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Originally published online as doi:10.1189/jlb.0604333 on October 28, 2004

Published online before print October 28, 2004
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(Journal of Leukocyte Biology. 2005;77:90-99.)
© 2005 by Society for Leukocyte Biology

MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells

P. Mukherjee*,1, T. L. Tinder*, G. D. Basu* and S. J. Gendler*,{dagger},1

* Department of Biochemistry and Molecular Biology and
{dagger} Tumor Biology Program, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, Arizona

1 Correspondence: Pinku Mukherjee, Cellular Immunology, and Sandra J. Gendler, Dept. of Biochemistry and Molecular Biology, Tumor Biology Program, Mayo Clinic College of Medicine, 13400 E. Shea Blvd., Scottsdale, AZ 85259. E-mail: mukherjee.pinku{at}mayo.edu and gendler.sandra{at}mayo.edu


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ABSTRACT
 
MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

Key Words: mucin 1 • p56lck • siRNA • ERK1/2 • calcium flux • T cell activation


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INTRODUCTION
 
MUC1 is a large mucin glycoprotein, which is expressed in more than 90% of all adenocarcinomas and plays an important role in tumor progression and metastasis [1 , 2 ]. Although the function of MUC1 is unknown, there is evidence that it may be involved in cell adhesion and signal transduction pathways as an adaptor protein (reviewed in refs. [2 , 3 ]). Recently, MUC1 has been implicated as an oncogene involved in the process of transformation [4 , 5 ]. MUC1 has a large, glycosylated extracellular domain with a variable number tandem repeat region (TR), a transmembrane region (TM), and a tyrosine phosphorylated cytoplasmic tail (CT). The TM and CT domains of MUC1 are highly conserved (88% identical in mouse), suggesting important functional roles [6 ]. The 72 amino acid tail has seven tyrosines, six of which are 100% conserved in the seven species studied, and contains a variety of kinase recognition sites. Tyrosine phosphorylation of MUC1 CT has been demonstrated in epithelial cell lines [7 8 9 10 11 ].

Historically, MUC1 expression was thought to be restricted to epithelial cells; however, nonepithelial expression of MUC1 has recently been described in myelomas, T and B cell lymphomas, normal proerythrocytes, and erythroblasts in bone marrow [12 13 14 15 16 17 18 19 ]. MUC1 is also expressed on normal hematopoietic cell types including T cells, dendritic cells, and bone marrow mononuclear cells. The CT is phosphorylated in some of the hematopoietic cells, indicative of a role in signal transduction [12 , 18 , 20 , 21 ]. When phosphorylated, these motifs provide potential sites for binding to Src homology 2 (SH2) domains of other kinases. MUC1 has previously been shown to interact with c-Src [22 ] and Grb2/SOS, which are signal transducers of a number of receptor kinase pathways [9 , 10 ]. The CT contains an immunoreceptor tyrosine-based activation-like motif (ITAM; YXXLX8YXXM) and kinase recognition sequences, which when phosphorylated, provide potential sites for binding to SH2 domains. It has recently been shown that lck phosphorylates MUC1 CT at Y46, and {zeta}-associated protein 70 (ZAP-70) phosphorylates MUC1 CT at the Y20 motif, which is located in the ITAM (unpublished data from our laboratory and ref. [23 ]). An indirect association of MUC1 with CD45, Yes, Fyn, and p56lck in membrane lipid rafts in a human T leukemia cell line, HUT78, has also been reported [24 ].

Signals from T cell receptor (TCR) trigger rapid tyrosine phosphorylation of src-related protein tyrosine kinases (PTKs), p56lck, and Fyn, followed by phosphorylation of ITAM. This creates docking sites for syk family tyrosine kinase ZAP-70, which phosphorylates linker for activation of T cells (LAT), and recruits other signaling molecules including phospholipase (PLC){gamma}1, Tec family kinases, SH2 domain-containing leukocyte protein of 76 kD, Grb2, and Gads to form a complex. The complex is aggregated in the specialized subdomains of the plasma membrane (lipid rafts) and amplifies the signal received from the TCR [25 26 27 ]. As MUC1 is implicated in signal transduction events in epithelial cells and as it contains tyrosine phosphorylation sites that could allow binding to SH2 domains, including the c-Src SH2 domain at the YEKV motif on MUC1 CT domain [22 ], we first determined if MUC1 is phosphorylated upon TCR ligation, followed by studying interactions of MUC1 CT with T cell-specific PTK p56lck. To further elucidate its role in signal transduction post-TCR ligation, we analyzed the downstream signaling events leading to proliferation in MUC1-deficient Jurkat cells. We describe for the first time that MUC1 is expressed in stimulated and unstimulated Jurkat cells and normal unstimulated T cells using specific antibodies directed not only against the TR extracellular protein core of MUC1 but also against the CT domain of MUC1. We demonstrate phosphorylation of MUC1 in response to TCR signaling and show a direct association of MUC1 with p56lck and localization with LAT in the low-density, detergent-insoluble membrane raft fractions. We demonstrate that MUC1-deficient Jurkats have reduced Ca2+ influx associated with reduced phosphorylation of extracellular signal-regulated kinase (ERK)1/2, reduced T cell proliferation, and cytokine production. This is the first study describing a direct and critical role of MUC1 during Jurkat cell activation and proliferation. Lack of MUC1 in Jurkat cells adversely affects the mitogen-activated protein kinase (MAPK) pathway and the Ca2+ release pathway, which ultimately control downstream proliferation and cytokine production.


