1 Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
Correspondence to: E. L. Reinherz; E-mail: ellis_reinherz{at}dfci.harvard.edu
Transmitting editor: S. Koyasu
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Abstract |
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Keywords: c-Cbl, cytoskeletal polarization, intramolecular regulation, RNA splicing, T cell
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Introduction |
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The role of CD2 in T cell motility and polarization was recently revealed using digitized time-lapse differential interference contrast and fluorescence microscopy to follow human T cell interaction on a cellular monolayer substratum consisting of dendritic cells or other APC (14). In this system, it was found that CD2 ligation induced T cell polarization. Such T cell polarization was not observed by cross-linking MHC I or TCR molecules by specific mAb. CD2 also facilitated the scanning movement of T cells along APC surfacesa process which is critically dependent on T cell ß integrin function. The accompanying T cell surface CD2 redistribution results in a 100-fold greater CD2 density in the uropod than the leading edge, compartmentalizing CD2, the TCR and GM-1 lipid rafts, largely in isolation from CD11a/CD18 and CD45. These data in conjunction with earlier findings emphasize how T cell adhesion, migration and immune activation functions mediated by CD2 and ß integrin molecules are distinct (15,16).
While CD2 surface redistribution is CD58 ligand dependent and occurs in T cells expressing tailless CD2 variants (11), other CD2 functions are intimately associated with the 118-amino-acid residue CD2 tail. In this regard, the CD2 intracellular domain contains five proline-rich segments responsible for the direct physical interaction of CD2 with various intracellular proteins. In particular, the protein CD2AP has been shown to be pivotal in T cell polarization and cytoskeletal rearrangements (17), while the two proteins CD2BP1 (18) and CD2BP2 (19) are implicated in cell adhesion and signal transduction respectively. CD2BP1 serves as an adaptor to recruit PTP-PEST to CD2, down-regulating focal adhesion and fostering T cell motility (18,2024). CD2BP2 contains, in lieu of an SH3 domain, a GYF recognition domain that interacts with two tandem CD2 PPPPGHR proline-rich regions proximal to the inner plasma membrane leaflet and enhances IL-2 production upon clustering of CD2 molecules (19,25). The src family tyrosine kinase p59fyn also interacts with this same proline-rich region, functionally linking CD2 signaling to the mitogen-activating protein kinase pathway (26).
The physiologic importance of such CD2 interactor proteins in the regulation of inflammatory responses is amply demonstrated by the recently described naturally occurring mutation of CD2BP1 observed in children suffering from PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum and acne) plus familial recurrent arthritis (27). These recurrent inflammatory disorders are linked to single amino acid substitutions within CD2BP1 which ablate PTP-PEST binding without altering the CD2 interaction site of the CD2BP1 SH3 domain. Such changes likely create a dominant-negative mutation, thereby accounting for the observed autosomal dominant inheritance pattern.
To identify other cytosolic interactor proteins, we have utilized yeast two-hybrid interaction cloning methods. We now identify CD2BP3 found in resting and activated human T lymphocytes as the major variant of the c-Cbl interactor protein CIN85 (28). While CD2BP3 bears a striking architectural similarity to human CMS (29) and the murine orthologue CD2AP (17), its functions are divergent. CD2BP3 is unable to directly interact with F-actin, and manifests a form of intramolecular regulation involving its own SH3 domain and proline-rich segment not observed in CMS. Moreover, CD2BP3 interaction with CD2 is of lower affinity than that of CIN85 and CMS, although all three proteins appear to bind to the same conserved distal proline-rich CD2 tail region. CIN85/CD2BP3 localizes in both the nucleus and cytoplasm, further suggesting potential involvement in the regulation of cytokinesis as well as the cytoskeleton of the cell. Unlike CIN85, however, CD2BP3 shows little capacity to facilitate down-modulation of surface CD2 following receptor ligation or disrupt cytoskeletal polarization. Our findings offer insight into the complexity of CD2 adhesion, motility and cytoskeletal regulation.
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Methods |
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Transient transfection of COS-7 cells and analysis of CD2 interaction
Full-length Flag-tagged CD2BP3 (Flag-FLCD2BP3) was generated by PCR appending a Flag sequence at the N-terminus of the CD2BP3 DNA sequence and cloned in pcDNA1.1. The CMS cDNA cloned in pFLAG-CMV-2 was kindly provided by Dr Kathrin Kirsch (Department of Biochemistry, Boston University, Boston, MA) while the CIN85 sequence cloned in FLAG2-pcDNA3 was kindly provided by Dr Sachiko Kajigaya (Hematology Branch, NHLBI/NIH, Bethesda, MD). Full-length (FL) and truncated () CD2 constructs were generated and cloned in pCDM8 as described in (11). COS-7 cells were seeded at 1 x 106 cells per 100 x 20 mm dish the day before the transfection. The cells were transfected with 20 µg DNA per dish using the calcium phosphate method (32). After 48 h, the cells were harvested, washed in PBS and lysed in 1% Triton X-100 lysis buffer [1% Triton X-100, 0.15 M KCl, 1 x TBS (10 mM Tris, pH 7.4, 0.15 M NaCl) supplemented with 1 mM PMSF and 0.35 trypsin inhibitor unit/ml aprotinin] in the presence of 0.25 mM ZnCl2 for 30 min 4°C at a concentration of 1020 x 106 cells/ml. Subsequently, the lysates were microcentrifuged for 15 min at 13,000 r.p.m. and precleared on CNBr-activated Sepharose 4B beads (Pharmacia Biotech, Piscataway, NJ) covalently coupled with mouse IgG (CNBrIgG, 10 µl beads/ml lysate) (33) overnight at 4°C. The cleared lysate was then immunoprecipitated with CNBrIgG (Control), an anti-CD2 (3T4-8B5, anti-T111) mAb, an anti-CD2BP3 (2C3 or 2B5 as indicated) mAb or an anti-Flag (M2) mAb (Sigma, St Louis, MO). The beads were then incubated 30 min at 65°C in non-reducing sample buffer, pelleted and the supernatants collected. After adding 2-mercaptoethanol and boiling for 4 min, the samples were analyzed by 8% SDSPAGE. Upon transfer of the proteins onto nitrocellulose membrane (Bio-Rad, Hercules, CA), the association with CD2 was evaluated by Western blotting the membrane with an M32B anti-CD2 rabbit polyclonal heteroantisera (34) [diluted 1:1000 in 5% milk in TBS-T (1 x TBS supplemented with 0.05% Tween 20)] for 23 h at room temperature followed by incubation with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (diluted 1:10,000 in 1% milk in TBS-T, 1 h at room temperature) (Santa Cruz Biotechnology, Santa Cruz, CA). The level of Flag-tagged protein was evaluated by Western blotting the membrane with an anti-Flag (M2) mAb (Sigma) (4 µg/ml in 5% milk in TBS-T; 23 h a room temperature) followed by incubation with HRP-conjugated anti-mouse IgG1 (1:5000 dilution) (Santa Cruz Biotechnology). The signal was developed with ECL (Perkin-Elmer Life Science, Gaithersburg, MD).
