Reports |
Address correspondence to Brian Seed, Dept. of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. Tel.: (617) 726-5975. Fax: (617) 726-5962. email: seed{at}molbio.mgh.harvard.edu
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Abstract |
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Key Words: Discs large; scaffold protein; PDZ domains; lymphocyte activation
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Introduction |
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Molecules that promote and regulate the association of membrane signaling with the cytoskeleton are expected to have, at minimum, a mechanism for association with the signaling molecule and/or membrane, as well as a mechanism for association with the cytoskeleton. Because such molecules are expected to coordinate the movement and regulation of large signaling assemblies, they would likely behave as molecular scaffolds and have mechanisms for interacting with one another as well as with signaling proteins.
Among candidate molecules with similar functions outside the immune system, the members of a class of proteins called PDZ proteins are of particular interest (Montgomery et al., 2004). The PDZ domains of these proteins form at least two kinds of proteinprotein contact (Songyang et al., 1997). The best studied is an interaction with the carboxy-terminal residues of proteins that terminate in a variably conserved sequence of form S/T-X-V or Y/F-X-V although considerable latitude is observed. PDZ domains also appear to be able to interact with internal residues on some proteins, including PDZ domains themselves. Thus, the PDZ domain itself has the attributes expected of a scaffolding structure supporting heterotypic and homotypic interactions.
Recent studies have described a role for membrane-associated guanylate kinases (MAGUKs) in T cell activation. Compromise of expression of the MAGUK protein, CARMA-1, by germline targeting, somatic mutation, or RNA interference inhibits antigen receptor mediated NF-B activation (Jun and Goodnow, 2003; Thome, 2004).
The human homologue of Drosophila Discs large (Dlg1) is a MAGUK found in postsynaptic densities in the central nervous system (Muller et al., 1995). Dlg1 has been implicated in the formation of tight junctions, in epithelial cell polarity, and in the control of proliferation of Drosophila imaginal discs. It consists of an amino-terminal proline-rich domain, multiple PDZ domains, an SH3 domain, HOOK domain, i3 domain, and a guanylate kinase (GK)like domain.
We present evidence that Dlg1 localizes with the actin cytoskeleton in T cells, associates with early participants in the signaling process and functions as a negative regulator of T cell activation.
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Results and discussion |
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The i3 domain of Dlg1 is thought to interact with ezrin-radixin-moesin family proteins, which couple membrane proteins to the actin skeleton (Lue et al., 1996; Wu et al., 1998); in T cells ezrin associates with the immunological synapse (Roumier et al., 2001). We analyzed the distribution of actin and Dlg1 in Jurkat cells allowed to settle on coverslips coated with anti-CD3 antibody OKT3 (Bunnell et al., 2001). As described previously (Bunnell et al., 2001), dramatic reorganization of the cellular architecture follows contact with the OKT3-coated surface, with prominent emergence of microspikes, filopodia, and lamellipodia. Fig. 1 A demonstrates that 2 min after contact endogenous Dlg1 was predominantly cytosolic, but by 5 min it had become codistributed with the cortical actin and lamellipodial structures. A more prominent concentric band of cortical actin was induced in Jurkat cells on adhering to immobilized anti-CD3 and anti-CD28.
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To study recruitment of Dlg1 to the T cell antigen-presenting cell (APC) interface we performed immunolocalization studies (Roumier et al., 2001; Lee et al., 2002) after stimulation with superantigen or antigenic peptide. Jurkat cells were incubated with Raji cells pulsed with staphylococcal enterotoxin E (SEE) and the distribution of actin and Dlg1 was analyzed by immunofluorescence microscopy. In the absence of SEE, Dlg1 and actin were evenly distributed in Jurkat cells, whereas in the presence of SEE, Dlg1 and actin were concentrated at the cell contact interface at 5 and 10 min (Fig. 2 A; not depicted). Within 5 min of exposure to SEE, TCR can be seen to translocate to the contact zone and colocalize with Dlg1 (Fig. 2 B). Dlg1 colocalization at the RajiJurkat interface was seen to diminish after prolonged association of the presenting and responding cells.