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MATERIALS AND METHODS
 
Jurkat cell activation
Jurkat cells (human T cell lymphoma) were maintained in RPMI-1640 media containing 10% fetal calf serum (FCS), 1% glutamax, and 1% penicillin and streptomycin. The cells were stimulated in vitro at 37°C with purified, plate-bound, anti-human CD3 antibody (1 µg/106 cells, clone UCHT1, BD PharMingen, San Diego, CA) for 5 and 15 min and 3, 6, and 24 h. Results following 15 min of stimulation were similar to the 5-min stimulation.

Antibodies used for detection of MUC1
Antibodies used include HMPV: MUC1 TR mouse monoclonal antibody (mAb) conjugated to fluorescein isothiocyanate (FITC; minimum epitope, APDTR, BD PharMingen) [28 ]; BC2: mouse mAb (minimum epitope, APDTR, provided by Dr. Michael McGuckin, Mater Medical Research Center, Australia); human milk fat globule-2 (HMFG-2): mouse mAb (minimum epitope, PDTRP, provided by Dr. Joyce Taylor-Papadimitriou, Imperial Cancer Research Fund, London, UK); and CT2: hamster mAb, made in our laboratory and directed against the last 17 amino acids of the CT.

Human T cells
The Mayo Clinic Institutional Review Board (Scottsdale, AZ) approved the research study. All healthy, age-matched volunteers signed informed consents for peripheral blood. Normal donors ranged from 35 to 55 years of age. Whole blood was obtained from the study subjects, and peripheral blood mononuclear cells were separated using a Ficoll-Paque density gradient centrifugation. Double-staining was performed using CD3 antibody conjugated to phycoerythrin (PE), and MUC1 TR mouse mAb conjugated to FITC. For lysates, buffy coat cells (1x107 cells) were incubated with 50 µl Miltenyi-CD3 microbeads (at 4°C for 20 min, Miltenyi Biotec, Auburn, CA) and positively sorted by magnetic activated cell sorting using the Mitenyi RS-columns and Vario MACs magnet as per the manufacturer’s recommendation. T cells were maintained in RPMI-1640 media containing 10% FCS, 1% glutamax, and 1% penicillin and streptomycin. The cells were stimulated in vitro at 37°C with purified, plate-bound, anti-human CD3 antibody and anti-human CD28 antibody (1 µg/106 cells, BD PharMingen) for 15 min.

Flow cytometric analysis
MUC1 expression was determined by flow cytometry using two mAb to MUC1, one detecting the extracellular TR domain (HMPV) and one detecting the CT domain (CT2 antibody). CD69 antibody was from BD PharMingen. Intracellular interleukin (IL)-2 levels were determined using the BD PharMingen protocol (BD Biosciences, San Jose, CA). Flow cytometric analysis was performed on the Becton Dickinson FACScan using the CellQuest program (Becton Dickinson, Mountain View, CA).

Immunoprecipitation and Western blot analysis
Lysates from Jurkat cells were prepared and immunoprecipitated with antibodies to MUC1 (CT2, BC2) and lck followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% resolving gels. Immunoprecipitations were performed on 1 mg of the protein. Gels were probed for CT2, BC2, lck (clone 3A5, Santa Cruz Biotechnology, Santa Cruz, CA), and phosphotyrosine (clone RC20, Transduction Laboratories, Lexington, KY). Lysates were made from 0.5–5 x 108 cells in cold buffer containing 20 mM HEPES, pH 8.0, 0.15 M NaCl, and 1% Triton X-100, supplemented with 80 µl/ml phosphatase inhibitor cocktail II (P-5726, Sigma Chemical Co., St. Louis, MO) and 10 µl/ml complete protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN). In brief, cells were incubated in lysis buffer for 15 min on ice and sonicated with Branson microtip, power 4, 3–10 x 10 s bursts (at 20%). Lysates were centrifuged (5000 rpm for 10 min in cold) to remove debris, and supernatants were collected, aliquoted, and stored in a –70°C freezer until further use. Other antibodies for Western blot analysis include LAT (Santa Cruz Biotechnology), ERK1/2 (Cell Signaling, Beverly, MA), and phosphorylated ERK1/2 (pERK1/2; Sigma Chemical Co.).