Generation of recombinant GST-fusion proteins, and analysis of the interaction of the individual SH3 domains with CD2 and c-Cbl in J77 cells
To create GSTSH3 fusion proteins, the cDNA sequences corresponding to the SH3 domains of CMS and CIN85 [CMS SH3A (bp 4180), CMS SH3B (bp 331507), CMS SH3C (bp 798990), CIN85 SH3A (bp 4177), CIN85 SH3B (bp 301483) and CIN85 SH3C (bp 793987)] were generated by PCR and inserted into pGEX-4T-1 (Pharmacia) at BamHI (or EcoRI) and XhoI sites in the polylinker region C-terminal to the GST-binding domain. The authenticity of these and all subsequent PCR products was verified by DNA sequencing. These constructs were used to transform Escherichia coli XL-2 blue and BL21(DE3) (Stratagene, La Jolla, CA) and the fusion proteins were generated by culturing the transformed E. coli under isopropyl-ß-D-thiogalactopyranoside (IPTG) inducing conditions (0.5 mM IPTG for 2 h at 37°C). The fusion proteins and the GST control protein were then purified using glutathioneSepharose 4B beads according to the manufacturers protocol (Pharmacia).
Jurkat J77 human T cells were cultured in RPMI medium 1640 (Gibco, Life Technologies, Rockville, MD) supple mented with 10% FCS (Sigma), 2 mM L-glutamine and penicillin/streptomycin (Gibco), and used to evaluate the binding of the individual GSTSH3 proteins (coupled to glutathioneSepharose beads at 5 mg/ml) to CD2 and c-Cbl. J77 cells were lysed in 1% Triton X-100 lysis buffer for 30 min at 4°C at 5060 x 106 cells/ml. The lysates were then precleared on GSTbeads (10 µl beads/ml lysate) overnight at 4°C. The precleared lysates were incubated for 24 h with GST alone or GSTSH3 beads in the presence of 0.2% BSA to reduce the non-specific binding and washed 3 times with lysis buffer (1 min, 6000 r.p.m.). The precipitates were subjected to 8% SDSPAGE followed by Western blotting, and the bound CD2 and c-Cbl proteins revealed by probing the membrane with M32B anti-CD2 rabbit polyclonal antibody (1:1000 dilution) or with anti-Cbl rabbit polyclonal antibody (1:1000 dilution) respectively, followed by incubation of the membranes with HRP-conjugated anti-rabbit antibody (1:10000 dilution) as described above. The signal was developed by ECL. To verify that the beads were coupled with an equal amount of GST and GST-fusion proteins, 5 µl of coupled beads were boiled in sample buffer and loaded on a 12% SDSPAGE and the gel was subsequently stained in 0.25% Coomassie blue.
Generation of Flag-PCC CIN85 and Flag-PCC CMS constructs, and analysis of the interaction with the GSTSH3 fusion proteins
To generate the Flag-PCC constructs, a Flag sequence was appended by PCR at the N-terminus of the cDNA sequences of CMS (bp 9791917) and CIN85 (bp 9821995), and the DNA fragments were cloned in pcDNA1.1 between BamHI and XhoI sites. COS-7 cells were transfected, lysed in 1% Triton X-100 lysis buffer (in presence or absence of 0.25 mM Zn2+ wherever indicated) and processed as described above. Western blotting was performed probing the nitrocellulose membrane with anti-Flag mAb for 2 h at room temperature. The signal was then developed by ECL.
Analysis of p130Cas association
COS-7 cells were transfected with p130Cas cloned in pEBB (kindly provided by Dr Ravi Salgia, DFCI) (10 µg) together with Flag-CMS, Flag-CIN85 or Flag FLCD2BP3 (10 µg). After 48 h, cells were harvested, lysed in 1% NP-40 buffer [1% NP-40, 20 mM TrisHCl pH8.0, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin] for 10 min in ice (35), and immunoprecipitated with an irrelevant mAb and with anti-Flag mAb. The proteins were separated on SDSPAGE, transferred onto nitrocellulose, and Western blotted first with anti-p130Cas rabbit polyclonal antibody (1:500; Santa Cruz) and then with anti-Flag mAb.
