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International Immunology, Vol. 15, No. 3, pp. 313-329, March 2003
© 2003 Japanese Society for Immunology

CD2BP3, CIN85 and the structurally related adaptor protein CMS bind to the same CD2 cytoplasmic segment, but elicit divergent functional activities

Elena V. Tibaldi1 and Ellis L. Reinherz1

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


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Interaction trap cloning was used to identify a CD2 cytoplasmic tail-binding protein termed CD2BP3. CD2BP3 is the major RNA splice variant of the CIN85 locus in human T lymphocytes, lacking SH3A, the first of three SH3 domains found in CIN85, but retaining SH3B, SH3C, a proline-rich domain and C-terminal coiled coil. CD2BP3 has 35% amino acid identity to CMS, a structurally related protein binding to the same highly conserved segment of the CD2 tail and known to be involved in T cell polarization/cytoskeletal interactions. Unlike CMS, however, CD2BP3 does not co-localize with F-actin and binds p130Cas weakly, if at all. Moreover, CIN85/CD2BP3 proteins are readily degraded by TCR cross-linking, consistent with the presence of a PEST sequence C-terminal to SH3C. CIN85 SH3A and CIN85/CD2BP3 SH3B bind to proline-rich segments within CIN85/CD2BP3 themselves as evidenced by mAb accessibility analysis and protein interaction studies including c-Cbl binding. This form of intramolecular regulation is not manifest by CMS. CMS and CIN85 activities are antagonistic, while the functions of CIN85 and CD2BP3 are also distinct. Thus, CD2-mediated adhesion, signaling and cell motility are regulated in a highly complex manner.

Keywords: c-Cbl, cytoskeletal polarization, intramolecular regulation, RNA splicing, T cell


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
CD2 is a transmembrane cell-surface glycoprotein expressed on T lymphocytes, thymocytes and NK cells, and functions to promote T cell adhesion, immune recognition/activation and motility (1,2). The human CD2 ligand, the counter-receptor CD58, is expressed on a diverse array of cell types including stromal cells and antigen-presenting cells (APC) (2). Dynamic binding between CD2 and CD58 counter-receptors on opposing cells optimizes immune recognition by approximating surface membranes at ~15 nm, a distance suitable for TCR–peptide–MHC ligand interaction (3–8). The weak affinity of the CD2–CD58 binding, resulting from rapid Kon and Koff parameters reminiscent of selectin interactions, is bolstered by CD2 redistribution at the cell–cell junction, augmenting T cell antigen responsiveness (9–12). Not surprisingly, disruption of CD2 gene function is detrimental to both CD4 and CD8 T lymphocyte function in mice (12,13).

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 surfaces—a 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 (18,20–24). 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.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
CD2BP3 cloning and characterization of the CD2BP3 binding site by a yeast trap interaction system
CD2BP3 was cloned from an activated human T cell cDNA library (18) using a previously described yeast two-hybrid system and defined interaction criteria (30). DNA sequencing was performed in the Molecular Biology Core Facility of DFCI using standard automated methods. To examine the interaction of different truncated or mutated versions of the CD2 tail with different variants of CD2BP3, the mutant cDNAs corresponding to each protein were generated from the original clones by PCR (31) and inserted into the relevant yeast expression vectors. For CD2 tail variants, the cDNAs were inserted in the pEG202 vector between EcoRI and SalI sites. For the CD2BP3 constructs, the cDNAs were inserted in the pJG4-5 between EcoRI and XhoI sites. Different combinations of CD2 tail and CD2BP3 constructs were used to transform yeast EGY48 pre-transformed with the pSH1834 vector. The triple transformants were selected for their growth in ura–, his–, trp– minimal medium. The colonies were then tested for lacZ expression under galactose-induced conditions.

