Linking the T Cell Surface Protein CD2 to the Actin-capping Protein CAPZ via CMS and CIN85*

Nicholas J. Hutchings {ddagger} §, Nicholas Clarkson {ddagger}, Robert Chalkley ¶ ||, A. Neil Barclay {ddagger} and Marion H. Brown {ddagger} **

From the {ddagger}Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, United Kingdom and the Ludwig Institute for Cancer Research, University College Branch of Cell and Molecular Biology, Royal Free and University College Medical School, London W1W 7BS, United Kingdom

Received for publication, March 12, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recruitment of CD2 to the immunological synapse in response to antigen is dependent on its proline-rich cytoplasmic tail. A peptide from this region (CD2:322–339) isolated CMS (human CD2AP); a related protein, CIN85; and the actin capping protein, CAPZ from a T cell line. In BIAcoreTM analyses, the N-terminal SH3 domains of CMS and CIN85 bound CD2:322–339 with similar dissociation constants (KD = ~100 µM). CAPZ bound the C-terminal half of CMS and CIN85. Direct binding between CMS/CIN85 and CAPZ provides a link with the actin cytoskeleton. Overexpression of a fragment from the C-terminal half or the N-terminal SH3 domain of CD2AP in a mouse T cell hybridoma resulted in enhanced interleukin-2 production and reduced T cell receptor down-modulation in response to antigen. These adaptor proteins are important in T cell signaling consistent with a role for CD2 in regulating pathways initiated by CMS/CIN85 and CAPZ.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD2 is a T and NK cell surface protein that mediates low affinity cell-cell interactions by binding to related immunoglobulin superfamily (IgSF) proteins, CD58 in humans and CD48 in rodents (reviewed in Ref. 1). In antigen-specific T cell activation, the interaction between CD2 and its ligand contributes toward lowering the threshold of activation by TCR in antigen-specific responses (2). Adhesion between the extracellular regions of CD2 and CD48 assists in the initial segregation of proteins in antigen-specific T cell activation (1). However, recruitment of CD2 into the central contact zone during formation of an immunological synapse is dependent on the cytoplasmic tail of CD2 (3). Among the proteins to which CD2 is related in its extracellular region (4), CD2 has a unique mechanism of engagement of intracellular machinery based on proline-rich motifs. The membrane-distal region of the cytoplasmic tail contains the sequence HQQKGPPLPRPRVQPKPP, which is conserved in CD2 from many species. Deletion in this region impairs signaling events induced by CD2 mAbs1 (5). CD2AP isolated in a yeast two-hybrid screen with the cytoplasmic tail of CD2 contains three SH3 domains (3). The N-terminal SH3 domain appeared to have a relatively high specificity and avidity for this conserved region of the CD2 cytoplasmic region (3). In cells, a truncated protein containing only the two most N-terminal SH3 domains of CD2AP disrupted T cell polarization, implicating CD2 in cytoskeletal rearrangement (3). Antigen-specific engagement was required for proper cluster formation by CD2, leading to speculation as to whether CD2 regulates the cytoskeleton or vice versa (3). There are data showing that other SH3 domains have the potential to bind the distal conserved region of CD2 (3, 6, 7). CD2BP2, a protein containing a novel GYF domain, is a candidate for interacting with a more membrane-proximal conserved motif PPPPGH, which is repeated exactly in human (8). A molecular interaction has not yet been defined to explain the role of the distal end of the CD2 tail in enhancing the avidity for its cell surface ligand (9).

We identified proteins that interact with the CD2 cytoplasmic region and show that there is a direct link through CMS and CIN85 with the actin capping protein, CAPZ. We show these proteins have a role in regulating signaling in T cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Peptides—The peptides were synthesized with an N-terminal biotin (Table I). CD2 peptide sequences are numbered according to Swissprot: P06729 [GenBank] . CD2:322–339* was used in peptide pull-down experiments. Position 14 differs from Swissprot:P06729. No difference in specificity of binding between CD2:322–339* (not shown) and CD2:322–339 (see Figs. 3 and 4) was observed in BIAcoreTM experiments.