Cytoskeletal polarization
Jurkat cells (107; kindly provided by Dr Andrey Shaw, Washington University, St Louis, MO) were transfected with 20 µg Flag-FLCD2BP3 or Flag-CIN85 by electroporation (250 V). After 24 h, 4 x 106 untransfected or transfected cells were plated on coverslips coated with anti-CD2 (anti-T112 + anti-T113) and incubated for 30 min at 37°C as described in (14). The coverslips were then fixed with 3.7% paraformaldeyde (1 h at 4°C), permeabilized in 0.1% saponin in 1% BSA/PBS (perm solution) (30 min at 4°C) and stained with 0.5 µM BODIPY TR Ceramide (Molecular Probes, Eugene, OR) (1 h at 4°C), mounted on microscope slides and observed under a Nikon Diaphot 300 fluoro-microscope. The efficiency of the transfection (30%) was evaluated by staining the cells with FITC-conjugated M2 anti-Flag mAb (Sigma) and fields with multiple transfectants selected for microscopical analysis.
Control of surface CD2 expression
COS-7 cells were transfected with FLCD2 (10 µg) and either Flag-FLCD2BP3 or Flag-CIN85 (10 µg), and cultured for 48 h. Cells were subsequently left unstimulated or treated with a combination of anti-T112 + anti-T113 mAb (ascites, diluted 1:100) in the absence or presence of epidermal growth factor (EGF; 50 ng/ml; Sigma) for the indicated time at 37°C. Cells were then washed, stained with an FITC-conjugated anti-CD2 (anti-T111) mAb and analyzed by FACS. Total cell lysates of the transfected cells were run in 8% SDSPAGE, separated proteins transferred onto nitrocellulose membrane, and probed with an anti-CD2 polyclonal antibody and an anti-Flag mAb to assess levels of protein expression.
Production and characterization of mouse mAb specific for human CIN85/CD2BP3
2C3/C7/E6 (2C3, IgG1) mAb was raised by immunizing mice with CIN85/CD2BP3 GSTSH3C, whereas all the other anti-CD2BP3 mAb [2B5/F2/C10/C8 (2B5, IgG2b), 10B6/G9/D3/E11 (10B6, IgG2b), 12A6/D11/C11 (12A6, IgG1), 23C9/G6/D8 or/G6/F5 (23C9, IgG2b)] were generated by immunizing mice with a histidine-tagged protein containing the N-terminal portion of CD2BP3 (HisSH3BC). The above HisSH3BC plasmid was generated by PCR, by placing a His tag (6 His) at the N-terminus of a truncated form of CD2BP3 (bp 4876), and inserting the DNA fragment into pET11a (Novagen, Madison, WI) between NheI and BamHI. These constructs were used to transform E. coli BL21(DE3) (Stratagene) and the fusion proteins were generated by culturing the transformed E. coli under IPTG-inducing conditions (0.4 mM IPTG for 3 h at 37°C). Histidine-tagged protein was purified by a Nickel column according to the manufacturers procedure (Pharmacia Biotech). The method of protein immunization, spleen fusion, selection of positive clones and purification of the mAb was performed as described in detail elsewhere (18). All the hybridomas selected were subcloned at least twice by the limiting dilution method. Each mAb was coupled to CNBr-activated Sepharose 4B beads at 5 mg/ml (33) and the ability to immunoprecipitate purified GSTSH3B, GSTSH3C and HisSH3BC proteins was examined. As a negative control, immunoprecipitations were performed using CNBr-beads coupled with an irrelevant mAb (W6/32 anti-MHC class I mAb). In this assay, the presence or absence of Coomassie blue staining fusion proteins on 12% SDSPAGE of immunoprecipitates was the readout. The CNBr-coupled mAb were also used to immunoprecipitate Flag-CMS, Flag-CIN85 and Flag-CD2BP3 from transfected COS-7 cells, and the captured proteins were separated by SDSPAGE and revealed by Western blotting with anti-Flag mAb or with anti-CD2BP3 mAb (ascites, 1:200) followed by incubation with HRP-conjugated anti-mouse IgG mAb
Analysis of CIN85/CD2BP3 and F-actin localization in HeLa cells
HeLa cells were cultured in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine and penicillin/streptomycin, and plated on sterile coverslips. HeLa cells were unstimulated or pretreated with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) for 10 min at 37°C, washed with PBS, fixed with 3.7% paraformaldeyde in PBS (10 min at room temperature) and permeabilized with 0.1% Triton X-100 in PBS (4 min at room temperature). Non-specific binding sites were blocked by incubating the cells in 1% BSA in PBS (30 min at 4°C). The cells were then stained with FITC-conjugated 23C9 anti-CD2BP3 mAb (1:250 dilution in 1% BSA/PBS) and with Alexa Fluor 568phalloidin (1:40 dilution in 1% BSA/PBS; Molecular Probes) for 30 min at 4°C. The coverslips were subsequently washed and mounted on microscope slides for analysis.