Purification of detergent-insoluble fractions
Lipid raft fractions were prepared as described by Zhang et al. [29 ]. Briefly, cells (5x107) were lysed in 1% Triton-X 100 in (25 mM 2-morpholinoethanesulfonic acid, pH 6.5, 150 mM NaCl, 5 mM EDTA) + phosphatase and protease inhibitors (MNE buffer), dounced 40 times in a manual homogenizer, and centrifuged at low speed (1000 rpm for 3 min) to remove nuclear fraction. Supernatant was subjected to sucrose gradient centrifugation [80% sucrose+1 ml lysate (mixed together), 30% sucrose in MNE buffer, 5% sucrose in MNE] in 11 x 60 mm polyallomer tubes (Beckman Instruments Inc., Palo Alto, CA) and ultracentrifuged in a Beckmann SW60 rotor for 18 h at 35,000 rpm at 4°C. Twelve fractions (~350 µl each) were collected from the top of the gradient such that the lower density fractions were at the top, and the higher density fractions were at the bottom and subjected to Western blot analysis.

Small interfering RNA (siRNA) treatment of Jurkats
To generate Jurkat cells that lack MUC1, we used siRNA oligos targeting MUC1 (Dharmacon Research, Lafayette, CO). Two 21-nucleotide siRNA sequences targeting MUC1 were selected from position 882–902 (oligo 1 sequence, 5'–3' ACC UCC AGU UUA AUU CCU C) and position 956–976 (oligo 2 sequence, 5'–3' AUG UUU UUG CAG AUU UAU A) relative to the start codon [30 ]. SiRNA, targeting luciferase as a negative control (sequence, CGU ACG CGG AAU ACU UCG A) as well as oligofectamine alone, was used in control transfections for all experiments. Cells were treated with MUC1- or luciferase-specific siRNA oligos using oligofectamine (Invitrogen, Carlsbad, CA) as described by the manufacturer of siRNA (Dharmacon Inc.). Briefly, cells were transfected with 75 µM siRNA oligo in serum-free Opti-MEM and allowed to remain in siRNA containing media for 48 h prior to harvesting and preparation of lysates.

Determination of free intracellular calcium (Ca2+)
We used the Ca2+ flux fluorometric assay as described previously [31 , 32 ]. In brief, 48 h post-siRNA transfection, Jurkat cells were loaded with 5 µM Fura-2AM (Molecular Probes, Eugene, OR) for 20 min in a 37°C water bath. Changes in intracellular calcium were monitored using the FluoroMax-3 spectrofluorometer (Spex, Edison, NJ). Once stable, basal intracellular Ca2+ levels were reached (~100 s), cells were stimulated with anti-CD3 antibody (1 µg/106 cells) in solution, and fluorescence was quantified. Emission was determined at 520 nm after excitation at 340 and 380 nm, and calcium concentration was calculated from the ratio of the two intensities [33 ]. The peak intracellular calcium concentration post-anti-CD3 antibody stimulation was used to establish the difference among the oligofectamine-transfected cells, nonspecific luciferase siRNA-transfected cells, and MUC1-specific siRNA-transfected cells.

Proliferation assay
Forty-eight hours post-siRNA transfection, cells were incubated for 24 h with or without anti-CD3 antibody and 3H-thymidine added 16 h prior to harvest. Cells were harvested using the Packard cell harvester, and incorporated thymidine was evaluated using the Topcount micro-scintillation counter (Packard Biosciences, Shelton, CT).

Statistics
One-way ANOVA with the Dunnet adjustment was used to assess the difference between the vehicle group and the MUC1 siRNA and luciferase siRNA groups. P values less than 0.05 are considered significant. Confidence intervals (95%) were generated around the difference in group means.