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TABLE I
Peptides

 


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FIG. 3.
CMS and CIN85 bind directly to CD2:322–339 with similar affinity. At 37 °C, the proteins were passed at indicated concentrations (µM) over peptides immobilized on streptavidin in a BIAcoreTM. The amount of peptide bound to the chip is given in RU in brackets, and the period of injection of protein indicated by the bars over the traces. A, CMS-SH3d1 was passed over CD2:322–339 (solid line; 116 RU) and control CD2:281–305 (dashed line; 240 RU). B, CIN85-SH3d1 was passed over CD2:322–339 (solid line; 110 RU) and control CD2:281–305 (dashed line; 101 RU. C and D, specific equilibrium binding values from A and B were plotted (circles) and KD calculated by nonlinear curve fitting (line) and Scatchard analysis (line) (insets). E, specific equilibrium binding values are plotted for CD2BP1-SH3 passed over CD2:322–339 (closed circle; 703 RU; closed square, 474 RU) and CD281–305 (open circle; 787 RU). F, cCAPZ (~46 µM) does not bind CD2:322–339 (solid line; 711 RU) or CD2:281–305 (dashed line; 811 RU) or ICOS-P (not shown; 699 RU) but (inset) (~4.6 µM) does bind anti-{alpha} (5B12.3) (solid line; 440 RU) and anti-{beta} (1E5.25.4) (dashed line; 558 RU) cCAPZ mAbs immobilized on anti-mouse Fc.

 


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FIG. 4.
CAPZ does not bind SH3 domains but does bind the C-terminal region of CMS. A, CIN85-SH3 (0.5 µM) did not bind immobilized cCAPZ (solid line; 2507 RU) or control streptavidin (dashed line; 1423 RU) but does bind CD2:322–339 (not shown). The inset shows CIN85-SH3 (2 µM) binds to immobilized CD2:322–339 (solid line; 711 RU) not CD2:281–305 (dashed line; 868 RU) or ICOS-P (dotted line; 699 RU). B, CMS-C (1.3 µM) bound to immobilized cCAPZ (1742 RU) but not to CD2:322–339 (150 RU) or to a blank flow cell. C and D, CMS-C (C) and CIN85-C (D) (1.3 µM) bound immobilized hCAPZ (4265 RU) not CD2:322–339 (640 RU), whereas (C, inset) CMS-CC (1.1 µM) did not bind immobilized hCAPZ (solid line; 2425 RU) or CD2:322–339 (dashed line; 640 RU). E and F, hCAPZ (0.42 µM) bound immobilized CMS-C (E, solid line; 1330 RU) and CIN85-C (F, solid line; 924 RU) but not CD2:322–339 (E and F, dashed lines; 664 RU).

 

Pull-down Experiments—The human T cell line Jurkat cells were washed twice in PBS and lysed at 108/ml in 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM EDTA, 10% glycerol, and 1% Brij-96 detergent. Protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 50 mM benzamidine, 1 mM sodium vanadate, 1 mM NaF were added immediately prior to use. The lysates were rotated at 4 °C for 60 min and then clarified at 10,000 x g (13,000 rpm) in a Microfuge or 2,000 x g (3,000 rpm) in a Beckman benchtop GPR centrifuge at 4 °C for 10 min and then filtered (0.45 µm). Streptavidin-coated Dynabeads (Dynal A.S., Oslo, Norway) were washed once in PBS before being saturated with biotinylated peptides. At least 20-fold excess peptide (16 µg) over the bead (25 µl at 10 mg/ml) capacity was used. After 30 min at 4 °C beads were washed twice in PBS. 25 µl of beads and 2.5 x 108 cell equivalents of lysate were used for each lane of the gel. The control beads were saturated with d-Biotin in at least 100-fold excess. The beads were rotated for 2 h at 4 °C in the lysate, transferred to new tubes, and washed three times with 200 µl of PBS in a Dynal MPC-E magnetic particle concentrator with a change of tube during each wash. Washed beads were resuspended in 10 µl of NuPAGE SDS sample buffer (Novex, San Diego, CA), reduced with 1% (w/v) dithiothreitol, vortexed, and incubated at 70 °C for 10 min. The beads were pelleted in a microcentrifuge, and supernatant was run on NuPAGE 4–12% Bis-Tris gels (Novex) in an X-Cell II gel tank (Novex) using MOPS SDS running buffer (Novex). The gels were stained with Coomassie, and the protein bands were excised from the gel and analyzed by trypsin digestion and mass spectrometry (10). After overnight trypsin digestion, the peptides were extracted twice with 30 µl of 50% acetonitrile, 5% trifluoroacetic acid. Finally, the dried peptide solution was resuspended in 10 µl of H2O.