Association between CIN85/CD2BP3 and c-Cbl
COS-7 cells were transfected with Flag-CIN85, Flag-FLCD2BP3, Flag-CD2BP3 and Flag-
ProCD2BP3 together with c-Cbl HA (kindly provided by Dr Hamid Band, BWH, Boston). Flag-
CD2BP3 was generated by PCR appending a Flag sequence at the N-terminus of a truncated form of CD2BP3 (bp 4876) while Flag-
ProCD2BP3 was generated by PCR deleting the region containing the proline-rich segments (bp 8961164) from the Flag-FLCD2BP3 construct. The cells were lysed in 0.5% NP-40 lysis buffer (0.5% NP-40, 50 mM Tris, pH7.5, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF supplemented with protease inhibitors and 0.25 mM Zn2+) and processed as described above. Immunoprecipitates were performed by incubating cleared lysate with CNBr-beads coupled to anti-Flag mAb, 2B5 anti-CD2BP3 mAb, anti-HA mAb (BAbCO, Richmond, CA) and irrelevant mouse IgG for 24 h at 4°C. The samples were run on 8% SDSPAGE, transferred to nitrocellulose and Western blotted using anti-Flag mAb or an anti-HA (diluted 1:1000; BAbCO) mAb.
CIN85/CD2BP3 degradation
Human peripheral blood mononuclear cells were purified using a Ficoll gradient from a healthy donor and T cells were further purified using nylon wool columns (36). T cells were cultured in complete medium [RPMI 1640 medium supplemented with 10% human AB serum (Nabi, Boca Raton, FL), 2 mM L-glutamine and penicillin/streptomycin (Gibco)] containing 25 ng/ml PMA and 1:200 anti-CD3 2Ad2 (IgM). After 48 h, the cells were diluted in complete medium supplemented with rIL-2 (112.5 U/ml) and kept in culture for an additional 4 days. The day before the experiment the medium was changed in order to remove the rIL-2. Cells (6 x 106/sample) were stimulated with PMA (100 ng/ml), 2Ad2 (1:200) or a combination of both at 37°C for the indicated time. The cells were subsequently washed, lysed in 1% Triton X-100 buffer, spun (10 min at 4°C), and the supernatants mixed with reducing sample buffer, boiled and loaded on 8% SDSPAGE. The proteins were transferred to nitrocellulose membranes and subsequently blotted with 23C9 anti-CD3BP3 mAb and with anti-ß-actin (1:5000; Sigma) [followed by incubation with HRP-conjugated anti mouse IgG2a mAb (1:10000; Santa Cruz Biotechnology)]. The signal was developed by ECL.
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Results |
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Sequence analysis of CD2BP3 and comparison with CIN85 and CMS
The CD2BP3 cDNA encodes a predicted 628-amino-acid protein with a calculated mol. wt of 68556.66 Da. Homology search shows that CD2BP3 represents a variant of a recently cloned adaptor protein, called CIN85 (mol. wt = 73129.49), characterized as a c-Cbl binding protein (28,37). Exon mapping of CD2BP3 and CIN85 in chromosome X [performed using TBLASTN of CD2BP3 and CIN85 sequences versus the human genome database (NCBI)] confirms that CD2BP3 and CIN85 proteins are splice variants, differing in their first identifiable exon (bp 471909471748 in CD2BP3 and bp 442800442642 for CIN85) and implying that these products are under the control of separate promoters. As shown in Fig. 1, CD2BP3 shares 92% amino acid identity with CIN85, but lacks the first SH3 domain (SH3A). Moreover, CD2BP3 shares a significant identity (35%) with another recently cloned protein named CMS (mol. wt = 71453.33), which has been identified as a p130Cas interactor and represents the human homologue of the previously identified mouse CD2AP (17,29). CMS shares 41% amino acid identity with CIN85. Note the striking similarity in overall domain organization of these molecules. Sequence alignment of individual SH3 domains shows that CIN85 SH3A (amino acids 557) shares 68% identity with CMS SH3A (amino acids 558), CIN85 SH3B (amino acids 105155) shares 72% identity with CMS SH3B (amino acids 115164) and CIN85 SH3C (amino acids 274326) shares 60% identity with CMS SH3C (amino acids 276328). In contrast, the linker regions between SH3 domains share lower identity: 18% between the two SH3ASH3B linkers and 32% between the two SH3BSH3C linkers. C-terminal to the SH3C domain, there is a region rich in proline residues which is likely to represent the target for SH3 domain binding. While in CIN85 there are four proline-rich stretches (P1 337343, P2 363369, P3 399406 and P4 419424), in CMS there are only three such stretches (P1 338344, P3 388395 and P4 410415), P2 being absent. Among the proline-rich stretches, the most conserved is P1 (100% identity), followed by P3 (75%) and P4 (67%).
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At the C-terminal end of this set of related proteins (CD2BP3 572628, CIN85 609665 and CMS 582639) there is a coiled-coil (CC) region whose involvement in oligomerization has been previously demonstrated (29,37). Sequence alignment shows a 37% overall identity between CIN85/CD2BP3 and CMS in this region. In CMS, four putative actin-binding sites (amino acids 534538, 599603, 610614 and 631635) with similarity to the LKKTET motif were described by Kirsch (29) consistent with the observation that CMS appears to be involved in cytoskeletal rearrangements. Three of the four sites lie within the CC region of CMS. In contrast, no identifiable actin-binding sites are present in CIN85/CD2BP3, with only a single marginally related sequence IKKA (658661) noted.
In Fig. 1(B) we show the sequence alignment of the human proteins together with related proteins cloned in other species: CD2AP (17) [also known as METS-1 (39)] represents the murine orthologue of CMS (29) and is involved in cytoskeletal polarization, while ruk-1 (40) [also known as SETA) (41)] is the rat orthologue of CIN85. Human SH3KBP1 is a splicing variant of CIN85 lacking both SH3A and SH3B, whose gene has been mapped on chromosome Xp22.1 p21.3 by in situ hybridization (42). Conserved residues are contained in the SH3 domains, in the proline-rich region and in the CC domain, arguing that these proteins are all members of the same family.