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 ({Delta}) 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 10–20 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 (CNBr–IgG, 10 µl beads/ml lysate) (33) overnight at 4°C. The cleared lysate was then immunoprecipitated with CNBr–IgG (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% SDS–PAGE. 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 2–3 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; 2–3 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 GST–SH3 fusion proteins, the cDNA sequences corresponding to the SH3 domains of CMS and CIN85 [CMS SH3A (bp 4–180), CMS SH3B (bp 331–507), CMS SH3C (bp 798–990), CIN85 SH3A (bp 4–177), CIN85 SH3B (bp 301–483) and CIN85 SH3C (bp 793–987)] 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 glutathione–Sepharose 4B beads according to the manufacturer’s 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 GST–SH3 proteins (coupled to glutathione–Sepharose 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 50–60 x 106 cells/ml. The lysates were then precleared on GST–beads (10 µl beads/ml lysate) overnight at 4°C. The precleared lysates were incubated for 2–4 h with GST alone or GST–SH3 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% SDS–PAGE 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% SDS–PAGE 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 GST–SH3 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 979–1917) and CIN85 (bp 982–1995), 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 Tris–HCl 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 SDS–PAGE, 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% SDS–PAGE, 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 GST–SH3C, 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 (His–SH3BC). The above His–SH3BC plasmid was generated by PCR, by placing a His tag (6 His) at the N-terminus of a truncated form of CD2BP3 (bp 4–876), 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 manufacturer’s 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 GST–SH3B, GST–SH3C and His–SH3BC 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% SDS–PAGE 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 SDS–PAGE 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 568–phalloidin (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-{Delta}CD2BP3 and Flag-{Delta}ProCD2BP3 together with c-Cbl HA (kindly provided by Dr Hamid Band, BWH, Boston). Flag-{Delta}CD2BP3 was generated by PCR appending a Flag sequence at the N-terminus of a truncated form of CD2BP3 (bp 4–876) while Flag-{Delta}ProCD2BP3 was generated by PCR deleting the region containing the proline-rich segments (bp 896–1164) 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 2–4 h at 4°C. The samples were run on 8% SDS–PAGE, 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{epsilon} 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% SDS–PAGE. 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.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Yeast two-hybrid screening of CD2 cytoplasmic tail interaction proteins and identification of CD2BP3
To identify proteins interacting with the CD2 cytoplasmic tail, we employed a yeast two-hybrid system using the full-length CD2 cytoplasmic tail cDNA sequence in pEG202 as bait and a T cell-derived cDNA library ligated into the pJG4-5 yeast vector as described in (18,30). Of ~7 x 106 yeast transformants screened in the CD2 tail interaction trap system, nine independent colonies containing distinct insert sizes were identified as positive. Within this group, CD2BP1 and CD2BP2 have been already characterized (18,19). Here we describe clone #30 (CD2BP3) which showed a strong and specific interaction in the yeast two-hybrid system. Under galactose induction, transformation of the CD2 tail/pEG202 and CD2BP3/pJG4-5 plasmid in yeast yielded double transformants positive for growth in leu– medium and lacZ expression in leu+ medium. In contrast, in the absence of induction (i.e. in the presence of glucose) doubly transformed colonies showed neither growth in leu– medium nor lacZ induction. These results indicate that the interaction between the hybrid CD2 tail protein and the CD2BP3 fusion protein is specific rather than a secondary effect of the CD2 tail fusion on yeast growth or lacZ expression.

