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Am J Physiol Lung Cell Mol Physiol 287: L809-L815, 2004. First published June 25, 2004; doi:10.1152/ajplung.00385.2003
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Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase

Erik P. Lillehoj,1 Hakryul Kim,1 Ellen Y. Chun,1 and K. Chul Kim1,2

1Department of Pharmaceutical Sciences, School of Pharmacy, and 2Division of Pulmonary and Critical Care Medicine, School of Medicine, University of Maryland, Baltimore, Maryland 21201

Submitted 12 November 2003 ; accepted in final form 3 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We reported previously that Muc1 on the surface of epithelial cells was a receptor for Pseudomonas aeruginosa (Lillehoj EP, Kim BT, and Kim KC. Am J Physiol Lung Cell Mol Physiol 282: L751–L756, 2002). Other studies showed that the Muc1 cytoplasmic tail (CT) contains multiple phosphorylation sites, some of which are phosphorylated constitutively and associated with signaling proteins. However, the relationship between extracellular P. aeruginosa binding and intracellular signaling is unknown. To investigate the signaling mechanism of Muc1, this study examined phosphorylation of its CT and activation of the extracellular signal-regulated kinase (ERK) in response to stimulation by P. aeruginosa or purified flagellin. Our results showed 1) the Muc1 CT was phosphorylated constitutively on serine and tyrosine, 2) serine phosphorylation was stimulated by bacterial cells or flagellin, and 3) binding of P. aeruginosa or flagellin to Muc1 induced phosphorylation of ERK. These results are the first to demonstrate Muc1 CT phosphorylation and ERK activation in response to a clinically important airway pathogen.

bacteria; flagellin; receptor; signaling; extracellular signal-regulated kinase; mitogen-activated protein

MUCUS COATS EPITHELIAL SURFACES and serves a major physical barrier against deleterious environmental agents. Airway mucus provides a front-line defense against inhaled particles and pathogens through its ability to physically trap and remove the foreign substances by mucociliary clearance. Both trapping and clearance rely on the viscoelastic and adhesive properties of mucus conferred by mucin glycoproteins. Eight mucin gene products (designated MUC in human, Muc in animals) are expressed by airway epithelial cells, five as secreted proteins (MUC2, -5AC, -5B, -7, and -8) and three membrane-bound molecules (MUC1, -4, and -13) (33). Secreted mucins are found in the mucus blanket overlying the airway epithelium, whereas membrane mucins act as receptors for intracellular signaling cascades (5, 13).

MUC1 is the best-characterized membrane mucin (14). It consists of an extensively O-glycosylated extracellular (EC) region with 20 amino acid tandem repeats, a transmembrane region, and a cytoplasmic tail (CT). MUC1 is initially synthesized as a single polypeptide chain but rapidly cleaved into two noncovalently associated subunits (39). The larger (>250 kDa) is derived from most of the EC region, whereas the smaller (25–30 kDa) contains a juxtamembrane segment of the EC region, the membrane-spanning region, and the CT (29). The intracellular CT contains multiple, evolutionarily conserved serine and tyrosine residues, some of which are phosphorylated and serve as docking sites for a variety of signaling proteins (2, 22–27, 35–37, 41, 43, 45, 47–49). Although MUC1 CT phosphorylation and association with signaling proteins have important implications for receptor-mediated signal transduction, the exact role of MUC1 in the airways remains to be elucidated.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that colonizes the lungs of cystic fibrosis (CF) and nosocomial pneumonia patients. In CF, PA infection and the ensuing inflammation are responsible for the majority of morbidity and mortality associated with the disease (34). However, the mechanism by which PA infects human airways and subsequent host responses are poorly understood. Recently, we found that PA binds to the hamster Muc1 EC region and identified flagellin as the cognate bacterial adhesin (31, 32). On the basis of these studies and the presence of signal transduction sequence motifs in its CT, it was proposed that Muc1 functions as an airway epithelial cell receptor for inhaled microorganisms (16). In the present study, we show that PA and flagellin stimulated phosphorylation of the Muc1 CT and activated the extracellular signal-regulated kinase (ERK). The functional significance of MUC1 signaling following PA binding to MUC1 on airway epithelial cells is discussed in relation to CF pathophysiology.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bacteria. PA strains PAK, PAK/NP, and PAK/fliC were kindly provided by Dr. Alice Prince (Columbia University, New York, NY). PA clinical isolates from CF patients (PA149, PA383) and an ulcerative keratitis patient (PA6294) were kindly provided by Dr. Gerald Pier (Harvard University, Boston, MA) (44).