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RESULTS
 
MUC1 is expressed in Jurkat cells and normal human T cells and is phosphorylated upon activation
MUC1 TR and MUC1 CT were detected in 50–70% of Jurkat cells using flow cytometry with fluorescence intensity of ~200 in unstimulated and stimulated cells (Fig. 1A ). Percent Jurkat cells expressing MUC1 increased to ~90% by 24 h using MUC1 TR (HMPV FITC) and MUC1 CT antibodies (Fig. 1A) . A similar increase has been reported in normal human T cells post-phytohemagglutinin stimulation [12 ]. These results were confirmed with other MUC1 TR antibodies including B27.29, HMFG-2, and BC2 (data not shown). Western blot analysis confirmed the flow cytometry data, using BC2 to detect the MUC1 TR domain (>220 kD) and CT2 to detect the MUC1 CT domain (~26 kD; Fig. 1B ). MUC1 is expressed as a heterodimer on the cell surface, which separates into a large, extracellular domain (>220 kD) and a short, hydrophobic TM and cytoplasmic domain of ~26 kD upon SDS-PAGE. Although little difference in MUC1 protein levels was observed by Western blot between unstimulated and stimulated cells (5 min stimulation, Fig. 1B ), there was increased phosphorylation of MUC1 CT upon activation by TCR ligation (Fig. 1C , left panel). To detect MUC1 phosphorylation, cell lysates were immunoprecipitated using CT2 antibody and probed with antiphosphotyrosine and CT2 antibodies. To confirm that Jurkat cells were activated post-TCR ligation, lysates from unstimulated and stimulated cells were analyzed for lck phosphorylation. In line with previous reports [34 , 35 ], activation of Jurkat cells led to an induction of lck phosphorylation (Fig. 1D) . MUC1 staining was also detected using MUC1 TR antibodies (HMPV and BC2) in patients with T cell lymphomas; three out of five patients showed positivity by immunohistochemistry (data not shown). Furthermore, other human T cell leukemia/lymphoma cell lines, including MOLT-4, HH, Loucy, and HUT78, expressed MUC1 by flow cytometry using MUC1 TR (HMPV) antibody (data not shown).



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  		Figure 1. MUC1 is expressed in unstimulated and stimulated Jurkat cells and is phosphorylated in stimulated cells. (A) Flow cytometric analysis of Jurkat cells, stained with MUC1 TR (HMPV) or MUC1 CT (CT2) antibody to detect MUC1 expression, was performed at 0 (unstimulated), 5 min, and 3, 6, and 24 h. Solid lines represent isotype controls, and stained cells are represented by different shades of gray histograms. (B) Western blot (IB) analysis of unstimulated (0') and stimulated (5') Jurkat cells. Protein (100 ug) was loaded per lane and probed for MUC1 TR (BC2, ~220 kD) and MUC1 CT (CT2, ~26 kD). (C) Immunoprecipitation (IP) and Western blot analysis of unstimulated (0') and stimulated (5') Jurkat cells for MUC1 CT and phospho-MUC1 (pTyr). IgG, Immunoglobulin G. (D) Western blot analysis of unstimulated (0') and stimulated (5') Jurkat cells for Lck and phosphor-lck (p Lck) using the lck antibody, which detects phosphorylated and nonphosphorylated lck equally well. Experiments were repeated five times with similar results.

In addition, MUC1 was detected in ~55% (11/20) of normal, unstimulated donor T cells in varying levels (Fig. 2A 2B 2C 2D 2E ; five representative T cell data are shown). Unstimulated T cells from donor B had no detectable MUC1. However, all normal T cells that were stimulated by TCR ligation or polyclonal activation for 24 h showed detectable levels of MUC1 by flow cytometry (data not shown). Flow cytometric data were further confirmed with Western blot analysis using CT2 antibody (Fig. 2F) . Similar to Jurkat cells, MUC1 CT was found to be phosphorylated upon TCR-mediated activation (Fig. 2F) .



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  		Figure 2. MUC1 is expressed in normal human T cells and is phosphorylated upon TCR ligation. (A–E) Flow cytometric analysis of five normal, unactivated human T cells stained with MUC1 TR (HMFG-2) antibody to detect MUC1 expression. Differential MUC1 TR staining pattern was observed. Solid lines represent isotype controls, and stained cells are represented by shaded histograms. (F) Immunoprecipitation and Western blot analysis of unstimulated (0') and stimulated (15') T cells from donor E are shown for MUC1 and phosphoMUC1. Similar results were obtained from donors C and D.

MUC1 CT associates with lck, a T cell-specific Src tyrosine kinase
To determine if MUC1 associates with the Src-related PTKs specific for T cells, lysates derived from unactivated and activated Jurkat cells were immunoprecipitated with MUC1 CT antibody (CT2), and immunoprecipitates were analyzed for lck kinase. The results demonstrate that lck coimmunoprecipitates with MUC1 in unactivated and activated cells (Fig. 3A ). Furthermore, MUC1 associates with the activated and unactivated forms of lck in the stimulated cells (Fig. 3A) . In the reciprocal experiment, analysis of anti-lck immunoprecipitates by immunoblotting with anti-MUC1 CT confirmed the association of MUC1 with lck kinase (Fig. 3B) . Similar MUC1-lck interactions were detected by coimmunoprecipitation in normal human T cells (Fig. 3C and 3D) , suggesting that the interactions may be physiologically relevant and are not an artifact of the Jurkat T lymphoma cell line. The apparent increase of MUC1 in the 15-min coimmunoprecipitation (Fig. 3D) is an artifact from scanning the smeary band of a heavily glycosylated protein. Three normal T cells were analyzed with similar results. Immunoprecipitation using the CT2 antibody pulls down MUC1 TR (data not shown) and MUC1 CT.