Localization of the CD2BP3-binding site on the CD2 cytoplasmic tail
As indicated in Fig. 2(A), within the 118-residue CD2 cytoplasmic tail, there are five proline-rich segments (PXXP). Previous studies indicated that the two most N-terminal sequences (PPPPGHR) are necessary for human CD2-triggered IL-2 production (43,44), representing the binding site for CD2BP2 via its GYF motif (19,25). The fourth proline-rich segment (PPLP) has been shown to represent the binding site for CD2BP1 (18). In order to localize the CD2BP3 binding site in the CD2 cytoplasmic tail, we utilized several CD2 constructs previously generated by PCR and cloned in pEG202 as described in (18) and graphically represented in Fig. 2(A). We also generated truncation variants of CD2BP3 representing the two SH3 domains plus the intervening sequence (SH3BC) or the individual SH3 domains (SH3B and SH3C) (Fig. 2B). Yeast were co-transformed with each CD2 tail segment cDNA construct as well as the CD2BP3 constructs and the strength of the interaction was determined by lacZ induction. From the results shown in Fig. 2(B, tabular inset), the binding of the entire CD2BP3 protein is stronger than that of the SH3BC fragment, suggesting that other portions of the molecule may contribute to the strength of binding, including the CC region which mediates oligomerization. While the strength of the bind ing is further reduced, the specificity pattern of the individual SH3B and SH3C domains is similar to that of the SH3BC, suggesting that in CD2BP3 these SH3 domains bind in a cooperative manner.
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In vivo association with CD2
To next investigate the association between CD2BP3 and CD2 proteins directly, we transiently transfected COS-7 cells with cDNAs corresponding to the full-length N-terminally Flag-tagged CD2BP3 (Flag-FLCD2BP3) and CD2 (FLCD2). After 48 h, the transfected COS-7 cells were lysed and immunoprecipitated with Sepharose bead-coupled anti-CD2 mAb (anti-T111), anti-CD2BP3 mAb (2C3) or anti-Flag mAb. The Western blotting of SDSPAGE separated proteins was then performed with polyclonal anti-CD2 heteroantisera (M32B) raised against the recombinant hCD2 ectodomain and developed by ECL (Fig. 2C). As shown, both anti-Flag and anti-CD2BP3 mAb (see below) co-immunoprecipitate a significant fraction of CD2 (relative to the anti-CD2 mAb immunoprecipitation control). This association is dependent on the interaction of the CD2 tail with CD2BP3 since there is no co-precipitation in COS-7 cells transfected with CD2, a human CD2-truncation variant
25-2 consisting of CD2 amino acids 1236 (11). Analogous experiments employing Flag-CMS and Flag-CIN85 constructs indicate that each associates with CD2 as well (Fig. 2C). In these latter experiments, we employed an anti-Flag mAb as well as two different anti-CD2BP3 mAb (2C3 and 2B5) which are described below. Note that 2C3 mAb, unlike 2B5, fails to recognize the CIN85CD2 complex.
Differential CD2 and c-Cbl binding activities of CMS and CIN85 SH3 domains
Prior analysis employing rodent CIN85 (SETA) and CMS (CD2AP) revealed a differential usage of their respective SH3 domains in CD2 binding: SETA employed SH3B (41) while CD2AP utilized SH3A (17). Moreover, from the above yeast complementation assay, it was evident that CIN85 SH3B and/or SH3C could be potentially involved in CD2 binding. To investigate the ability of the individual SH3 domains of CIN85 and CMS to pull-down CD2 from Jurkat lysates, purified GSTSH3A, SH3B or SH3C of CIN85 and CMS proteins (Fig. 3A) as well as GST alone (negative control) were coupled to glutathioneSepharose beads and incubated with cell lysates. The associated cellular proteins were eluted from the Sepharose-coupled beads, subjected to SDSPAGE and associated CD2 revealed by Western blotting with M32B anti-CD2 polyclonal antisera. As shown in Fig. 3(B), the GST fusion proteins of CMS SH3A and CIN85 SH3A domains (GSTSH3A) each pull-down a greater amount of CD2 than the corresponding GSTSH3B domains. In contrast, neither CMS- or CIN85-derived GSTSH3C proteins are able to associate with CD2 as detected in this pull-down assay. Note that the amount of GST and GST-fusion proteins coupled to the beads as shown by Coomassie stain is equivalent. We can conclude that both CMS SH3A and CIN85 SH3A have a stronger affinity for CD2 than CMS SH3B and CIN85/CD2BP3 SH3B, and that the SH3C domains of these proteins bind too weakly to CD2 to be detected.