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 471909–471748 in CD2BP3 and bp 442800–442642 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 5–57) shares 68% identity with CMS SH3A (amino acids 5–58), CIN85 SH3B (amino acids 105–155) shares 72% identity with CMS SH3B (amino acids 115–164) and CIN85 SH3C (amino acids 274–326) shares 60% identity with CMS SH3C (amino acids 276–328). In contrast, the linker regions between SH3 domains share lower identity: 18% between the two SH3A–SH3B linkers and 32% between the two SH3B–SH3C 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 337–343, P2 363–369, P3 399–406 and P4 419–424), in CMS there are only three such stretches (P1 338–344, P3 388–395 and P4 410–415), 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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  		Fig. 1. Primary amino acid sequence comparison of CD2BP3, CIN85 and CMS proteins. (A) Schematic representation. A graphical representation is shown with the tandem SH3 domains at the N-termini of the proteins, the proline (pro-rich) domains, the putative PEST sequence (based upon the PESTfind search) and the CC region at the C-terminal ends of the proteins. Note the absence of a comparable PEST sequence in CMS. Potential PKC (closed circle), CK2 (open circle) (http://www.expasy.ch) and tyrosine phosphorylation sites (Y) (NetPhos 0.2) are indicated. Actin-binding sites (A) represent motifs similar to the LKKTET sequence found in several actin-binding proteins. The numbers refer to the amino acid residue of each protein. (B) Protein sequence alignment of CMS/CIN85 family members. CD2BP3 and SH3KBP1 represent two different human splicing variants of CIN85 characterized by the absence of SH3A or the SH3A plus SH3B N-terminal SH3 domains respectively. Ruk-1 (SETA) is the rat orthologue of CIN85. CD2AP represents the murine orthologue of CMS. Highlighted in black are residues that are identical in all six sequences; the residues that are identical in five of the six sequences are denoted by boxes. The numbering refers to the CIN85 sequence with the alignment performed by the Clustal X method (identity 0.5).

 
In the C-terminal portion of CIN85 (amino acids 490–516) and CD2BP3 (amino acids 453–479) there is a PEST sequence [identified by PESTfind (http://us.expasy.org/tools); score = 17.07] that could represent a potential target for protein degradation (38). Of note, there is no similarly localized PEST sequence in CMS, suggesting that CMS may be less prone to proteolysis than either CIN85 or CD2BP3. It is known that PEST sequences are the target of phosphorylation, in particular induced by casein kinase II (CK2): interestingly, in CIN85/CD2BP3 there are two potential phosphorylation sites for CK2 contained in the PEST sequence. Moreover, in the CIN85 sequence there are 13 other putative sites for CK2 and 15 for protein kinase C (PKC), suggesting that the protein could be highly phosphorylated. Of note, CD2BP3 possesses two extra putative CK2 phosphorylation sites within the first 17 residues in lieu of SH3A. In the CMS sequence, a PEST motif may be contained in the SH3B–SH3C linker region (amino acids174–208) as suggested by a marginal PEST final score (6.96), so that CMS might be prone to some degree of proteolysis. On the other hand, by comparison to CIN85, CMS has a significantly reduced number of phosphorylation sites (10 for CK2 and 10 for PKC). Five potential tyrosine phosphorylation sites are noted in CMS, whereas only one site exists in CIN85 SH3A (Fig. 1A).

At the C-terminal end of this set of related proteins (CD2BP3 572–628, CIN85 609–665 and CMS 582–639) 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 534–538, 599–603, 610–614 and 631–635) 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 (658–661) 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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  		Fig. 2. Identification of CD2 cytoplasmic tail–CD2BP3 interaction sites by yeast two-hybrid complementation analysis. (A) Schematic representation of CD2 cytoplasmic tail variants. D1 and D2, domain 1 and domain 2 of the extracellular segment of CD2; TM, transmembrane segment of CD2; MPR, membrane-proximal region of CD2 tail. The numbers in each segment refer to the amino acid residues of the mature protein. The five proline-rich segments are underlined. All CD2 variants are depicted from residue 221 (*). (B) Schematic representation of CD2BP3 constructs used in yeast two-hybrid interaction assays. The numbers in each segment represent the amino acid residues. The SH3 domains, the four pro-rich segments (open rectangles) and the CC region are indicated. The tabular insert summarizes binding results based on yeast two-hybrid analysis with ‘+’ denoting strength of interaction as scored previously (18). (C) Association of CD2 and CD2BP3 proteins in COS-7 cells. Flag-tagged full-length CD2BP3 (Flag-FLCD2BP3) was co-transfected with full-length CD2 (FLCD2) or with the cytoplasmic tail deletion variant {Delta}CD2 into COS-7 cells. FLCD2 was also co-transfected with Flag-CMS or Flag-CIN85. Transfected cells were lysed; the proteins were immunoprecipitated with the indicated mAb coupled to CNBr–Sepharose 4B beads, separated on 8% SDS–PAGE under reducing conditions (here and in all subsequent gels) and Western blotted with an anti-CD2 (M32B) polyclonal heteroantisera or with an anti-Flag mAb.

