated in response to CD8 Ab treatment. (A) Equal protein aliquots of cell lysates from HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were immunoblotted with CD8 Ab. (B) HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A as described in Materials and methods and equal protein aliquots of lysates were immunoprecipitated with CD8 Ab B followed by immunoblotting with pTyr Ab. (C) HEK293-CD8/MUC1-WT, DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and lysates were immunoblotted with c-tubulin Ab. These results are representative of 3–5 experiments.
in a dramatic increase in Tyr phosphorylation compared with IgG-treated cells. In contrast, CD8 Ab treatment of HEK293-CD8/MUC1-DCT and -7YF cells revealed no evidence of Tyr phosphorylation. To control for equal gel loadings and immunoblot transfers, all transfected cells had equivalent amounts of c-tubulin (Fig. 1C). HEK293-CD8/MUC1-WT cells exhibit activation of ERK1/2, but not p38, SAPK/JNK, or ERK5, in response to CD8 Ab treatment MAP kinases are a conserved family of enzymes involved in a variety of cellular activities such as proliferation, differentiation, and apoptosis. To investigate which MAP kinase pathway(s) are involved in MUC1 signaling, we activated HEK293-CD8/MUC1-WT cells with normal IgG or CD8 Ab and probed cell lysates for phosphorylated forms of ERK1/2, p38, SAPK/ JNK, and ERK5. As shown in Fig. 2A, increased levels of pERK1/2 were detected following treatment with CD8 Ab compared with IgG. Levels of total ERK2 protein were equal in the two treatment conditions indicating that CD8 Ab did not increase overall ERK expression (Fig. 2B). In contrast, phosphorylation of p38 (Fig. 2C), SAPK/JNK (Fig. 2D), and ERK5 (data not shown) was not observed. Positive controls obtained by treatment with pervanadate (0.6 mM Na3VO4 and 0.6 mM H2O2) confirmed the ability of HEK293-CD8/ MUC1-WT cells to activate p38 and SAPK/JNK.
Fig. 2. HEK293-CD8/MUC1-WT cells exhibit activation of ERK1/2, but not p38 or SAPK/JNK, in response to CD8 Ab treatment. HEK293-CD8/MUC1-WT cells were treated with normal IgG or CD8 Ab and equal protein aliquots of lysates were analyzed by immunoblotting with Abs to pERK1/2 (A), total ERK2 (B), phospho-p38 (C), phospho-SAPK/JNK (D), or c-tubulin (E). Positive control lysates were prepared from cells treated with 0.6 mM Na3VO4 and 0.6 mM H2O2. These results are representative of three experiments.
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Identical results were seen in three independent clones of HEK293-CD8/MUC1-WT cells. HEK293-CD8/MUC1-DCT and -7YF cells do not exhibit ERK1/2 activation in response to CD8 Ab treatment Next, we used biochemical and visual approaches to determine whether CD8 Ab-stimulated Tyr phosphorylation of the MUC1 CT was required for ERK1/2 activation. HEK293-CD8/MUC1-WT, -DCT, or -7YF cells were treated with IgG or CD8 Ab and pERK1/2 were detected by immunoblotting. As seen previously, CD8 Ab treatment of HEK293-CD8/MUC1-WT cells increased pERK1/2 levels compared with IgG-treated cells (Fig. 3A). In contrast, pERK1/2 were not detected in HEK293-CD8/MUC1-DCT- or -7YF cells. As controls, total ERK2 (Fig. 3B) and c-tubulin (Fig. 3C) levels were equivalent in all samples. To confirm that Tyr phosphorylation of the MUC1 CT was necessary for ERK1/2 activation, we performed immunofluorescence analysis of IgG- or CD8 Ab-treated HEK293-CD8/ MUC1-WT, -DCT, and -7YF cells (Fig. 4). CD8 Ab treatment of HEK293-CD8/MUC1-WT cells induced pERK1/2 expression and nuclear localization of the MAP kinase. In contrast, pERK1/2 were not seen in IgG-treated HEK293-CD8/MUC1-WT cells or CD8 Ab-treated CD8/MUC1-DCT or -7YF cells. Identical immunoblot and immunofluorescence results were obtained using three independent clones of HEK293CD8/MUC1-WT, -DCT, and -7YF cells.