Antibodies. Rabbit antiserum to P. aeruginosa strain PAK flagellin was kindly provided by Dr. Dan Wozniak (Wake Forest University, Winston-Salem, NC). Rabbit antiserum to PAK pilin was kindly provided by Dr. Randy Irvin (University of Alberta, Edmonton, Canada). Mouse monoclonal antibody (M4E8) to PAK LPS (serotype 6) was kindly supplied by Dr. Joseph Lam (University of Guelph, Guelph, Canada). CT33 rabbit antiserum was prepared against a synthetic peptide corresponding to the COOH-terminal 17 amino acids of the human MUC1 CT protein. As previously reported (30), CT33 is identical in specificity to the CT1 antiserum described by Pemberton et al. (40) and displays cross-reactivity with hamster Muc1 where 15 of the 17 residues are conserved with the human sequence. Antiphosphorylated ERK1/2 (pERK1/2) antibody (mouse monoclonal, #M8159) and anti-{gamma}-tubulin antibody (mouse monoclonal, #T9028) were from Sigma (St. Louis, MO). Anti-ERK2 antibody (rabbit polyclonal, #C-14) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Purification of flagellin. PAK flagellin was purified by differential centrifugation as described (32). A mock preparation was similarly prepared with the flagella-deficient PAK/fliC– mutant strain. Aliquots were assayed for protein content, and purity was checked by electrophoresis and demonstration of a single 50-kDa protein band on an SDS-polyacrylamide gel stained with Coomassie blue. Western blotting with antiflagellin antiserum identified purified flagellin and confirmed the absence of pilin and LPS contaminants using antipilin and anti-LPS antibodies.

CHO-Muc1 and CHO-X cells. We constructed the pcDNA3/Muc1 expression plasmid by inserting a full-length hamster Muc1 cDNA (38) into the pcDNA3 vector (Invitrogen, San Diego, CA) as described (31). The pcDNA3/Muc1 plasmid and pcDNA3 vector alone were transfected into Chinese hamster ovary (CHO) cells (CCL61; American Type Culture Collection, Manassas, VA) with Lipofectin (Life Technologies, Rockville, MD), and stable clones were isolated. Independent clones expressing hamster Muc1 or empty vector were isolated and are referred to as CHO-Muc1 and CHO-X, respectively. Both cell lines were maintained in F-12/Dulbecco's modified Eagle's medium (DMEM) containing 200 µg/ml G418, 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies).

[32P]orthophosphate and [3H]glucosamine metabolic labeling. CHO-Muc1 or CHO-X cells (2 x 106 cell/100-mm dish) were incubated for 4 h in phosphate-free DMEM (Life Technologies) containing 5% FBS and labeled for 2.5 h in phosphate-free, serum-free DMEM containing 200 µCi/ml of H3[32P]04 (9,000 Ci/mmol; DuPont-NEN, Boston, MA). To label the Muc1 EC protein, we incubated CHO-Muc1 cells in F-12/DMEM containing 0.4 mg/ml of glucose and 100 µCi/ml of D-[6-3H]glucosamine (20 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) for 24 h as previously described (31). Labeled cells were incubated for 10 min with 2 x 108 colony-forming units/100-mm dish of PA, 10 µg/ml of purified flagellin, or an equivalent amount of a mock flagellin control. Flagellin and the mock preparation were prepared from the PA wild-type and flagellin-deficient PAK/fliC bacterial strains as described (4, 32). Cells were extracted at 4°C with 50 mM Tris·HCl, pH 7.4, 0.5% Triton X-100, 1.0% protease inhibitor cocktail, 1.0% phosphatase inhibitor cocktail, and 1.0 mM Na3VO4 (Sigma), and lysates were centrifuged at 18,000 g for 10 min at 4°C. Protein concentrations were determined by the method of Bradford (3) with bovine serum albumin as standard (Bio-Rad, Hercules, CA).