 
To pinpoint the CD2BP3-binding site in the CD2 tail, we analyzed the strength of the interaction between CD2BP3 and progressively larger C-terminal CD2 tail truncation variants lacking the segment downstream of the fifth proline-rich segment (ET1), the fifth proline-rich segment (K) and the fourth proline-rich segment (L). From the results in the Fig. 2(B, tabular inset), it is clear that the absence of the last 13 residues in construct ET1 does not affect the interaction, while the deletion of the fifth proline-rich segment (PKPP) in construct K attenuates the strength of the interaction compared to the full-length CD2. More importantly, the further deletion of the fourth proline-rich segment in construct L completely abolishes the interaction. These results suggest that the distal tandem proline-rich segments are critically involved in CD2BP3 binding. We also confirmed that the other three N-terminal proline-rich segments are not involved: the strength of the binding of CD2BP3 with 3KD, a construct lacking these segments, is comparable to that of the full-length CD2. The presence of the fourth proline-rich segment is most critical as (i) deletion of proline residues 302 and 303 in the LP construct abolishes binding, while the deletion of Pro313 and 314 in the PK construct does not have an effect, and (ii) the 3D construct which eliminates the fourth proline-rich segment yields no binding activity.

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 SDS–PAGE 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 {Delta}CD2, a human CD2-truncation variant {Delta}25-2 consisting of CD2 amino acids 1–236 (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 CIN85–CD2 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 GST–SH3A, –SH3B or –SH3C of CIN85 and CMS proteins (Fig. 3A) as well as GST alone (negative control) were coupled to glutathione–Sepharose beads and incubated with cell lysates. The associated cellular proteins were eluted from the Sepharose-coupled beads, subjected to SDS–PAGE 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 (GST–SH3A) each pull-down a greater amount of CD2 than the corresponding GST–SH3B domains. In contrast, neither CMS- or CIN85-derived GST–SH3C 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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  		Fig. 3. Binding activities of individual CMS and CIN85 SH3 domains. (A) Schematic representation of CIN85 and CMS constructs. The individual SH3 domains of CIN85 and CMS were cloned C-terminal to the GST sequence in pGEX-4T-1, expressed in E. coli and purified as described. N-terminal Flag-tagged PCC CIN85 (Flag-PCC CIN85) and Flag-tagged PCC CMS (Flag-PCC CMS) constructs were generated by PCR and transfected into COS-7 cells. (B) Differential binding of the individual CMS and CIN85 SH3 domains. Equal amounts of GST and GST–SH3 fusion proteins were coupled to glutathione–Sepharose beads as shown by Coomassie stain of the proteins (top panel). The coupled beads were used to immunoprecipitate CD2 and c-Cbl from J77 cell lysates. The indicated molecules were revealed by Western blotting with anti-CD2 and anti-c-Cbl polyclonal antisera respectively. The coupled beads were also employed in the immunoprecipitation of Flag-PCC CMS and Flag-PCC CIN85 from transfected COS-7 cells, and associated proteins detailed in Western blotting using an anti-Flag mAb. Association of Flag-PCC CIN85 with GST–SH3B is revealed in the presence of Zn2+ (0.25 mM) (lower panel).

 
To evaluate the ability of the individual SH3 domains of CIN85 and CMS to differentially associate with other ligands, additional assays were performed. Given that CIN85 was previously found to interact with c-Cbl, an important modulator of signaling attenuation (28,45), the capacity of the SH3 domains to pull-down c-Cbl from Jurkat cell lysates was assessed by Western analysis with an anti-c-Cbl polyclonal sera. As shown in Fig. 3(B), CMS SH3B reveals a stronger affinity for c-Cbl compared to CMS SH3A, while the CMS SH3C domain does not bind at all. Conversely, CIN85 SH3A binds more strongly to c-Cbl than CIN85 SH3B, whereas the CIN85 SH3C shows detectable binding, even if to a lesser degree than CIN85 SH3B. From these results we conclude that both CIN85 and CMS bind CD2 and c-Cbl, but display differential SH3 domain usage in these interactions.