Fig. 4. Immunofluorescence analysis of ERK1/2 activation in HEK293-CD8/MUC1-WT, -DCT, and -7YF cells in response to CD8 Ab treatment. HEK293-CD8/MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and immunostained with CD8 Ab B plus Alexa Fluor 546-labeled goat anti-rabbit IgG Ab (green) and pERK1/2 Ab plus Alexa Fluor 488-labeled goat antimouse IgG Ab (red). Nuclei were visualized by DAPI staining (blue). These results are representative of three experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Discussion
Fig. 3. HEK293-CD8/MUC1-DCT and -7YF cells do not exhibit ERK1/2 activation in response to CD8 Ab treatment. HEK293-CD8/ MUC1-WT, -DCT, and -7YF cells were treated with normal IgG or CD8 Ab A and equal protein aliquots of lysates were analyzed by immunoblotting with Abs against pERK1/2 (A), total ERK2 (B), or ctubulin (C). These results are representative of three experiments.
Previously, we reported that HEK293 cells expressing the chimeric membrane protein CD8/MUC1 and stimulated with CD8 Ab exhibited increased Tyr phosphorylation of the MUC1 CT [10]. Additionally, Schroeder et al. [14] observed elevated ERK1/2 activation in transgenic mice expressing human MUC1, although phosphorylation of the CT was not reported by these investigators. Therefore, we performed the current studies to address the following questions: (1) Is ERK1/2 activation dependent upon MUC1 CT phosphorylation? (2) Are other MAP kinases activated through MUC1 phosphorylation? (3) What is the physiological role for CT phosphorylation? The information presented in this report clearly establish that Tyr phosphorylation of the MUC1 CT is required for activation of the downstream ERK1/2 pathway and this pathway, as opposed to those involving the p38, SAPK/JNK, or ERK5 kinases, appears to be preferentially activated. While discovering the function of MUC1 remains an intense effort of several laboratories, some studies have implicated roles for this membrane-tethered glycoprotein in cell–cell and cell–matrix adhesion, proliferation, oncogenesis, immunity, and apoptosis mediated through
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CT phosphorylation [6,9,14–17,21–24]. Zrihan-Licht et al. [12] were the first to observe Tyr phosphorylation of MUC1 although the specific sites of modification were not reported. Pandey et al. [13] subsequently identified phosphorylation at Tyr60 leading to MUC1 association with the adaptor proteins Grb2 and Sos. Studies by Li et al. [9,15,17] identified a Tyr46–Glu–Lys–Val tetrapeptide motif in the MUC1 CT that was phosphorylated by the epidermal growth factor (EGF) receptor (ErbB1), c-Src, and Lyn kinases. Using the CD8/MUC1 chimera, we recently confirmed phosphorylation at Tyr46 and Tyr60 and identified Tyr20 and Tyr29 as phosphorylation sites previously unreported. Much interest has focused on identifying the downstream manifestations of MUC1 CT phosphorylation. Schroeder et al. [14] reported that the CT of human MUC1 expressed in transgenic mice was phosphorylated and interacted with all four members of the ErbB receptor family. Activation of MUC1 transgenic mouse mammary glands also led to elevated pERK1/2 activity compared with Muc1 null and wild type animals suggesting a functional link between the introduced membrane protein and MAP kinase signaling. In other studies, Tyr46 phosphorylation by EGF receptor, c-Src, or Lyn increased the interaction between MUC1 and b-catenin, a component of the adherens junctions of mammalian epithelial cells [9,15]. b-Catenin translocates to the nucleus and forms a transcriptional activator in association with LEF-1/TCF [25] and nuclear localization of the MUC1 CT and b-catenin, as well as the catenin-like protein p120ctn, was reported by several investigators [8,25,26]. These studies suggest that overexpression of MUC1 by cancer cells could potentially contribute to oncogenesis by disruption of b-catenin/ p120ctn function during cell–cell interactions and/or transcriptional regulation. The MUC1 CT also has been shown to be constitutively Tyr-phosphorylated in