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  		Figure 3. MUC1 associates with T cell tyrosine kinase, lck. Lysates from unstimulated (0') and stimulated Jurkat cells or normal T cells were immunoprecipitated with antibodies to MUC1 CT (CT2) or Lck, and precipitates were analyzed for (A and C) Lck or (B and D) MUC1 (CT2). Coimmunoprecipitation of MUC1 CT and lck is observed in both directions. Immunoprecipitations from T cells from donor E are shown. Experiments were repeated five times for Jurkat cells and three times for normal T cells with similar results.

MUC1 is associated with the low-density, detergent-insoluble membrane fractions (lipid rafts) with activated LAT and Lck
The identification of phosphorylated MUC1 in Jurkat lymphoma cells and its association with lck led us to examine its accumulation within lipid rafts, where many of the signal transduction events are amplified during T cell activation. Membrane proteins from unstimulated and stimulated Jurkat cells were separated by sucrose gradient ultracentrifugation, and the first 12 fractions were analyzed by Western blot analysis for MUC1, lck, and LAT, where LAT and lck were used as markers for the T cell activation [29 ]. In unstimulated Jurkat cells, lck was detected in fractions 7–12, LAT was in fractions 6–10, and MUC1 CT (Fig. 4A 4B 4C , left panels) and MUC1 TR (data not shown) were found in fractions 4–12. Upon stimulation, lck was phosphorylated and was now predominantly detected in fractions 3–7, with low levels in fractions 8–12 (Fig. 4A , right panels). Similarly, activated LAT was predominantly detected in fractions 3–7, with low levels in fractions 8–11 (Fig. 4B , right panels). However, MUC1 in activated cells was detected only in fractions 4–6 and was not detected in any other fractions (Fig. 4C , right panels). Figure 4 indicates that MUC1 is expressed in the lower density fractions, where signal transducers characteristic of T cells, LAT, and lck are clustered but not restricted.



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  		Figure 4. MUC1 is detected in the low-density, detergent-insoluble membrane fractions. Western blot analysis of the first 12 sucrose gradient fractions for (A) Lck, (B) LAT, and (C) MUC1 CT. Left panels, Fractions from unstimulated cells; right panels, fractions from stimulated cells. Protein (20 µg) was loaded per lane. Experiment was repeated three times with similar results.

Efficient knock-down of MUC1 using siRNA
To further evaluate the functional role of MUC1 in Jurkat cells, we generated cells that lack MUC1 using two different siRNA oligos targeting MUC1. As controls, siRNA-targeting luciferase was used. Both MUC1 siRNA oligos gave similar results with respect to knocking down MUC1. Western blot analysis shows the low level of MUC1 protein expression induced by oligo 1 and oligo 2 at 48 h post-siRNA transfection (Fig. 5A ). Stimulation with immobilized anti-CD3 antibody did not reinstate MUC1 expression in these cells (Fig. 5A) , enabling us to evaluate the downstream effects of TCR ligation in the MUC1 siRNA-treated Jurkat cells. All other experiments were conducted with cells treated with siRNA oligo 1. Flow cytometric analysis confirmed the Western blot analysis and allowed determination of the kinetics of the MUC1 knock-down. Results demonstrate that by 24 h, there is a 50% reduction in MUC1 surface expression (Fig. 5B , upper left), and the most effective (>95%) knock-down is observed at 48 h (Fig. 5B , upper right), where MUC1 expression is nearly undetectable. This effect persisted for 72 h (Fig. 5B , lower left); by 96 h, MUC1 expression reappears (Fig. 5B , lower right).



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  		Figure 5. MUC1-specific siRNA is efficient in knocking down MUC1 protein expression in Jurkat cells. (A) Western blot analysis of MUC1 CT, 48 h post-treatment with MUC1-specific siRNA 1 (oligo 1: 882–902) or siRNA 2 (oligo 2: 956–976). Controls cells were treated with luciferase-specific siRNA (Luc siRNA) or oligofectamine (Vehicle). (B) Flow cytometric analysis of MUC1 TR (HMPV) surface expression at 24, 48, 72, and 96 h post-siRNA treatment. Individual histograms of isotype control-, MUC1 siRNA-, vehicle-, and Luc siRNA-treated cells are shown. Experiments repeated three times with similar results.