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 328–665) 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 GST–SH3 proteins (CMS SH3A, SH3B, SH3C and CIN85 SH3A, SH3B, SH3C) coupled to beads for pull-down assays. The bound proteins were subjected to SDS–PAGE 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 327–639), 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 CMS–CIN85 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 protein–protein 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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  		Fig. 4. Differential association of p130Cas with CMS, CIN85 and CD2BP3. COS-7 cells were transfected with p130Cas and the indicated Flag-tagged proteins. Subsequently, either control (–) or anti-Flag (+) mAb were used for immunoprecipitation. Association was revealed by first Western blotting with an anti-p130Cas polyclonal antibody and then with an anti-Flag mAb and signal revealed by ECL. The graph below represents the ratio of the band density in anti-Flag versus control immunoprecipitates in the anti-p130Cas Western blot. Densitometry scanning of ECL-derived bands used the public domain NIH Image software.

 
CIN85 is more efficient than CD2BP3 at blocking cytoskeletal polarization
The murine orthologue of CMS, CD2AP has been shown to be involved in cytoskeletal rearrangement. Not surprisingly, therefore, an experimentally created form of CD2AP containing its first two SH3 domains (SH3AB) alone inhibited cytoskeletal polarity (17). Given that CIN85 SH3A and B share 68 and 72% amino acid identity respectively with the corresponding CMS SH3 domains, CIN85 could represent a physiological (i.e. naturally occurring) dominant-negative regulator. To test this possibility, we analyzed the influence of CIN85 and CD2BP3 proteins on the polarization of the Golgi apparatus in Jurkat cells upon CD2 cross-linking (14). To this end, Jurkat T cells were either left untransfected or transiently transfected with Flag-CIN85 or Flag-FLCD2BP3 as indicated and then plated on coverslips coated with a combination of anti-CD2 mAb. The staining with the BODIPY TR Ceramide in the untransfected cells shows a clear Golgi polarization towards the center of the cells (75%) (Fig. 5). Conversely, a significant reduction in the Golgi polarization was observed in the CIN85-transfected cells (12%), while only a marginal effect was observed in the CD2BP3-transfected cells (56%). From these results we conclude that CIN85 antagonizes strongly T cell polarization initiated by CD2 cross-linking, whereas, in contrast, CD2BP3 has little effect.



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  		Fig. 5. CIN85 is more efficient than CD2BP3 at preventing cytoskeletal polarization. Jurkat cells, untransfected or transiently transfected with Flag-CIN85 or Flag-CD2BP3, were plated on coverslips pre-coated with a pair of anti-CD2 mAb (anti-T112 + anti-T113) for 30 min at 37°C and then stained with BODIPY TR Ceramide to detect the Golgi apparatus. The indicated panels represent immunofluorescence photos of representative fields (x40 magnification). Between 100 and 150 cells were examined to determine the percent polarization.

 
Role of CIN85 in CD2 cell-surface expression
Upon surface receptor ligation, CIN85 has been shown to form a ternary complex with endophilin and tyrosine-phosphorylated c-Cbl that down-regulates EGF and C-met receptors via ubiquitination (46,47). In this regard, prior studies have shown that CD2 clustering triggers the phosphorylation of several intracellular components including c-Cbl (48) suggesting, by analogy, possible formation and recruitment of a related CIN85–endophilin–c-Cbl complex with the CD2 molecule. To investigate the role of CIN85 and CD2BP3 in controlling CD2 surface expression, COS-7 cells were transfected with CD2 and either Flag-CIN85 or Flag-FLCD2BP3, and triggered with anti-CD2 mAb. In some cultures, EGF was added as a further stimulus for c-Cbl phosphorylation. Figure 6 shows that in CIN85-transfected cells the triggering via anti-CD2 mAb results in a greater loss of surface CD2 molecules compared to similarly triggered CD2BP3-transfected cells. Significant CD2 down-modulation occurs after 1 h, with a 50% reduction in the surface CD2 expression, increasing to 70% after 3 h. EGF addition does not alter CD2 expression further, indicating that down-regulation does not involve EGF receptor–CD2 ‘cross-talk’. It is noteworthy that basal CD2 cell surface expression in CIN85-transfected cells is higher than in CD2BP3-transfected cells given that Western blotting of cell lysates with an anti-CD2 polyclonal antibody shows no difference in total CD2 in the two cell populations (Fig. 6 and data not shown). This finding suggests that CIN85 stabilizes CD2 expression on the resting T cell plasma membrane.