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Intramolecular interaction between the CIN85/CD2BP3 proline-rich region and the CIN85/CD2BP3 SH3 domains
The presence of a proline-rich region downstream of the SH3 domains in CD2BP3 raised the possibility that a self-regulating form of intramolecular interaction might exist. To evaluate the possibility of such an intramolecular association in CIN85/CD2BP3 and CMS molecules, we generated a truncated form of CIN85/CD2BP3 (Flag-PCC CIN85) containing an N-terminal Flag tag appended to the C-terminal portion of the protein comprising the proline-rich region (amino acids 328665) and a comparable CMS construct (Flag-PCC CMS) (Fig. 3A). We subsequently transfected COS-7 cells with Flag-PCC CIN85 or Flag-PCC CMS and used the GST-alone or GSTSH3 proteins (CMS SH3A, SH3B, SH3C and CIN85 SH3A, SH3B, SH3C) coupled to beads for pull-down assays. The bound proteins were subjected to SDSPAGE and Western blotting using anti-Flag mAb. The result in Fig. 3(B) shows that CIN85 SH3A can bind to the Flag-PCC CIN85, confirming the possibility of CIN85 self-ligation and attendant intramolecular regulation. In contrast, when we performed the same experiment by transfecting the COS-7 cells with Flag-PCC CMS (amino acids 327639), we failed to observe any association, supporting the previous observations by Kirsch (29). This result clearly demonstrates the existence of a unique interaction between CIN85 SH3A and the proline-rich CIN85 segment which is not found in CMS. The CMS SH3A does weakly interact with the CIN85 proline-rich segment, suggesting the possibility of an intermolecular CMSCIN85 interaction, but this finding is currently of unknown significance. Interestingly, as shown in the Fig. 3(B, bottom panel), in the presence of Zn2+ we observed that CIN85/CD2BP3 SH3B can also bind to the CIN85/CD2BP3 proline-rich region, even if to a lesser extent than CIN85 SH3A.
Differential association of CIN85 and CD2BP3 with p130Cas
CMS was initially cloned as an interactor with p130Cas (29). This proteinprotein interaction is mediated by the SH3 domain of p130Cas and the proline-rich region of CMS. Given that CIN85/CD2BP3 also contain a proline-rich region, we investigated their potential association with p130Cas in Flag-FLCD2BP3, Flag-CIN85 and Flag-CMS transfected COS-7 cells. As shown in Fig. 4, p130Cas clearly associates with CMS and, to a lesser extent, with CIN85. Interestingly, almost undetectable levels of p130Cas are associated with CD2BP3. In Fig. 4, we graphically represent the relative amount of p130Cas associated with the indicated Flag-tagged proteins. Thus, p130Cas, an important protein involved in focal adhesions, associates differently with each of the three CD2 interactors.
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Intramolecular regulation of CIN85/CD2BP3 probed by specific mAb and c-Cbl interaction
Since mAb offer excellent probes to assess protein conformational change, we employed these reagents to examine putative intramolecular regulation of CIN85/CD2BP3 via endogenous SH3proline-rich segment interactions. As schematically represented in Fig. 7(D), Flag-tagged versions of CIN85, CD2BP3 and two proline-rich segment deletion mutants were employed. All cDNA constructs were transfected in COS-7 cells and subsequently the cell lysates were immunoprecipitated with anti-Flag, 2C3 anti-CD2BP3 or control mAb. The resulting proteins were separated on SDSPAGE and Western blotted with anti-Flag mAb (Fig. 7E). In the case of the full-length CD2BP3 molecule, the amount of protein reactive with the 2C3 mAb is >3-fold lower (2C3/Flag ratio = 0.3, as calculated using the public domain NIH Image software) than the pull-down by the anti-Flag mAb, indicating that the 2C3 epitope is largely inaccessible to antibody binding. On the other hand, in the case of Flag-CD2BP3 molecule, the 2C3 mAb could bind as efficiently as anti-Flag mAb (2C3/Flag ratio = 0.99). Transfection results with the Flag-
ProCD2BP3 construct independently confirm that the proline-rich region is responsible for obscuring the mAb binding site (2C3/Flag ratio = 0.93).
As c-Cbl interacts with CIN85/CD2BP3 SH3 domains (28), its binding could also be affected by intramolecular regulation. To investigate this possibility, cDNAs encoding Flag-CIN85 or Flag-FLCD2BP3 were co-transfected in COS-7 cells with HA-tagged c-Cbl. After 48 h, equivalent aliquots of the cell lysates were immunoprecipitated in parallel with anti-Flag mAb, 2B5 anti-CD2BP3 mAb, anti-HA mAb or an irrelevant mAb (Control). Subsequently, the amount of associated CIN85/CD2BP3 was detected by anti-Flag Western blotting, while the amount of c-Cbl was detected by anti-HA mAb. The results are shown in Fig. 7(F). As evident from the first two sets of panels, only a fraction of c-Cbl is associated with CIN85 and CD2BP3 (29 and 21% respectively, assessed as the ratio of anti-HA Western blot signal intensity in anti-CD2BP3 versus anti-HA immunoprecipitates). In contrast, with Flag-CD2BP3 and Flag-
ProCD2BP3, the c-Cbl protein is virtually entirely bound to the CD2BP3 variants (100 and 83% respectively). We conclude that the CIN85/CD2BP3 intramolecular association involving SH3 domainproline segment interaction is one mechanism by which the binding of CIN85 and CD2BP3 to other molecules is regulated. Whether phosphorylation of CIN85/CD2BP3 affects this intramolecular conformation is unknown, but appears likely.
Inducible degradation of CIN85/CD2BP3 proteins
The presence of the PEST sequence in the C-terminal portion of CIN85/CD2BP3 raised the possibility that these proteins might be prone to proteolysis. To test this hypothesis, activated human T cells were treated with PMA and the anti-CD3 mAb, 2Ad2, alone or together. As shown in Fig. 8(A), the combination of PMA + 2Ad2 treatment induces a significant decrease in the amount of CIN85/CD2BP3 protein within 10 min (
50% reduction). After 30 min, the amount of protein is further reduced to 30%. At later time points the amount of protein rebounds, suggesting initiation of de novo synthesis. In the case of PMA treatment alone, induced degradation is slower than that of PMA + 2Ad2 treatment and the steady-state protein level does not appear to be restored, at least within the 3 h observation period. Figure 8(B) provides a graphical representation of the CIN85/CD2BP3 degradation kinetics, indicating CIN85/CD2BP3 protein levels following treatment as a function of the percentage of protein present in untreated cells. Although not shown, 2Ad2 treatment alone also reduces CIN85/CD2BP3 protein levels but to a lesser extent than that of the 2Ad2 + PMA combination.