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To better understand the association patterns of Dlg1 with other molecules, we immunoprecipitated Dlg1 from the cytosolic and membrane fractions of Jurkat T cells that had been exposed to agonistic antibody (anti-CD3 and anti-CD28) stimulation. We first confirmed that Dlg1 specifically interacts with Lck in Jurkat cells (Hanada et al., 1997). We next showed that endogenous Dlg1 specifically affiliates with the signaling molecules TCR and Cbl. The TCR
species complexed with Dlg1 contains both phosphorylated and nonphosphorylated components, and the phosphorylation status depends on the activities of Lck and Zap-70 (Fig. 3, A and B; not depicted).
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The interpretation that Dlg1 acts as an activation antagonist is consistent with findings from other systems. The phenotype that originally led to the discovery of Dlg1, grossly enlarged imaginal disks in Drosophila, suggested a role for the protein in restraint of imaginal disk cell proliferation, and Dlg1 has been considered to be a kind of tumor suppressor in Drosophila (Dimitratos et al., 1999). The connection with the cortical actin skeleton suggests that one important role of Dlg1 may be to promote receptor internalization and/or recycling after the initial phase of contact and engagement.
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Materials and methods |
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Expression constructs
Construction of rat Dlg1-GFP and deletion constructs have been described (as SAP97-GFP) elsewhere (Wu et al., 1998). These constructs were provided by C. Garner (Stanford University, Palo Alto, CA). For reporter assays carboxy-terminal GFP tag sequences were introduced in-frame with full-length SAP97 and carboxy-terminal deletion constructs consisting of residues, 1909, 219906 (PDZ2-3-SH3-I3-GK), 406906 (PDZ3-SH3-I3-GK), and 540906 (SH3-I3-GK). Human Dlg1 was isolated by PCR amplification from a Raji cell library and inserted between HindIII and NotI sites downstream of an EF1 promoter.
Immunoprecipitations
107 Jurkat cells were stimulated with 2 µg/ml of anti-CD3 antibody (OKT3) and 2 µg/ml of anti-CD28 (clone 9.3), washed twice in 1-ml of ice cold PBS and resuspended in 750 µl of hypotonic lysis buffer (20 mM Tris HCl, pH 7.5, 10 mM NaCl, 2 mM EDTA, 5 mM EGTA, 10 mM ß-ME, one protease inhibitor cocktail tablet (Roche) per 10 ml, 1 mM Na3VO4, 20 mM NaF), transferred to a 1-ml syringe, and sheared by passing 30 times through a 25-gauge needle. The lysates were centrifuged at 1,000 rpm for 7 min to precipitate nuclei and the supernatant was collected. 50 µl of the total cell extract was saved, and the remainder was centrifuged at 16,000 rpm for 20 min. The supernatant was collected and the pellet resuspended in lysis buffer with 1% NP-40 for 30 min with gentle vortexing every 5 min on ice. The supernatant and membrane fractions were immunoprecipitated for 4 h at 4°C with Dlg1 (Santa Cruz Biotechnology, Inc.) specific antisera. Isotype controls were performed for each immunoprecipitation. Immune complexes were collected with goat antimouse beads (Amersham Biosciences) and washed three times with lysis buffer. Bound proteins were eluted with 50 µl SDS sample buffer and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes, which were blocked overnight and probed with indicated antibody. After washing, filters were incubated with a 1:5,000 dilution of antimouse or antirabbit HRP conjugate (DakoCytomation) in TBST for 1 h. Protein bands were detected by ECL (Amersham Biosciences).
Luciferase assays
Determination of NFAT activities was performed as described previously (Wu et al., 1995; Roumier et al., 2001). In brief, 5 x 106 Jurkat cells were sedimented, resuspended in 0.25 ml IMDM, and electroporated (250 V/960 µF) with 10 µg of NFATx3-luciferase reporter construct, 10 µg of full-length or fragments of Dlg1-GFP or1 µg pcDNA3-Vav1, and 1 µg pEAK12-GFP. Upon electroporation, cells were diluted in 2 ml IMDM, placed in wells of a 6-well plate, and incubated for 16 h. 0.5-ml aliquots were then transferred to wells of a 24-well plate and exposed to 5 µg/ml OKT3. Alternatively, Raji cells pulsed with SEE for 30 min were incubated with T cells overexpressing or knockdown of Dlg1 for 6 h at 37°C. Luciferase assays were performed according to the manufacturer's instructions (Promega). The electroporation efficiency was normalized by assessing GFP expression by flow cytometry. Expression of various constructs was confirmed by immunoblots.