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  		Fig. 6. Surface CD2 down-modulation upon CD2 triggering in CIN85- but not CD2BP3-transfected cells. COS-7 cells were transfected with CD2 in combination with Flag-CIN85 or Flag-CD2BP3. After 48 h the cells were treated for the indicated time with anti-CD2 mAb in the presence or absence of EGF. The cells were then stained using FITC-conjugated anti-CD2 mAb (T111) and analyzed by FACS. The numbers represents the mean fluorescence intensity of the peak channel prior to or after cross-linking [untreated, black; CD2 cross-linked (CD2xL), gray; EGF + CD2xL, dotted].

 
Generation of CIN85/CD2BP3 specific mAb
To further probe aspects of CIN85/CD2BP3 biochemistry, subcellular localization and function, a panel of mAb was raised against recombinant CD2BP3 fragments. These antibodies were tested for their ability to recognize native protein by immunoprecipitation and denatured CIN85/CD2BP3 molecules in Western blotting or by immunofluorescence microscopic analysis of fixed and permeabilized cells. As summarized in Fig. 7(A), the five mAb define three binding patterns specific for CIN85/CD2BP3. 2B5 and 10B6 are excellent immunoprecipitating mAb while 23C9 and 12A6 represent good Western blotting reagents. 2C3 mAb differs from the four other mAb, immunoprecipitating CIN85 better than CD2BP3 and weakly identifying CD2BP3 in Western blots. Epitope specificity was further defined by investigating the capacity of each mAb to immunoprecipitate recombinant proteins representing the individual CD2BP3 SH3 domains (GST–SH3B and GST–SH3C) or a construct comprising the tandem domains including the intervening sequence (His–SH3BC). The 2C3 mAb reacts with CD2BP3 SH3BC as well as both individual SH3B and SH3C domains, suggesting recognition of a shared SH3 domain epitope. 23C9 and 12A6 mAb react with SH3BC and SH3C, but not SH3B, indicating their specificity for the CD2BP3 SH3C domain. By contrast, 2B5 and 10B6 mAb recognize the SH3BC protein, but neither individual SH3B or SH3C domains, implying that the epitope must be localized elsewhere, presumably in the intervening linker segment.