Immunoprecipitation of the Muc1 CT protein. Equal amounts (0.5 mg) of 32P- or 3H-labeled cell lysates were reacted overnight at 4°C with 10 µl of CT33 or preimmune normal rabbit serum (NRS). Antibody-bound proteins were isolated with 0.1 ml of protein A-agarose (Life Technologies), washed three times with lysis buffer, and resolved on 15% (for the CT protein) or 6% (for the EC protein) SDS-polyacrylamide gels as described (18). 32P-labeled proteins were transblotted to polyvinylidene difluoride (PVDF) membrane (Bio-Rad) as described (32) and exposed to BioMax MS film (Kodak, Rochester, NY) at –80°C. Gels containing 3H-labeled proteins were soaked in Amplify fluorographic reagent (Amersham Pharmacia Biotech, Piscataway, NJ), dried under vacuum, and exposed to X-ray film at –80°C.

Phosphoamino acid analysis. The 32P-labeled CT protein was excised from the PVDF membrane, hydrolyzed for 1 h in 6 N HCl at 110°C, evaporated to dryness, and redissolved in water containing 35 mM each of phosphotyrosine, phosphoserine, and phosphothreonine standards. Hydrolysates (2.0 µl) were resolved by high-voltage electrophoresis in 10 mM pyridine, 0.1 M acetic acid, pH 3.5, on cellulose plates (EM Science, Gibbstown, NJ) at 1,000 V for 40 min with a Hunter Thin Layer Peptide Mapping apparatus (C.B.S. Scientific, Del Mar, CA) as described (6), visualized by autoradiography at –80°C, and unlabeled phosphoamino acids identified with 0.2% ninhydrin.

Quantification of 32P-labeled CT protein and amino acids. We determined the intensity of the phosphorylated CT (pCT) protein band in autoradiograms using a GS-700 imaging densitometer and quantified using Quantity One software version 4.1 (Bio-Rad). 32P-labeled phosphoamino acids were scrapped from the cellulose plate in the areas of the ninhydrin-stained phosphoserine and phosphotyrosine standards and were quantified by Cerenkov counting. Values were corrected for background radioactivity obtained from a nonradioactive area of the plate equal in size to the phosphoamino acid spots. The results were expressed as the mean values ± SE of three experiments. The significance of the difference between groups was calculated by the Student's t-test and considered significant at P < 0.05.

ERK activation and immunoblotting. CHO-X and CHO-Muc1 cells were seeded at 8 x 105 cells/60-mm dish and incubated in F-12/DMEM containing 5% FBS and 200 µg/ml of G418. Nontransfected CHO cells were seeded at the same density, incubated 24 h in F-12/DMEM containing 5% FBS, and transfected with an expression plasmid encoding Muc1 with a deletion of its entire EC region (Muc1/NEC) as described (31). At 48 h postseeding, the cells were washed with PBS and incubated for 1 h in 1.0 ml/dish of serum-free DMEM containing PA, PAK/NP, or PAK/fliC (100:1 PA/CHO cell ratio), purified flagellin (1.0 or 10 µg/ml), an equivalent amount of the mock flagellin preparation, or PA-purified LPS (serotype 10, 1.0 µg/ml; Sigma). After incubation, the cells were immediately extracted with lysis buffer at 4°C and equal protein quantities (20 µg) resolved on 12.5% SDS-polyacrylamide gels as described above. Proteins were transferred to PVDF membrane, and the membrane blocked for 30 min in 10 mM Tris·HCl, pH 7.5, containing 0.05% Tween 20 and 5% nonfat dry milk, and reacted 16–24 h at 4°C in anti-pERK1/2 antibody (diluted 1:1,000) or anti-ERK2 antibody (1:1,000). Anti-{gamma}-tubulin antibody (1:5,000) was used as a control to confirm equal protein loading of the samples. Immunoblots were washed with 50 mM Tris·HCl, pH 7.5, containing 0.15 M NaCl and 0.1% Tween 20 for 30 min, incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (KPL, Gaithersburg, MD), and developed with enhanced chemiluminescence reagents (Amersham Pharmacia). The bands corresponding to the pERK1, pERK2, and ERK2 proteins were identified by comigration of prestained protein size markers (Bio-Rad) and quantified as described above.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Muc1 CT is phosphorylated constitutively on tyrosine and serine. The amino acid sequence of the MUC1 CT is highly conserved among different species and contains a relatively high percentage of tyrosine, serine, and threonine residues as potential phosphorylation sites (40). To examine phosphorylation of the hamster Muc1 CT, we immunoprecipitated 32P-labeled CHO-Muc1 cell lysates using rabbit antiserum CT33 against the Muc1 COOH terminus. As shown in Fig. 1A, CT33 reacted with a 29.0-kDa phosphoprotein. This protein was not present in CHO-Muc1 lysates immunoprecipitated with NRS or CHO-X precipitates using CT33. The 29.0-kDa band was identified as the Muc1 pCT since immunoprecipitation was blocked by the synthetic peptide used to prepare CT33 but not by an unrelated peptide (data not shown). Moreover, Quin and McGuckin (41) and Zrihan-Licht et al. (49), using different antibody reagents, reported an identical molecular mass of the human MUC1 pCT. Phosphoamino acid analysis of the hamster pCT identified phosphoserine and phosphotyrosine (Fig. 1B).