Reduced intracellular Ca2+ flux coupled with reduced proliferation and intracellular IL-2 levels in MUC1-deficient Jurkat cells
To determine if MUC1 siRNA-treated Jurkats have defects in TCR signaling, we measured downstream Ca2+ flux and proliferation in MUC1 siRNA and control cells pre- and post-TCR ligation. Results clearly indicate that cells knocked down for MUC1 exhibited reduced increase in intracellular calcium levels upon stimulation with anti-CD3 antibody (Fig. 6A ; 500 nM in luciferase siRNA-transfected and oligofectamine-treated wild-type cells vs. 329 nM in MUC1 siRNA-treated cells). Figure 6B shows reduced Ca2+ release as a percent of oligofectamine control cells in MUC1 siRNA-treated cells and luciferase siRNA-treated cells (P=0.01, Student’s two-tailed t-test). We next evaluated proliferation, expression of CD69, and IL-2 production in MUC1 siRNA-treated cells as compared with control cells. A significant decrease in proliferation determined by 3H-thymidine uptake was observed in MUC1 siRNA-treated cells following stimulation (Fig. 6C , P<0.001). This profound reduction in proliferation was accompanied by a significantly lower percentage of cells expressing CD69 surface receptor (Fig. 6E , 18% in control cells vs. 2% in MUC1 siRNA-treated cells, P<0.01). Surface expression of the CD69 receptor is a known, endogenous marker of Ras activation, which is critical for proliferation of Jurkat cells [36 ]. As transcription of the IL-2 gene is dependent on intracellular calcium and ras activation [36 ], we next analyzed IL-2 production in MUC1 siRNA-treated cells. Consistent with the results observed with CD69, we found significant reduction in intracellular IL-2 levels in MUC1 siRNA-treated cells (Fig. 6F , 76% in control cells vs. 3% in MUC1 siRNA-treated cells, P<0.001). These effects were not a result of cell death or apoptosis (data not shown). At 96 h post-transfection, when MUC1 protein reappears in the Jurkat cells, the altered phenotype is completely restored to that observed in wild-type Jurkat cells (data not shown), further confirming the specificity of MUC1 knock-down.



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  		Figure 6. (A) MUC1 regulates TCR-induced calcium fluxes. Representative graph of Ca2+ release post-anti-CD3 activation in MUC1 siRNA-, luciferase siRNA-, and vehicle-treated cells. Forty-eight hours post-siRNA transfection, Jurkat cells were loaded with Fura-2AM, and intracellular calcium was monitored before and after stimulation using a spectrofluorometer. The ratio of bound Fura-2AM/free Fura-2AM is shown on the representative graph. The arrow corresponds to the time at which the anti-CD3 antibody was added. Cells were observed for 250 s. (B) Ca2+ release as percent of vehicle following anti-CD3 activation in MUC1 siRNA- and Luc siRNA-treated Jurkat cells (n=3 experiments; P=0.01 using the Student’s two-tail t-test). (C) MUC1 regulates TCR-induced proliferation. 3H-Thymidine uptake assay of vehicle-, MUC1 siRNA-, and luc siRNA-treated cells unstimulated (open bars) or stimulated (solid bars). A significant decrease in proliferation was observed in MUC1 siRNA-treated cells following stimulation (P<0.001). (D) A representative profile of forward- (FSC) and side-scatter (SSC) of Jurkat cells. (E) MUC1 regulates expression of early activation marker CD69. Flow cytometric analysis of CD69 surface expression in vehicle-, MUC1 siRNA-, and luc siRNA-treated cells, which were unstimulated (open histograms) or stimulated for 48 h (shaded histograms). (F) MUC1 regulates intracellular IL-2 levels. Flow cytometric analysis of intracellular IL-2 in vehicle-, MUC1 siRNA-, and luc siRNA-treated cells, which were unstimulated (open histograms) or stimulated for 48 h (shaded histograms). A significant decrease in CD69 (P<0.01) and intracellular IL-2 (P<0.001) was observed in MUC1 siRNA-treated cells compared with vehicle-treated cells following 48 h stimulation.

Decreased phosphorylation of ERK1/2 MAPK in MUC1-deficient Jurkat cells
As an additional indicator of T cell activation, we examined the inducible phosphorylation of ERK1/2 MAPK, a serine/threonine kinase whose activation in T cells is critical for proliferation and cytokine production. Since MUC1 is an oncogene, which impacts tumor growth and metastasis through activation of the Ras/MAPK pathway [4 , 5 , 9 , 10 ], we compared the phosphorylation status of ERK1 and ERK2 in MUC1 siRNA-treated cells. Cells treated with MUC1 siRNA show negligible levels of pERK1 (p44) and ERK2 (p42) in unstimulated cells (Fig. 7 , upper panel). Even upon TCR-mediated activation, phosphorylation of ERK2 remained low in the MUC1 siRNA-treated cells (Fig. 7) , clearly suggestive of defective MAPK activation. Unlike ERK2, ERK1 phosphorylation showed normal levels of phosphorylation upon activation (Fig. 7) . Low phosphorylation level was not a result of lower protein expression, as expression of ERK1/2 protein was similar in control and MUC1 siRNA-treated cells (Fig. 7 , lower panel). Thus, MUC1 influences the Ras/MAPK pathway directly or indirectly in Jurkat cells, which modulates cell proliferation, CD69 expression, and IL-2 production.