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Discussion |
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CD2BP3 represents an RNA splice variant of the previously described human protein CIN85, itself cloned by yeast two-hybrid analysis as a c-Cbl interactor protein (28). Unlike CIN85, however, CD2BP3 lacks the first of three N-terminal SH3 domains (SH3A), but retains SH3B, SH3C, four tandem proline-rich stretches, a PEST sequence and a C-terminal CC (Fig. 1). In B cells, CIN85 was found to be associated with B cell linker protein (BLNK), Grb2, Sos1, p130Cas and the p85 subunit of phosphatidylinositol-3-kinase (37). Additionally, CIN85 associates through its own proline-rich region with endophilin via the endophilin SH3 domain (46,47). The CIN85endophilinc-Cbl complex associates with activated EGF and c-Met receptors, thereby controlling receptor internalization. Tyrosine phosphorylation of residues on the distal C-terminal segment of c-Cbl influences accessibility of the c-Cbl proline-rich segments association with CIN85 SH3 domains and, hence, modulates CIN85c-Cbl binding. As the ubiquitin ligase function of c-Cbl resides in its RING-finger domain (52), the CIN85c-Cbl binding activity is separable from the ligase enzymatic activity that mediates ligand-dependent receptor down-regulation via ubiquitination.
The rat orthologue of CIN85, termed Ruk or SETA, forms a complex with the phosphatidylinositol-3-kinase holoenzyme via an interaction involving the proline-rich segment of Ruk and the phosphatidylinositol-3-kinase p85 regulatory subunits SH3 domain (40). This interaction is of functional significance given that Ruk inhibits the enzymes ligand binding activity. Because the phosphatidylinositol-3-kinase signaling pathway mediates survival effects of certain growth factors (5355), it is not surprising that Ruk regulates survival of neuronal cells. Moreover, Ruk interacts directly with apoptosis-linked genes (40). Also noteworthy is the observation that the rat CIN85 orthologue has been shown to exist in three isoforms termed RukL, RukM and RukS which are splice variants encoding the full-length protein, a protein lacking SH3B and SH3C or a molecule retaining only the C-terminal CC respectively (56).
CMS was originally cloned from a yeast two-hybrid human placental library as a p130Cas ligand with multiple SH3 domains (29). In addition to molecular architectural similarities with CIN85, CMS has been shown to bind to the p85 subunit of phosphatidylinositol-3-kinase, c-Cbl and Grb2 (57). However, CMS expression is cytosolic, co-localizing with F-actin and p130Cas in membrane ruffles. The murine CMS homologue, termed CD2AP, was cloned as a CD2 cytoplasmic tail binding protein, mapping to the same region as CIN85/CD2BP3 found herein (17). In T cells, cytoskeletal polarization is linked to the interaction of CD2AP with CD2. In this regard, a dominant-negative version of CD2AP containing the SH3A and SH3B domains fused to GFP strongly inhibited the ability of CD2 to stimulate T cell polarization. Collectively, these results with human CMS in non-T cells and rodent CD2AP in T cells argue persuasively for a specialized role of the CMS/CD2AP scaffolding molecule in regulation of the actin cytoskeleton. Development of nephrotic syndrome in CD2AP-deficient mice due to renal epithelial cell defacement of foot processes is also consistent with this notion (58).
Differences between CIN85 and CMS are significant, however. CMS lacks the second of four proline-rich segments found in CIN85, contains fewer PKC and CK2 phosphorylation sites, and has no PEST sequence proximal to the C-terminal CC. In addition, CMS possesses four putative actin-binding sites absent from CIN85/CD2BP3, consistent with the lack of detectable association of the latter with F-actin. The inability of any of the three CMS SH3 domains to ligate the CMS proline-rich segment also contrasts with the measurable interaction observed between CIN85/CD2BP3 SH3 domains and the corresponding CIN85/CD2BP3 proline-rich region. The intramolecular ligation function, greater number of phosphorylation sites and PEST sequence argue that CIN85/CD2BP3 is highly regulated by intracellular biochemical processes in a fashion distinct from CMS.
The CMS interaction with p130Cas is an important clue to molecular function. p130Cas represents a key element in the regulation of cellular adhesion and migration processes (22,59) being a major substrate for the focal adhesion kinase p125FAK as well as the protein tyrosine phosphatase (PTP-PEST) (60). The p130Cas phosphorylation state determines the formation or disruption of the focal adhesion complex. For example, constitutive hyperphosphorylation of focal adhesion proteins such as p130Cas, FAK and paxillin in PTP-PEST/ fibroblasts results in an increased number of focal adhesions and cells spreading on the extracellular matrix with a subsequent reduction in migration (61). During conjugation of T cells with CD58-expressing APC, CD2 is redistributed to the cell contact area (10,11,14,17). Ligand-induced clustering of CD2 on the T cell surface determines the recruitment of CMS/CD2AP, reorganizing the p130Cas at the site of T cell contact and thereby directing this important focal adhesion component (17). CD2 redistribution also recruits the CD2BP1 adaptor which is constitutively associated with PTP-PEST (18). CD2BP1 associates with the same region of the cytoplasmic tail of CD2, but only upon CD2 clustering, hence providing an inducible association mechanism. As PTP-PEST dephosphorylates p130Cas, it would appear that CD2 molecules function in a pivotal manner to regulate focal adhesion processes through cytoplasmic tail adaptors.