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  		Fig. 1. The Muc1 cytoplasmic tail (CT) is phosphorylated constitutively on tyrosine and serine. A: Chinese hamster ovary (CHO)-Muc1 or CHO-X cells were labeled with 32P for 2.5 h, and equal protein amounts of cell lysates were immunoprecipitated (IP) with CT33 antiserum or normal rabbit serum (NRS). Precipitated proteins were resolved on a 15% SDS-polyacrylamide gel, transferred to polyvinylidene difluoride, and visualized by autoradiography. The Muc1 phosphorylated cytoplasmic tail (pCT) was identified on the basis of comigration of prestained protein size markers indicated on the right. B: pCT protein from A was excised from the membrane and hydrolyzed in 6 N HCl, and amino acids were separated by high-voltage electrophoresis on cellulose plates. The migration positions of phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) were identified using unlabeled amino acids and staining with ninhydrin. PO4, free radioactive phosphate.

 
Muc1 CT phosphorylation is stimulated by PA. To determine the effect of PA binding to the Muc1 EC region on phosphorylation of the CT, CHO-Muc1 cells were labeled with 32P and treated for 10 min with PBS (negative control) or PA (strain PAK); cell lysates were immunoprecipitated with CT33 and analyzed for the pCT protein. As shown in Fig. 2A, cells stimulated with PA showed a more prominent pCT band compared with PBS-treated cells. Identical results were obtained with three additional, independently isolated clones of CHO-Muc1 cells (data not shown). Quantification of band intensities demonstrated that, compared with a normalized value of 100% for PBS-treated cells, PA stimulation of CHO-Muc1 cells significantly increased the intensity of the pCT band by an average of 212 ± 45% (n = 3, P < 0.05; Fig. 3). CHO-Muc1 cells treated with PA and lysates immunoprecipitated with NRS did not contain pCT. In addition, CT33 immunoprecipitation of [3H]glucosamine-labeled CHO-Muc1 cells did not reveal any difference in expression of the EC protein in the absence or presence of PA, thus ruling out the possibility that PA-stimulated phosphorylation was due to a higher quantity of Muc1 protein (Fig. 2B).



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  		Fig. 2. Pseudomonas aeruginosa (PA) stimulates phosphorylation of the Muc1 CT. A: CHO-Muc1 cells were labeled with 32P as described in Fig. 1 and treated for 10 min with PBS or PA strain PAK at a 100:1 PA/CHO cell ratio. Equal protein amounts of cell lysates were immunoprecipitated with CT33 antiserum or NRS; precipitated proteins resolved on a 15% SDS-polyacrylamide gel and visualized by autoradiography. B: CHO-Muc1 cells were labeled with [3H]glucosamine for 24 h and treated with PBS or PAK as in A; cell lysates were immunoprecipitated with CT33, resolved on a 6% SDS-polyacrylamide gel, and visualized by fluorography. C: CHO-Muc1 cells were treated with PBS, clinical isolates of PA (PA149, PA383, or PA6294) or PAK, and CT phosphorylation was determined as above.