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  		Fig. 7. Intramolecular regulation of CIN85/CD2BP3 proteins revealed by mAb binding analysis. (A) Characterization of the binding specificity of a panel of anti-CD2BP3 mAb. In immunoprecipitation experiments (IP), each of the indicated mAb was coupled to CNBr-beads and incubated either with His–SH3BC or CIN85/CD2BP3 SH3B (GST–SH3B) and CIN85/CD2BP3 SH3C (GST–SH3C) and the associated proteins were separated in SDS–PAGE and revealed by Coomassie blue staining. Solid circles represent detectable binding, whereas open circles represent lack of binding. The same anti-CD2BP3 mAb-coupled beads were also used to immunoprecipitate the indicated full-length Flag-tagged proteins transfected in COS-7 as revealed by Western blotting (WB) with an anti-Flag mAb and interaction graded (++, + and +/–) relative to the signal resulting from anti-Flag mAb immunoprecipitation and Western blot. In the Western blotting experiments, the indicated molecules were immunoprecipitated with anti-Flag mAb from transfected COS-7 cells and the association was revealed by probing with anti-CD2BP3 mAb. Immunofluorescence experiments (IF) were performed in HeLa cells using FITC-conjugated anti-CD2BP3 antibodies. In the upper panel, cell lysates from J77 cells or primary T cells were immunoprecipitated using anti-CD2BP3 mAb-coupled CNBr beads, run in 8% SDS–PAGE and Western blotted with 23C9 anti-CD2BP3 mAb. (B) Microscopic analysis of CIN85/CD2BP3 and F-actin intracellular localization. HeLa cells were left untreated or treated with 10 ng/ml PMA for 10 min at 37°C, washed, fixed, permeabilized and subsequently stained with FITC-conjugated 23C9 anti-CD2BP3 and with Alexa Fluor 568–phalloidin to detect F-actin. (C) Staining of HeLa cells using FITC-conjugated anti-CD2BP3 mAb (23C9) reveals a co-localization with the mitotic spindle as shown in the images of two dividing cells. (D) Schematic representation of the CD2BP3 constructs. All constructs were generated by PCR, appending a Flag-tag at the N-terminus. (E) COS-7 cells were transfected with Flag-FLCD2BP3, Flag-{Delta}CD2BP3 and Flag-{Delta}proCD2BP3, lysed, and immunoprecipitated with beads coupled with an irrelevant IgG (control), anti-CD2BP3 (2C3) mAb or anti-Flag mAb. The samples were run in 8% SDS–PAGE and Western blotted with anti-Flag mAb. (F) COS-7 cells were co-transfected with c-Cbl HA and either Flag-CIN85, Flag-FLCD2BP3, Flag-{Delta}CD2BP3 or Flag-{Delta}proCD2BP3. Cell lysates were immunoprecipitated with anti-Flag, anti-CD2BP3 (2B5), anti-HA and irrelevant IgG (Control), and Western blotted with anti-Flag and anti-HA mAb as indicated.

 
Figure 7(A, top panel) shows the utility of the 23C9 mAb in Western blotting. Note that two bands at 83 and 90 kDa in Jurkat lysates, representing CD2BP3 and CIN85 proteins respectively, display somewhat anomalous mobility compared to their predicted mol. wt, perhaps as a consequence of phosphorylation. These bands are approximately equal in intensity. In contrast, in human resting and activated T cells the lower CD2BP3 band dominates (Fig. 7A). This observation suggests that in normal human T cells, the amount of CD2BP3 is greater than that of CIN85, possibly explaining why CD2BP3, but not CIN85, was isolated by yeast two-hybrid analysis, given that the cDNA library used in cloning was derived from activated T cells. Figure 7(B) shows immunofluorescence analysis of permeabilized HeLa cells stained with the FITC-conjugated 23C9 mAb and Alexa Fluor 568–phalloidin to localize CIN85/CD2BP3 and F-actin respectively. Note that, unlike the overlapping distribution pattern previously observed with CMS and F-actin (29), there is no analogous co-localization of CIN85/CD2BP3 and actin, consistent with the absence of putative actin-binding sites in the CIN85/CD2BP3 protein sequences. Note how 23C9 mAb also stains the cell nucleus, but not the nucleolus. The 23C9 mAb reacts with mitotic spindles as well (Fig. 7C). Identical patterns of CIN85/CD2BP3 intracellular staining were observed with the 12A6 mAb (data not shown).