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  		Figure 7. MUC1 regulates ERK1/2 phosphorylation. Western blot analysis of ERK1/2 protein (lower panel) and pERK1/2 (upper panel) of MUC1 siRNA, Luc siRNA, and vehicle-treated cells. All experiments were repeated three times with similar results.


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DISCUSSION
 
We demonstrate for the first time the potential role of MUC1 in signal transduction events during Jurkat lymphoma cell and normal T cell activation and proliferation. We show that MUC1 is phosphorylated in Jurkat T cells and normal human T cells upon TCR ligation and associates with lck in coimmunoprecipitation experiments (Figs. 1 2 3) . In addition, MUC1 localizes with lck and LAT in lipid rafts, further suggesting a role for MUC1 during T cell activation (Fig. 4) . It is important that upon down-regulation of MUC1 (Fig. 5) , Jurkat cells undergo several physiological alterations including reduction in Ca2+ influx followed by subsequent reduction in MAPK phosphorylation, especially of ERK2 (Figs. 6A and 6B and 7) . Along with alterations in the proximal signals, we observed reduced CD69 surface expression, reduced proliferation, and reduced IL-2 production (Fig. 6C 6E and 6F) .

MUC1 is recognized as an important tumor antigen in epithelial and hematopoietic malignancies including T cell lymphomas. We show for the first time that in a human T cell lymphoma line, MUC1 plays a critical role in activation and proliferation, and absence of MUC1 may be associated with marked reduction in proliferation upon TCR ligation. Reduced proliferation correlates with reduced Ca2+ influx and reduced ERK phosphorylation, events that are critical in the progression of cancer. In the absence of MUC1, activation of ERK2 was almost completely blocked, even upon stimulation, linking MUC1 activation to the Ras/MAPK signaling pathway. As the sequence that binds the c-Src tyrosine kinase has been identified as the YEKV motif in the CT of MUC1 [22 ], it is plausible that lck binds and phosphorylates MUC1 at the same site in activated Jurkat cells. At the same time as our studies were under way to determine phosphorylation by lck and to map the site on MUC1 CT, it was reported that lck phosphorylates MUC1 CT on the YEKV motif, which in turn stimulated the binding of MUC1 to ß-catenin in 293 cells (ref. [23 ] and unpublished data). These results confirm the interaction of MUC1 with lck kinase and suggest integration with the ß-catenin pathway.

One possibility for the downstream signaling defects in MUC1-deficient Jurkat cells is that MUC1 may be involved in the transport/localization of lck to the TCR signaling complex, where activated LAT forms the platform to recruit other signal transduction molecules. This is probably mediated through interaction of MUC1 with activated lck, presumably at the SH2 domain (Fig. 3) . The suggestion that MUC1 may be involved in lck localization is exemplified in Figure 4 (right panels), where upon TCR-mediated stimulation, MUC1 is detected in the same low-density membrane fractions as phosphorylated lck and LAT. Thus, we speculate that the signaling defect may be a result of the loss of recruitment of lck to the TCR signaling complex, where downstream signaling is amplified. Studies are under way to evaluate more definitively the role of MUC1 in localization of lck to the appropriate signaling compartment in specific T cells including T cells that lack lck. In our preliminary data, we have observed that MUC1 coprecipitates with LAT (unpublished data). Whether the interaction or binding is direct is not yet clear; however, the functional significance of the interaction may be profound with regard to TCR-mediated activation, as data suggest profound downstream, functional impairment in the absence of MUC1 (Fig. 6) . As ZAP-70 phosphorylates LAT, we have determined that MUC1 (CD227) coprecipitates with ZAP-70 (unpublished data and ref. [23 ]) and therefore, may form a complex with LAT during TCR-mediated activation.

In the absence of MUC1, ERK1/2 phosphorylation is reduced significantly in resting Jurkat cells (Fig. 7) . Upon TCR-mediated activation, ERK1 becomes phosphorylated, but ERK2 remains largely nonphosphorylated, leading to a defect in cell-cycle progression of Jurkat cells in the absence of MUC1. It has been shown previously that phosphorylated MUC1 CT leads to activation of a MAPK pathway through the Ras-MAPK kinase (MEK)-ERK2 pathway in some carcinomas [4 , 9 , 10 , 37 ]. One of the suggested roles of MUC1 may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2/Sos-Ras-MEK-ERK2 pathway. Thus, we suggest that the decrease in proliferation of MUC1-deficient Jurkat cells may be a result of a direct effect of MUC1 on the Ras-MEK-ERK2 pathway or an indirect effect on proximal signaling via altering lck phosphorylation and relocation or by both. Eventually, this defect in activation of the Ras-MEK-ERK2 pathway may lead to reduced expression of CD69 and production of IL-2. The data, however, do not indicate that MUC1 is absolutely required for lymphoma cells to proliferate, as siRNA knock-down of MUC1 did not completely block proliferation but instead, inhibited anti-CD3-induced proliferation of these cells (Fig. 6B) , suggesting that MUC1 may not function as an oncogene in these particular cells, as has been recently reported in the literature for epithelial cell carcinomas [4 , 5 ]. Whether overexpressing MUC1 in Jurkat cells may cause increased proliferation without CD3 activation is not yet evaluated and is an area of active investigation.