 


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  		Fig. 3. PA stimulates serine phosphorylation of the Muc1 CT. The immunoprecipitated pCT bands from Fig. 2A (n = 3) were quantified by densitometry. Subsequently, the pCT bands were hydrolyzed in 6 N HCl and analyzed for phosphoamino acids, and pSer and pTyr were quantified as described in MATERIALS AND METHODS. In each case, the values for PBS-treated cells were adjusted to 100%, and the values for PA-stimulated cells were normalized accordingly. *Significantly increased pCT band or pSer residue following PA stimulation (P < 0.05).

 
Schroeder et al. (44) observed that whereas laboratory strains of PA, including PAK, adhered to epithelial cells, specific binding above background levels was not seen with fresh clinical isolates. Therefore, we tested the ability of PA clinical isolates to stimulate phosphorylation of the Muc1 CT. As shown in Fig. 2C, increased CT phosphorylation occurred following treatment of CHO-Muc1 cells with three PA clinical strains, including respiratory isolates from two CF patients (PA149 and PA383) and a corneal isolate from an ulcerative keratitis patient (PA6294). In all three cases, the PA-induced pCT band was comparable in intensity to that produced by PAK.

In three separate experiments using CHO-Muc1 cells treated with PBS or PAK, the pCT bands were excised from the membrane, subjected to acid hydrolysis, and analyzed for phosphoamino acids. As shown in Fig. 3, PA treatment increased the level of phosphoserine by 167 ± 14% compared with control-treated cells, and this increase was statistically significant (P < 0.05). In contrast, the level of phosphotyrosine following PAK stimulation was not significantly greater than control-treated cells (P > 0.05).

Muc1 CT phosphorylation is stimulated by flagellin. Because previous studies demonstrated PA binding to Muc1 was mediated by flagellin (32), we investigated the effect of flagellin on CT phosphorylation. Flagellin was purified from PAK bacteria, producing a single Coomassie blue-stained band on SDS-polyacrylamide gels. Immunoblotting with antibodies specific for PAK pilin or LPS demonstrated that the flagellin was not contaminated with these bacterial components (see Ref. 32). As shown in Fig. 4A, 10 µg/ml of purified flagellin stimulated Muc1 CT phosphorylation approximately twofold compared with cells treated with a mock flagellin preparation isolated from the flagellin-deficient PAK/fliC strain. Treatment of CHO-X cells with the same concentration of flagellin did not reveal a phosphoprotein corresponding to pCT. Phosphoamino acid analysis of the constitutive pCT band (i.e., mock-treated) demonstrated the presence of phosphotyrosine and a minor amount of phosphoserine (Fig. 4B). After flagellin treatment, however, this pattern was reversed, and predominantly phosphoserine was observed with a minor amount of phosphotyrosine.



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  		Fig. 4. Flagellin stimulates serine phosphorylation of the Muc1 CT. A: CHO-Muc1 or CHO-X cells were labeled with 32P as indicated in Fig. 1 and treated for 10 min with 10 µg/ml of purified flagellin or an equivalent amount of mock flagellin prepared from PAK/fliC. Equal protein amounts of cell lysates were immunoprecipitated with CT33 antiserum, precipitated proteins resolved on a 15% SDS-polyacrylamide gel, and visualized by autoradiography. B: pCT proteins from mock- and flagellin-treated CHO-Muc1 cells in A were analyzed for phosphoamino acids as described in Fig. 1.