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 SH3–proline-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 SDS–PAGE 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-{Delta}CD2BP3 molecule, the 2C3 mAb could bind as efficiently as anti-Flag mAb (2C3/Flag ratio = 0.99). Transfection results with the Flag-{Delta}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-{Delta}CD2BP3 and Flag-{Delta}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 domain–proline 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{epsilon} 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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  		Fig. 8. Degradation of CIN85 and CD2BP3 by TCR cross-linking. (A) Reduction in CIN85/CD2BP3 protein levels. Activated human T cells were stimulated with PMA or PMA + 2Ad2 (anti-CD3{epsilon}) mAb for the indicated time. Subsequently, the cells were washed, lysed and the detergent soluble fraction run on 8% SDS–PAGE. The proteins were transferred onto a nitrocellulose membrane and Western blotted first with 23C9 anti-CD2BP3 mAb and then with an anti-actin mAb as a loading control. (B) Graphic representation of CIN85/CD2BP3 protein degradation. The x-axis represents the duration of the treatment in minutes and the y-axis represents the percentage of CIN85/CD2BP3 proteins at any given time of the treatment (PMA, open circles; PMA + 2Ad2, solid circles) relative to the amount of those proteins in the absence of treatment. The amount of protein in the Western blot was measured by densitometry scanning of ECL-derived bands using the public domain NIH Image software. Due to the close approximation of CIN85 and CD2BP3 bands, the proteins were scanned together.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, we cloned by yeast two-hybrid interaction a human CD2 adaptor protein, termed CD2BP3, which binds to the human CD2 cytoplasmic tail in the region containing the C-terminal proline-rich segments. This gene is widely expressed in various cell types [(28) and our unpublished results]. In T lymphocytes, as well as in fibroblasts and epithelial cells, the encoded protein displays a curious distribution, being cytosolic as well as nuclear but exclusive of nucleoli. Analysis of mitotic cells shows strong reactivity of the mitotic spindle with CIN85/CD2BP3 mAb. Known involvement of members of the Pombe cdc15 homology family, including CD2BP1, in cytokinesis and actin distribution implies that this localization is significant (49–51). The differential expression of cell cycle regulators in the nucleolus and the exclusion of CIN85/CD2BP3 from this organelle are also likely more than fortuitous.

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 CIN85–endophilin–c-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 segment’s association with CIN85 SH3 domains and, hence, modulates CIN85–c-Cbl binding. As the ubiquitin ligase function of c-Cbl resides in its RING-finger domain (52), the CIN85–c-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{alpha} regulatory subunit’s SH3 domain (40). This interaction is of functional significance given that Ruk inhibits the enzyme’s ligand binding activity. Because the phosphatidylinositol-3-kinase signaling pathway mediates survival effects of certain growth factors (53–55), 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{alpha} 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 protein–protein 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 SH3–proline intramolecular associations has been demonstrated for the Tec tyrosine kinase (64) as well as the Bruton’s 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 protein–protein 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 E2–ubiquitin 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-Cbl–CD2BP3 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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  		Fig. 9. Schematic representation of the relation between CIN85, CD2BP3 and CMS in resting and activated T cells. In resting T cells (left), CD2 molecules are uniformly distributed on the cell surface. The cytoplasmic portion of a fraction of the CD2 molecules associates with the adaptor proteins CIN85 and CD2BP3. The level of CIN85 expression is lower than CD2BP3 as suggested by Western blotting analysis of total cell lysates (see text), but the affinity for CD2 interaction is greater for CIN85 than for CDBP3. The model also shows the adaptor protein, CMS, which has been previously found to be associated with CD2 in activated but not in resting T cells (17). When the T cell encounters an antigen-laden APC and is activated (right), CD2 molecules are recruited in the contact area through the interaction with CD58. Such clustering of CD2 mediates CMS association (17) and subsequent polarization of the Golgi apparatus towards the contact region (14) as TCR triggering by specific pMHCII (or I, not shown) ligand initiates a signaling cascade resulting in degradation of CIN85 and CD2BP3 by c-Cbl mediated ubiquitination. This process permits CMS to bind to CD2 in the absence of competition from CIN85/CD2BP3.

 

   Acknowledgements
 
This work was supported by NIH grant A121226. E. V. T. is presently a student of the PhD Program ‘Biomedical Biotechnologies’, University of Ancona Medical School, Ancona, Italy. We thank Drs Hailin Yang and Linda Clayton for careful review of the manuscript.


   Abbreviations
 
APC—antigen-presenting cell

CC—coiled-coil

CK2—casein kinase II

EGF—epidermal growth factor

FL—full length

HRP—horseradish peroxidase

IPTG—isopropyl-ß-D-thiogalactopyranoside

PKC—protein kinase C

PMA—phorbol 12-myristate 13-acetate

PTP—protein tyrosine phosphatase


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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