activated T lymphocytes and dendritic cells [16,27]. Li et al. [27] showed that MUC1 was a potential downstream effector of Lck and ZAP-70, tyrosine kinases essential for T cell activation. Whereas Lck phosphorylated Tyr46, ZAP-70 phosphorylated Tyr20. In both cases, however, Lck- or ZAP-70-mediated phosphorylation stimulated binding of MUC1 to b-catenin. The Tyr20-His-Pro-Met site also fits the canonical sequence motif for binding of the SH2 domain of phosphatidylinositol 3-kinase (PI3K) and over-expression of MUC1 activated the anti-apoptotic PI3K/Akt pathway [24]. Collectively, these studies suggest not only that MUC1 contributes to normal processes of intercellular adhesion, proliferation, and apoptosis, but also inappropriate MUC1 expression may induce a transformed phenotype by dysregulation of these, or other, cellular activities. Based on our prior report that intracellular Tyr phosphorylation of the MUC1 CT was stimulated by
extracellular stimulation [10], we were interested in determining whether this process initiated MAP kinase signaling. In particular, we focused on the major MAP kinase families, including ERK1/2, p38, SAPK/ JNK, and the big mitogen kinase1 (BMK1/ERK5), mediators of a diverse array of cell functions [28,29]. MAP kinase pathways are not mutually exclusive and, in general, an initially specific extracellular signal may be amplified or attenuated by intracellular crosstalk between different pathways. In the present study, we observed activation solely of ERK1/2 following treatment of HEK293-CD8/MUC1-WT cells with CD8 Ab and pERK1/2 were detected only if Tyr phosphorylation of the MUC1 CT was permissible. Moreover, by immunocytochemistry, pERK1/2 were localized in the nucleus. Nuclear translocation of ERK1/2 is critical for activation of other protein kinases (e.g., RSK1, RSK2, and RSK3) involved in transcriptional activation of the cAMP response element binding protein (CREB), the co-activator CBP, and c-Fos [30]. pERK1/2 also phosphorylates c-Jun, activating transcription factor-2 (ATF-2), and Elk-1, an ETS family domain protein that mediates transcription from serum response elements [31–33]. Interestingly, some of these factors induce expression of proinflammatory cytokine genes including interleukin-1b and tumor necrosis factor-a [34,35]. What could be the relationship between MUC1 Tyr phosphorylation and inflammation? Recent studies in animal models suggest that MUC1 is a cell surface receptor mediating innate immunity at epithelial surfaces. For example, hamster Muc1 expressed by epithelial cells was shown to be an adhesion site for Pseudomonas aeruginosa, an opportunistic pathogen primarily responsible for morbidity and mortality associated with cystic fibrosis [36–38]. P. aeruginosa binding to hamster Muc1 resulted in phosphorylation of its CT and activation of ERK1/2 [39]. Furthermore, Muc1 null mice exhibited increased bacterial conjunctivitis due to Staphylococcus, Streptococcus, and Corynebacterium spp. compared with wild type mice [40]. DeSouza et al. [22] observed that female Muc1 null mice housed under normal conditions displayed chronic infection and inflammation of the uterus as a result of infection by the normal bacterial flora of the reproductive tract. This phenotype was not observed in mice raised in a pathogen-free environment. In vitro studies also have suggested that MUC1 plays a role in protecting the endometrium from microbial attack [23]. Our current working model hypothesizes that binding of microorganisms to MUC1 expressed on mucosal surface stimulates phosphorylation of its CT, activation of a MAP kinase signaling pathway, elaboration of proinflammatory cytokines, and induction of innate immune responses ultimately leading to clearance of the offending microbes. Research to
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further validate this theory is currently ongoing in our laboratory. [16]
Acknowledgments This work was supported by grants from NIH (RO1 HL-47125) and the Cystic Fibrosis Foundation.
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