 
PA stimulates ERK phosphorylation. Using a recombinant CD8/MUC1 fusion protein containing the EC region of CD8 and the human MUC1 CT, we previously demonstrated that CD8 antibody treatment of CD8/MUC1-expressing cells stimulated phosphorylation of the MUC1 CT and activation of a Ras-MEK1/2-ERK2 mitogen-activated protein (MAP) kinase pathway (35). To determine whether a similar signaling pathway was activated in response to stimulation of the Muc1 EC region, we treated CHO-Muc1 and CHO-X cells with PA and examined cell lysates for pERK1 and pERK2. As shown in Fig. 5A, PA stimulated predominantly pERK2 formation. pERK2 activation in CHO-Muc1 cells treated with PA was about threefold greater compared with PBS treatment. Basal pERK2 activation in the absence of PA was not an unexpected result based on the data presented above, demonstrating constitutive Muc1 CT phosphorylated without stimulation. Control immunoblotting experiments demonstrated equal sample loadings ({gamma}-tubulin) and equal levels of total ERK2 (Fig. 5B). PA treatment of CHO-X cells also activated pERK2, but, compared with CHO-Muc1 cells, the level of pERK2 in PA-treated CHO-Muc1 was ~2.5 times greater compared with CHO-X. Because LPS is known to stimulate the ERK MAP kinase (20), we treated CHO-X and CHO-Muc1 cells with purified PA LPS and analyzed ERK activation. As shown in Fig. 5C, the level of pERK2 was increased in LPS-stimulated CHO-X cells, indicating that the ability of PA to stimulate ERK phosphorylation in CHO-X cells was accounted for, at least in part, by LPS. Furthermore, since pERK2 levels in LPS-treated CHO-X and CHO-Muc1 cells were equivalent, the increased MAP kinase activation seen in PA-stimulated CHO-Muc1 cells was likely a result of the interaction between bacterial flagellin and Muc1.



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  		Fig. 5. PA stimulates ERK phosphorylation. CHO-Muc1, CHO-X, or CHO-Muc1/NEC cells were treated for 60 min with PBS or PAK at a 100:1 PA/CHO cell ratio, and cell lysates resolved on a 12.5% SDS-polyacrylamide gel and immunoblotted (IB) with anti-pERK1/2 antibody plus anti-{gamma}-tubulin antibody (A) or anti-ERK2 antibody (B). The pERK1 (44 kDa) and pERK2 (42 kDa) bands were identified by comigration of prestained protein markers. The {gamma}-tubulin band (50 kDa) was identified by immunoblotting with anti-{gamma}-tubulin antibody alone. C: CHO-Muc1 or CHO-X cells were treated with PBS or 1.0 µg/ml of purified PA LPS, and pERK2 levels were determined as above.

 
To confirm these results, we analyzed CHO cells expressing Muc1/NEC. Muc1/NEC is a construct with a deletion of the EC region that retains the transmembrane and CT regions that exhibited significantly reduced PA binding compared with the full-length Muc1 (31). As shown in Fig. 5A, CHO-Muc1/NEC cells demonstrated equivalent pERK2 levels following treatment with PBS or PA. These results were interpreted to indicate that CHO-Muc1/NEC cells treated with the buffer control underwent a basal level of ERK2 activation due to constitutive Muc1/NEC CT phosphorylation but were incapable of PA-stimulated pERK2 activation since Muc1-mediated bacterial binding did not occur.

PA stimulates ERK phosphorylation through flagellin. Because flagellin stimulated Muc1 CT phosphorylation, the role of PA flagellin in ERK activation was next examined. First, we utilized the PAK wild type and the PAK/NP and PAK/fliC bacterial strains. PAK/NP is a derivative of PAK in which the pilA gene encoding the pilin protein was replaced by homologous recombination with a mutant gene interrupted by a tetracycline resistance cassette (42). PAK/fliC is a nonmotile strain in which the fliC gene encoding flagellin was replaced with a homologous gene interrupted by a gentamicin resistance cassette (12). Treatment of CHO-Muc1 cells with PAK/fliC resulted in drastically reduced pERK2 levels compared with PAK wild type or PAK/NP treatment (Fig. 6A). In CHO-X cells, a minor level of pERK2 activation was detected following treatment with PAK wild type or PAK/NP, and pERK2 was not apparent after PAK/fliC treatment. Second, we tested the ability of flagellin to activate ERK and observed a dose-dependent increase in pERK2 levels following treatment of CHO-Muc1 cells with 1.0 or 10 µg/ml of flagellin (Fig. 7A). pERK2 was not seen in CHO-X cells treated with flagellin (Fig. 7A) or CHO-Muc1 treated with the mock flagellin preparation (data not shown). Together, these results indicate that the interaction of PA or its flagellin with the Muc1 EC region stimulated phosphorylation of the ERK2 MAP kinase.