MALDI Mass Spectrometry—0.5 µl of saturated 2,5-dihydroxybenzoic acid in H2O was mixed on-target with 0.5 µl of sample and dried. Peptide mass fingerprints were acquired on a REFLEX III mass spectrometer (Bruker Daltonics, Coventry, UK). The spectra were internally or externally calibrated (Calibration Mixture 2 from a Sequazyme kit, Applied Biosystems, Warrington, UK).

Data interpretation was carried out using the Prospector Suite of search programs (prospector.ucsf.edu/). Peak lists from MALDI spectra were input into MS-Fit, and the NCBI nonredundant protein data base was searched.

Constructs—Maltose-binding protein fusion proteins were constructed using the pMAL-c2X vector (www.neb.com). Templates CMS (11) and CIN85 (12) for amplification by PCR using cloned Pfu polymerase were provided by K. Kirsch and S. Kaijigaya, respectively. The fragments were cloned in-frame into vector cut with BamHI and SalI. SH3 domains with 5' BamHI and 3' SalI sites lacked the initiator Met and contained a stop codon after the following C-terminal sequences CIN85-SH3, FVKLLPP; CMS-SH3d1, KEIKRE; and CIN85-SH3d1, REIKKE. C-terminal regions were cloned using an engineered 5' BamHI site for CIN85-C, a natural BglII site for CMS-C, and a XhoI site after the stop codon producing the following N-terminal sequences after the junction with pMAL: CMS-CC, IVEALK; CMS-C, SGTVYP; and CIN85-C, GALPPR. CD2BP1-SH3 was amplified from human PBL cDNA and fused to pMAL, the sequence codons for SPAQEY following the BamHI site and with a SalI site after the stop codon. In the T cell hybridoma, PLEGFP-C1 (www.clontech.com) was used for expression of EGFP fused to CD2AP-C, a HindIII (1078 bp)-XhoI (2426 bp) restriction fragment of CD2AP (M. Dustin and A. Shaw, St. Louis, MO), CD2AP-SH3d1-CC (CMS and CD2AP SH3d1 are identical, CD2AP terminology is used in the mouse experiments) constructed in two steps using a 3' primer containing adjacent BglII and HindIII sites. The intermediate vector was cut with BglII and SalI, and the CMS-CC fragment was inserted, which produced the joining sequence ERSIVE. CMS-CC was also inserted into pLEGFP-C1 to provide a dimeric EGFP control. A mouse CD2 pBabe construct was made by Anna Cambiaggi.

Proteins—pMAL fusion proteins were expressed and affinity-purified using standard procedures with amylose resin (www.neb.com). SH3 constructs were further purified by gel filtration using Superdex 75 (Amersham Biosciences). Chicken CAPZ {alpha}1{beta}1, which was the first CAPZ available to us was expressed using a construct provided by T. Obinata (13) and purified from bacterial lysates containing 0.15 M NaCl by ion exchange on Q Sepharose using a binding buffer containing 10 mM Tris-HCl, pH 8.0, 0.05 M NaCl and eluted with the same buffer and increasing NaCl concentration, with CAPZ eluting at about 0.4 M NaCl. A single peak containing the heterodimeric CAPZ was isolated by gel filtration on Superdex 200. Human erythrocyte CAPZ was provided by P. Khulman (14). Attempts to produce full-length CMS and CIN85 in bacteria as pMAL fusion proteins and in insect cells with a His-biotin tag were hampered by low level expression and degradation and not pursued.

Confirmatory sequencing of all constructs was obtained using Big-DyeTM (ABI) Sanger dideoxynucleotide method on a Prism 377 DNA or 3100 Genetic Analyzer sequencer (ABI). Purified proteins were analyzed by SDS-PAGE and Coomassie staining. The concentrations were estimated by absorption at 280 nm in a 1-cm flow cell using theoretical extinction coefficients for the pMAL fusion proteins: CMS-SH3d1 = 74,250 M1 cm1 and CIN85-SH3d1 = 77,380 M–1 cm1. Experimentally determined extinction coefficients determined in triplicate for CMS-SH3d1 (76,973, 79,501, and 88,107 M–1 cm1) are similar to the theoretical value.