Upon T cell migration, CD2 localizes to the uropod of polarized T cells (14). Movement resulting from complex regulation of adhesion and de-adhesion cycles (62,63) correlates with the accumulation of CD2 in the uropod and recruitment of p130Cas and PTP-PEST via the above adaptor protein linkages. Interestingly, our analysis of p130Cas association clearly indicates a preference for CMS interaction, with a smaller fraction interacting with CIN85 and virtually no association with CD2BP3. Thus, even if highly structurally related, CMS, CIN85 and CD2BP3 are functionally distinct. CD2BP3 and CIN85 can act as a physiologic dominant-negative regulator of CMS by binding to CD2 without efficiently recruiting p130Cas, acting in a fashion analogous to the aforementioned experimentally created CD2AP variant (17). Additionally, CD2BP3 must antagonize CIN85 by competing for the same cellular interactors, but ligating CD2 with weakened affinity.
Both CIN85/CD2BP3 antibody accessibility studies and proteinprotein association analysis offer evidence for intramolecular association mediated by binding of SH3A or SH3B domains to the proline-rich region. This regulatory feature is not present in CMS, but has significant implications for phosphatidylinositol-3 kinase and endophilin SH3 domains which bind to the CIN85/CD2BP3 proline-rich region, and, conversely, the proline-rich segment of c-Cbl which binds to CIN85/CD2BP3 SH3 domains. Moreover, the existence of analogous SH3proline intramolecular associations has been demonstrated for the Tec tyrosine kinase (64) as well as the Brutons tyrosine kinase (65) whose mutation causes X-linked agammaglobulinemia. For these kinases, intramolecular association is in competition with intermolecular binding mediated by the same region. Likewise, in the case of the adaptor proteins such as CIN85/CD2BP3, the significance is related to modulation of proteinprotein co-association. In this paper, we show that the intramolecular CIN85/CD2BP3 binding regulates c-Cbl association to this adaptor and, by inference, to CD2 itself. c-Cbl is a negative regulator of immune receptor signal transduction involved in down-modulation of surface expression upon ligand binding (45). Constitutive association of CIN85 with endophilin, an adaptor linked to the endocytic pathway (46,47), and the ability of the co-associated c-Cbl to activate E2ubiquitin conjugating enzymes induces the receptor degradation process. In this regard, CIN85, but not CD2BP3, controls CD2 surface levels, augmenting basal CD2 expression, but reducing CD2 expression upon CD2 cross-linking, presumably by receptor internalization. Since CD2BP3 has a weaker affinity for CD2 than CIN85, it is probably less efficient at recruiting c-Cbl, particularly in view of a weaker c-CblCD2BP3 interaction (Fig. 7F). Although not shown, c-Cbl tyrosine phosphorylation is comparable in CD2-triggered CD2BP3 and CIN85 transfectants.
The presence of an intramolecular regulation in CIN85/CD2BP3 could serve as a mechanism to further reduce or otherwise regulate the amount of c-Cbl recruited to CD2, thereby stabilizing CD2 molecules on the cell surface. Phosphorylation at one or more CK2 or PKC sites may influence the intramolecular regulation process. The weakened CD2 association of CD2BP3 relative to CIN85 may further attenuate this degradation mechanism. Previously described Ruk ubiquitination is consistent with its c-Cbl association (56), but appears to require additional modification to cause protein degradation. As shown in the current study (Fig. 8), activation of PKC alone or in conjunction with TCR cross-linking initiates the CIN85/CD2BP3 degradation process.
The hypothesis that regulatory mechanisms related to phosphorylation, protein degradation and RNA splicing may control the above processes underscores the complexity of cellular control mechanisms. That CD2BP3, CD2BP1, CIN85, CD2AP and by inference CMS all bind to the same CD2 tail segment implies either that kinetic differences in binding may occur and/or that differential cellular localization events are involved. With regard to compartmentalization, we have observed that CD2BP1, CIN85/CD2BP3 and CMS are non-lipid raft associated (data not shown), and, hence, competitive binding to the CD2 cytoplasmic tail is likely. Given that CD2 clustering is driven by CD58 binding, a patch of CD2 will be capable of interacting with multiple adaptors, however. That CIN85, CD2BP3 and CMS are each oligomerized by a CC region argues that heterologous oligomers may also be formed. CD58 ligand-driven movement of human CD2 from non-raft to lipid raft compartments (66) likely also fosters exchange, dynamically shifting the balance of CD2 tail interactors and the linked intracellular signaling molecules. Details regarding control mechanisms governing the overall balance are important to ascertain. If CIN85 and CD2BP3 proteins are induced to undergo proteolysis upon TCR cross-linking, whereas CMS is less prone to degradation, then antigen recognition may favor CMS binding to CD2, leading to T cell polarization (Fig. 9). CIN85 prevents CD2-triggered T cell polarization, while CD2BP3 is minimally inhibitory (Fig. 5). The greater protein expression of the CD2BP3 versus CIN85 isoform may also favor CMS complex formation with CD2. The regulatory nature of these protein interactions remains to be fully explored, but is undoubtedly linked to functional processes of T cell adhesion, migration and activation.
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Acknowledgements |
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Abbreviations |
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CCcoiled-coil
CK2casein kinase II
EGFepidermal growth factor
FLfull length
HRPhorseradish peroxidase
IPTGisopropyl-ß-D-thiogalactopyranoside
PKCprotein kinase C
PMAphorbol 12-myristate 13-acetate
PTPprotein tyrosine phosphatase
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References |
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