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  		Fig. 6. PA stimulates ERK phosphorylation through flagellin. CHO-Muc1 or CHO-X cells were treated with PAK, PAK/NP, or PAK/fliC and immunoblotted with anti-pERK1/2 antibody plus anti-{gamma}-tubulin antibody (A) or anti-ERK2 antibody (B) as described in Fig. 5.

 


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  		Fig. 7. Purified flagellin activates ERK phosphorylation. CHO-Muc1 or CHO-X cells were treated with PAK or 1.0 or 10 µg/ml of purified flagellin and immunoblotted with anti-pERK1/2 antibody plus anti-{gamma}-tubulin antibody (A) or anti-ERK2 antibody (B) as described in Fig. 5.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Given the receptor-like structure of MUC1 as revealed by the amino acid sequence of its CT, it is not surprising that numerous studies showed it to be constitutively phosphorylated and associated with signaling proteins (2, 22–27, 35–37, 41, 43, 45, 47–49). Because our previous study demonstrated that expression of hamster Muc1 on the surface of CHO cells significantly increased adhesion of PA (31, 32), this investigation was undertaken not only to verify constitutive CT phosphorylation but also to determine the effects of PA and flagellin on induced phosphorylation and cell signaling. The results confirm that the Muc1 CT was phosphorylated constitutively on tyrosine and serine and that serine phosphorylation, in particular, was significantly augmented after treatment with PA or flagellin. PA-stimulated CT phosphorylation was universal to laboratory and clinical strains of bacteria. In addition, both treatments activated the pERK2 MAP kinase. Although we strongly suspect that PA-induced pCT formation was responsible for downstream pERK2 activation, we cannot exclude the possibility that they were independent events.

Two possible explanations may account for the altered phosphorylation pattern following PA or flagellin treatment: 1) increased serine phosphorylation and/or 2) increased tyrosine dephosphorylation. Although tyrosine phosphatase inhibitors were present during cell stimulation and detergent lysis, some degree of tyrosine dephosphorylation was evident following flagellin treatment (Fig. 4). Interestingly, a similar effect was not apparent in PA-treated cells where the amount of Muc1 CT phosphotyrosine was similar to control-treated cells (Fig. 3). The different responses of PA- and flagellin-stimulated cells may have been a manifestation of Muc1-independent binding of bacteria to CHO-Muc1 cells leading to increased tyrosine kinase activity that counterbalances flagellin-induced tyrosine phosphatases. In support of this speculation, several groups have demonstrated that epithelial cells treated with PA exhibit higher c-Src tyrosine kinase activity (10, 11, 22).

Using a CD8/MUC1 chimeric protein containing the EC region of CD8 and the CT of MUC1, Meerzaman et al. (36) previously observed increased MUC1 tyrosine phosphorylation following CD8 antibody treatment of CD8/MUC1-transfected cells. Therefore, we were initially intrigued to observe increased serine, but not tyrosine, phosphorylation of the full-length hamster Muc1 in response to PA or flagellin treatment. However, the previous report did not specifically address the effect of CD8 antibody-induced CD8/MUC1 serine phosphorylation, and it remains to be determined if this residue also undergoes stimulated phosphate modification in the chimeric protein. In other experiments, preliminary results indicate that a CD8/MUC1 construct devoid of all cytoplasmic tyrosines by site-directed mutagenesis retained the ability to activate pERK2 in response to CD8 antibody stimulation suggesting that serine phosphorylation of the chimera may occur (Wang H, Lillehoj EP, and Kim KC, unpublished observations).

The current results confirm previous reports describing tyrosine (2, 25, 27, 36, 37, 41, 43, 45, 47, 49) and serine (2, 22, 49) phosphorylation of the human MUC1 CT. At least four sites of tyrosine phosphorylation were identified: Tyr20, Tyr29, Tyr46, and Tyr60 (numbered according to the 72-amino acid human MUC1 CT) (23, 25, 27, 37, 45). Baruch et al. (2) and Zrihan-Licht et al. (49) demonstrated serine phosphorylation of MUC1/Y, a membrane-bound splice variant devoid of the tandem repeat region, that was stimulated by binding of MUC1/Y to MUC1/SEC, a soluble MUC1 splice variant lacking the transmembrane and CT regions. Li et al. (22) observed phosphorylation of a specific residue, Ser44, by glycogen synthase kinase 3{beta} (GSK3{beta}). Because species-specific sequence variations in the MUC1 CT may affect the kinases and/or phosphatases that act on MUC1, some of the observed effects could result from species-related differences and weaken the conclusion that infection with PA in human airway epithelia would have similar signaling effects. To our knowledge, however, the results presented in this report are the first to identify stimulated phosphorylation of the CT by a biological agent relevant to airway pathophysiology.