BIAcore Analysis—The experiments were carried out using a BIAcore 2000 at 25 and at 37 °C (15, 16) for affinity measurements with SH3d1 proteins using buffers, anti-mouse Fc{gamma}, and amine coupling to research grade CM5 chips (BIAcore AB) using flow rates of 10 µl/min and for equilibrium binding 20 µl/min. Chicken CAPZ mAbs 5B12.3 and 1E5.25.4 were obtained from Development Studies Hybridoma Bank (www.uiowa.edu/~dshbwww). Biotinylated peptide solutions were injected in HEPES-buffered saline, pH 7.4, at 400 ng/ml over immobilized streptavidin (3000–4000 RU). CMS, CIN85, and cCAPZ were directly immobilized by amine coupling in 10 mM NaAc buffer, pH 5.0. The response units immobilized are in the relevant figure legends. An extra washing step after blocking with ethanolamine was avoided to avoid possible inactivation of immobilized material. For affinity measurements, 5-µl injections of increasing and decreasing concentrations of monomeric SH3d1 proteins were passed over immobilized peptides at 20 µl/min at 37 °C, and the data were analyzed as described (15, 16).

Antigen-specific T Cell Hybridoma assays—2B4 mouse T cell hybridoma and Chinese hamster ovary cells expressing mouse I-Ek were used as model T and antigen presenting cells, respectively, as described (17). CD2+ 2B4 T cell hybridoma cells were made by Anna Cambiaggi (Oxford, UK) using calcium phosphate transfection of the BOSC 293T packaging cell line, retroviral transduction, and selection using puromycin at 1 µg/ml. Several clones were pooled to produce a polyclonal population. PLEGFP-C1 constructs were introduced into the CD2+ and CD2– T cell hybridomas by retroviral transduction, with recombinant virus being produced by transfection using FuGENE (Roche Applied Science) into the Phoenix packaging cell line. Antigen-specific IL-2 production and TCR down-modulation experiments were essentially as previously described (17) with the following differences. For both antigen stimulation and IL-2 production, round bottomed plates were used in the initial experiments (i.e. Fig. 6), and subsequently, flat bottomed plates were used. To measure IL-2 production, 8,000 HT2 cells (50 µl) were incubated with supernatants (50 µl: usually 1:1 and 1:5) for 16 h followed by a 6-h pulse with 0.8–1 µCi of [3H]thymidine. IL-2 production of triplicate or quadruplicate cultures was calculated using a standard of recombinant human IL-2 and curve fitting in PRISM. Down-modulation experiments were initially carried out in plates, with triplicate cultures being pooled into one fluorescence-activated cell sorter tube (Fig. 7B), but subsequently the whole assay was performed in fluorescence-activated cell sorter tubes (Fig. 7C). Optimal staining was obtained using a three-step staining procedure with KT3 (anti-mCD3), biotinylated anti-rat Ig (Jackson Labs), and Streptavidin-Quantum Red (Sigma). The stained cells were fixed with 2% paraformaldehyde and read the following day. The median fluorescence was determined from cells gated for the same levels of EGFP.



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FIG. 6.
Overexpression of CD2AP-C in a T cell hybridoma enhances IL-2 production in response to antigen. A–C, EGFP fusion proteins containing CD2AP-C (A) and CMS-CC localized to the plasma membrane of 293T cells (B), whereas EGFP remained cytoplasmic (C). D and E, level of expression of EGFP (D) and CD3 (E) in CD2+ hybridoma cells expressing no (thin line) or EGFP fusion proteins of CD2AP-C (thick solid line), CMS-CC (dashed line), and EGFP control (dotted line). F and G, IL-2 production in response to with (+Ag) and without (–Ag) antigen (moth cytochrome peptide; mcc), dose dependence (F) and constant 0.023 µM moth cytochrome c (G) by cells expressing CD2AP-C, CMS-CC, or control vector (EGFP). Raw data are shown in F, and IL-2 production calculated from the linear part of the IL-2 response curve is shown in G. Inset in F, enhanced IL-2 production was also observed in response to concanavalin A.