RNA interference
The vector pSuppress consisting of the RNA polymerase IIIdependent H1-RNA gene promoter was provided by D. Billadeau (Division of Oncology Research, Mayo Clinic, Rochester, MN). Complementary oligonucleotides designed against sequences in the SH3 domain shRNA#1 (5'-GATCCCCgactaaagacagtgggcttTTCAAGAGAaagcccactgtctttagtcTTTTTGGAAA-3' and 5'-TCGATTTCCAAAAAgactaaagacagtgggcttTCTCTTGAAaagcccactgtctttagtcGGG-3') and shRNA#2 designed against sequences in the GK domain (5'-GATCCCCgagagcagatggaaaaagaTTCAAGAGAtctttttccatctgctctcTTTTTGGAAA-3' and 5'-TCGATTTCCAAAAAgagagcagatggaaaaagaTCT-CTTGAAtctttttccatctgctctcGGG-3') were cloned into unique BglIIXhoI site downstream of HI RNA promoter and inserts were confirmed by sequencing. Stable Jurkat cells were generated by transfecting shRNA expressing vector: pEAK12-GFP puromycin plasmid at 10:1 ratio. Stables were expanded for an additional 4 wk before immunoblot analysis for Dlg1 expression and NFAT reporter analysis. Reporter analysis was performed as described above.
Fluorescence microscopy
Jurkat T cells were stimulated with anti-CD3 antibody (OKT3) on coverslips as described previously (Bunnell et al., 2001). Coverslips were coated with 10 µg/ml anti-CD3 antibody and 10 µg/ml anti-CD28 for 2 h at 37°C in a moist chamber. The slides were washed with PBS four times. Spreading assays were initiated by placing 10 µl of a concentrated Jurkat cell suspension on anti-CD3coated coverslips. After incubation at 37°C for 2 or 5 min, the cells were fixed for 10 min with 3.5% PFA and 0.1% Tween-20 in PBS at RT.
Jurkat T cells were stimulated with antibody-coated beads and APCs (Raji cells) pulsed with SEE as described previously (Roumier et al., 2001). In brief, the cells were mixed with beads coated with CD3/CD28 (Dynal) at a ratio of two beads/cell. After centrifugation for 5 min at 100 g, the cellbead mixture was incubated for an additional 515 min at 37°C. Conjugates were then resuspended, plated onto poly-L-lysinecoated slides, and fixed for 10 min as above. To distinguish APCs from Jurkat cells, APCs were loaded with cell tracker CMAC blue (CT blue molecular probes).
T cells were obtained from DO11.10 transgenic mice as described previously (Lee et al., 2002). DO11.10 transgenic T cells were induced with OVA peptide in bulk splenocyte cultures for 56 d, washed extensively and spun over Lympholyte M to remove dead cells and debris. T cells were rested in media supplemented with IL2 for several hours or overnight. To initiate conjugate formation, T cells were centrifuged briefly with LPS-induced B cell blasts (used as APCs) pulsed with 110 µM OVA peptide for 23 h at 37°C and incubated for 1, 2, 5, or 10 min. Conjugates were examined for endogenous Dlg1 recruitment to the immune synapse by immunofluorescence microscopy.
After blocking with PBS including 1% BSA, the cells were labeled with rabbit anti-Dlg1, mouse anti-CD3 antibody in PBS including 1% BSA, followed by three washes of 5 min each. After labeling with secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes), phallotoxin conjugated with Alexa Fluor 594 (Molecular Probes) and DAPI, the slides were mounted with Aqua Poly/Mount (Polyscience). F-actin polymerization was analyzed by immunofluorescence microscopy using an Axioscope (Carl Zeiss MicroImaging, Inc.) microscope, with focus adjusted to the plane of maximum staining intensity. More than 100 cells were analyzed for each condition (either resting or after TCR activation).
Online supplemental material
Fig. S1 is an analysis of Dlg1 expression in B cell developmental subsets by real-time PCR. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200309044/DC1.
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Acknowledgments |
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This work was supported by grants AI27849, AI46731 (to B. Seed), and CCFA, Development Funds Gastroenterology and Surgery (to R.J. Xavier). S. Rabizadeh was supported by Damon Runyon CRF Fellowship DRG-1620.
Submitted: 8 September 2003
Accepted: 27 May 2004
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