What could be the functional significance of Muc1 phosphorylation in response to PA? On the basis of prior studies, we speculate that PA-induced CT phosphorylation may differentially modulate the barrier function of the airway epithelium during early- and late-stage bacterial infection. An SAGNGGSSL sequence in the MUC1 CT binds to {beta}-catenin, a component of the adherens junction mediating epithelial cell-cell adhesion through association with E-cadherin (48). Phosphorylation of Ser44 in the adjacent SPY sequence by GSK3{beta} decreased the interaction of MUC1 with {beta}-catenin (22). In contrast, phosphorylation of Tyr46 by c-Src or the epidermal growth factor receptor promoted MUC1-{beta}-catenin association (25, 27). These studies indicated that MUC1 and E-cadherin compete for binding to the same pool of {beta}-catenin with consequences for increasing or decreasing cell-cell interactions (17, 22). Thus PA-induced MUC1 serine phosphorylation at an early stage following bacteria inspiration may dissociate the MUC1-{beta}-catenin complex, thereby promoting {beta}-catenin-E-cadherin association and strengthening the integrity of the epithelial barrier against further bacterial insult. Conversely, intracellular invasion of epithelial cells by PA activated c-Src tyrosine kinases (10, 11, 22), suggesting that MUC1 CT tyrosine phosphorylation late in the course of infection may produce the opposite effect, leading to dissociation of adherens junctions, desquamation, and apoptosis of infected cells (15).

In addition to its role in cell adhesion, {beta}-catenin colocalizes in the nucleus with the MUC1 CT (23, 24, 26, 28, 46). Interestingly, {beta}-catenin is a transcriptional activator of interleukin (IL)-8 (19), a proinflammatory cytokine responsible for transepithelial efflux of neutrophils during lung injury (21), suggesting an MUC1-dependent mechanism of {beta}-catenin-induced IL-8 gene activation and neutrophil recruitment. This proposed pathway could account, at least in part, for the excessive lung inflammation characteristically seen in CF patients (34). Supporting this model, other studies revealed that CF airway inflammation was mediated by a pathway utilizing the NF-{kappa}B transcription factor. For example, DiMango et al. (7, 8) showed that PA binding to CF airway epithelial cells led to increased production of IL-8 through activation of NF-{kappa}B. Eidelman et al. (9) identified a subset of genes from the tumor necrosis factor-{alpha}/NF-{kappa}B pathway expressed in concert with IL-8 secretion from CF epithelial cells.

MAP kinase signaling pathways also were shown to be involved in PA-stimulated IL-8 production by lung epithelial cells (1). NF-{kappa}B activation occurred downstream of a Ras-MEK1/2-ERK1/2-pp90rsk pathway following PA binding to airway epithelial cells (20). Activation of Ras, MEK1/2, and ERK2 was demonstrated using the CD8/MUC1 chimeric protein (35). Subsequently, Schroeder et al. (43) reported that expression of the full-length MUC1 glycoprotein not only led to association of the CT with Grb2 and Sos adapter proteins but also activated the ERK1/2 kinases. Taken together in the context of the current studies, these observations suggest that binding of inspired pathogens to the MUC1 EC region on airway epithelial cells stimulates phosphorylation of its CT, activation of a downstream Grb2-Sos-Ras-MEK1/2-ERK1/2 signaling pathway, and initiation of an inflammatory response. Ongoing studies in our laboratory are directed at defining in more detail the epithelial cell signaling pathways emanating from PA binding to MUC1 during conditions of normal or pathophysiological outcomes to bacterial exposure of the airways.


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This work was supported by National Heart, Lung, and Blood Institute Grant RO1 HL-47125 and by the Cystic Fibrosis Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 Penn St., Baltimore, MD 21201.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.


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