 


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FIG. 7.
Interactions of the N-terminal SH3 domain of CD2AP are important in regulating T cell signaling, and the mechanism involves TCR down-modulation. A, IL-2 production in response to antigen (30 nM moth cytochrome c) by cells expressing CD2AP-C, CD2AP-SH3D1-CC, and CMS-CC. B and C, TCR down-modulation by cells expressing CD2AP-C, CD2AP-SH3D1-CC, and CMS-CC in response to antigen (1 µM moth cytochrome c). Two (of >5) representative experiments are shown.

 


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CMS, CIN85, and CAPZ Were Isolated from Jurkat Cell Lysates by CD2 Peptide 322–339 —To identify proteins interacting with the cytoplasmic tail of CD2, synthetic peptides were coupled to magnetic beads and used as an affinity matrix in "pull-down" experiments to purify associated proteins from extracts of the Jurkat T cell line. Four bands seen in analysis by SDS-PAGE and Coomassie Brilliant Blue staining (Fig. 1) with peptide CD2:322–339 but not with other peptides including CD2:281–305 (data not shown) were identified (Table II). Band 2 represents CMS, the human homologue of CD2AP (11). Band 1 is a closely related protein CIN85 (12), which has the same overall structure as CMS. Bands 3 and 4 contained the {alpha} and {beta} subunits of the actin capping protein, CAPZ. CAPZ{alpha} exists in two closely related isoforms ({alpha}1 and {alpha}2) that differ at the C termini (18). Band 3 contained both CapZ {alpha}1 and {alpha}2 isoforms, because peptides unique to {alpha}1 and {alpha}2 were present (Table II). Peptide data from band 4 matched the human CAPZ{beta}2 subunit (19) (Table II), which is the dominant isoform in nonmuscle cells (20).



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FIG. 1.
CMS, CIN85, and heterodimeric capping protein CAPZ are isolated from Jurkat cell lysates by CD2:322–339 peptide. Coomassie-stained SDS-PAGE reducing gel showing Jurkat cell lysate and bands specifically isolated by beads coated with CD2:322–339 peptide but not with biotin alone. See Table II for identification of bands 1–4 as CIN85, CMS, CAPZ{alpha}1 and CAPZ{alpha}2, and CAPZ{beta}2.

 

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TABLE II
Identities of bands 1–4 in Fig. 1

The bands were digested with trypsin and analysed by MALDI mass spectrometry. The percentage sequence coverage observed in the MALDI mass fingerprints is indicated. A contaminating protein, peanut glycinin was identified in band 1.

 

CMS and CIN85 but Not CAPZ Bind Directly to CD2:322–339 Direct interaction between the mouse homologue of CMS, CD2AP, and the cytoplasmic region of CD2 contained in CD2: 322–339 has been previously demonstrated (3). The N-terminal SH3 domain of CD2AP mediated binding. Both this SH3 domain and the CD2:322–339 peptide have identical sequences in mice and humans. To confirm that CMS was binding directly to CD2:322–339 via its N-terminal SH3 domain and to ask whether CIN85 had comparable specificity, we tested binding of recombinant forms of CMS-SH3d1 and CIN85-SH3d1 (Fig. 2) to peptides using a BIAcoreTM. The N-terminal SH3 domains of CMS and CIN85 are the most similar, being 68% identical. Care was taken to conduct experiments with monomeric material within hours of elution from a gel filtration column. CMS-SH3d1 was injected over immobilized peptide, and the data are shown for five concentrations in Fig. 3A. CD2:281–305, which did not bind pMAL fusion proteins containing all three SH3 domains of CMS (not shown) or CIN85 (Figs. 2 and 4A, inset), was used as a negative control peptide. CMS-SH3d1 bound to CD2:322–339 and not to CD2:281–305 (Fig. 3A). CIN85-SH3d1 bound in a similar fashion (Fig. 3B) The signals for CMS-SH3d1 and CIN85-SH3d1 passing over the control CD2:281–305 are due simply to the high concentration of protein necessary for detection of weak interactions. Binding was characteristic of a low affinity monomeric interaction with rapid kinetics enabling a quantitative comparison between the two proteins to be made by calculating the KD from equilibrium binding values over a range of concentrations of soluble CMS or CIN85 SH3d1 (Fig. 3, C and D). Fitting binding data to a nonlinear hyperbole and Scatchard analysis (Fig. 3, C and D, insets) showed that CMS-SH3d1 and CIN85-SH3d1 bound CD2:322–339 according to a Langmuir model and with similar dissociation constants, 105 and 76 µM, i.e. in the order of 100 µM. Duplicate sets of data gave comparable results: CMS, 76 µM and CIN85, 72 µM. Slightly lower levels of immobilized peptide resulted in KD values of 129 and 127 µM for 127 and 76 and 77 µM for CIN85.