Although Ca2+ influx and proliferation are affected when MUC1 is knocked down, we observed no change in levels of PLC{gamma}1 phosphorylation (data not shown). Although it is possible that PLC{gamma}1 is involved in mitogenic signaling under certain circumstances, studies have demonstrated that in carcinoma cells, mitogenesis and proliferation can occur despite blockade of PLC{gamma}1 signaling [38 39 40 ]. Thus, other alternative pathways may be involved in Ca2+ flux and proliferation in Jurkat cells, which are not dependent on PLC{gamma}1 activation.

The physiological relevance of MUC1 expression in the low-density membrane fractions (Fig. 4) is unknown at this time, but it has been described previously in another human T cell line, HUT78 [24 ]. In this cell line and in the EL4 mouse thymoma cell line, MUC1 and P-selectin glycoprotein ligand-1 (PSGL-1) were enriched in the low-density fraction [glycosylation-enriched microdomain (GEM)] together with glycosphingolipids (GSL), CD45, Yes, fyn, and lck. The authors suggested that MUC1 and PSGL-1 make up another glycosyl cluster (glycocluster) in addition to the previously well-established GSL cluster, which is organized with signal transducer molecules in the GEM fraction. Components of the glycocluster complex with signal transducers in the GEM are neither invaginations nor moving platforms but rather rigid adhesion sites ("dock" rather than "raft") on cells from which phenotypic changes can be initiated. It is suggested that MUC1 associates with the GEM as a result of its interaction with cholesterol, presumably through its hydrophobic TM region. Thus, we speculate that the MUC1-associated glycocluster, like GSLs, is organized with the cytoplasmic signal transducers such as the src kinase lck and LAT. Ligands binding GSLs initiate signaling through these signal transducers, affecting the downstream MAPK pathway and leading to phenotypic changes in Jurkat T lymphoma cells [41 , 42 ]. Thus, enrichment of MUC1 in the low-density membrane fractions may suggest a functional role for this protein in recruiting various signal transducers in T cells. As is documented, glycoclusters in leukocytes mediate adhesion, and this adhesion is highly dependent on O-glycosylation [43 ]. Thus, O-linked glycoclusters consisting of MUC1 and PSGL-1 in lymphocyte microdomains may play crucial roles in cell adhesion coupled with signaling. Preliminary data suggest that MUC1 colocalizes with caveolin-1 and GM1 in anti-CD3-activated Jurkat cells by confocal microscopy (data not shown); however, further studies will address the localization of MUC1 in lipid rafts in T cells more specifically and its potential function in the immunologic synapse.

In summary, a role for MUC1 in signal transduction in normal T cells and Jurkat T lymphoma cells is proposed. MUC1 appears to be important in TCR-mediated activation, as evidenced by the inhibition of T cell function when its expression is down-regulated by siRNA. Binding to MUC1 may cause recruitment of signaling molecules such as lck kinase to the plasma membrane, resulting in molecules in correct proximity for propagation of signals. Through such a mechanism, MUC1 could bridge TCR activation to downstream signaling events, in particular, intracellular calcium flux, proliferation, and productive Ras activation, leading to downstream events such as expression of CD69 and production of the cytokine IL-2. Extensive studies are under way to understand the interaction of MUC1 with multiple biochemical pathways in T cell activation mediated through the TCR.


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ACKNOWLEDGEMENTS
 
This work was funded by National Institutes of Health CA64389 (S. J. G.) and Mayo Foundation. We are grateful to Dr. Michael McGuckin, Mater Medical Research Center, Australia, for the BC2 monoclonal antibody; Biomira, Inc., Edmonton, Alberta, Canada, for the B27.29 antibody; and Dr. Joyce Taylor-Papadimitriou, Cancer Research, UK, for the HMFG-2 antibody. We thank Dr. Kaleeckal Harikumar for assistance with the calcium influx assay. We also thank Jim Tarara for helping with the confocal studies and flow cytometry, Marvin H. Ruona for help with the graphic production, and Carol Williams for preparation of the manuscript. We thank Dr. Kandavel Shanmugam for critical comments on the manuscript.

Received June 9, 2004; revised October 4, 2004; accepted October 7, 2004.


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