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FIG. 2.
Purified proteins used to characterize interactions with peptides from CD2 cytoplasmic region. A, CMS and CIN85 are structurally similar, each comprising three SH3 domains (rectangles), numbered from the N terminus 1, 2, and 3 followed by a prolinerich terminal half (C) and a predicted coiled-coil (CC) region at the C terminus. pMAL (represented by an oval) fusion proteins were engineered from fragments of CMS and CIN85. CMS-SH3d1 and CIN85-SH3d1, pMAL fused to the N-terminal SH3 domain of CMS and CIN85; CIN85-SH3, the three SH3 domains of CIN85; CMS-C and CIN85-C, the C-terminal half of CMS and CIN85; CMS-CC, the coiled-coil region of CMS. B, purified proteins. Lanes 1–7, pMAL fusion proteins shown in A. Lane 1, CIN85-SH3; lane 2, CMS-SH3d1; lane 3, CIN85-SH3d1; lane 4, CD2BP1-SH3; lane 5, CMS-CC; lane 6, CMS-C; lane 7, CIN85-C. Lanes 8 and 9, heterodimeric CAPZ marked with arrows. Lane 8, human erythrocyte CAPZ hCAPZ; lane 9, recombinant chicken CAPZ, cCAPZ.

 

We did not detect in our pull-down experiments another SH3 domain containing protein, CD2BP1, previously reported to interact with the region of CD2 covered by CD2:322–339 in either the absence or the presence of presence of 200 µM Zn2+ ions (7). This may have been due to the sensitivity of the assay, and this putative interaction was tested using purified monomeric pMAL fusion protein containing the SH3 domain of CD2BP1, CD2BP1-SH3 (Fig. 2B, lane 4). An interaction was detected at 37 °C, but levels of binding were low despite the high levels of peptide immobilized, and the interaction showed less specificity than CMS or CIN85 because CD2BP1 bound to both CD2:322–339 and CD2:281–309 peptides (Fig. 3E). In experiments using immobilized peptide levels that gave clear CMS-SH3 binding at 4 µM, no binding was detectable with CD2BP1-SH3 at 40 µM even in the presence of 2 mM MgCl2 (data not shown).

CAPZ does not contain domains that are known to bind proline-rich sequences (21) and was not predicted to bind directly to the CD2 cytoplasmic tail. To distinguish between direct and indirect interactions, recombinant chicken CAPZ {alpha}1{beta}1, which was available to us (cCAPZ; Fig. 2B, lane 9), CAPZ being highly conserved among species, was tested for interaction with CD2 peptides. Chicken CAPZ (46 µM) (Fig. 2) did not bind CD2 peptides (Fig. 3F) but was antigenically active because it bound mAbs specific for either chicken CAPZ {alpha}1 and {beta}1 (Fig. 3F, inset).

CAPZ Binds the C-terminal Region of CMS and CIN85 but Not the Coiled-coil Region of CMS—The lack of binding of CD2 peptides by cCAPZ was consistent with CAPZ being associated with the CD2 peptide via CMS and/or CIN85. The lack of proline-rich sequence in CAPZ suggested the mechanism of interaction between CAPZ and CIN85 or CMS would not involve the SH3 domains. This prediction was correct because CIN85-SH3d1–3 did not bind to immobilized cCAPZ (Fig. 4A), whereas CIN85-SH3d1–3 bound CD2:322–339 peptide (inset). A C-terminal fragment of CMS (CMS-C; Fig. 2, A and B, lane 6) was tested for interaction with immobilized cCAPZ. It bound avidly (Fig. 4B) to cCAPZ but not to CD2:322–339. This result with cCAPZ {alpha}1{beta}1 heterodimer shows that the interaction is not CAPZ {beta} isoform-specific because the human CAPZ (hCAPZ) identified in the pull-down experiments contained the {alpha}1{beta}2 heterodimer. The avid binding is consistent with CMS-C being dimeric as shown by gel filtration (data not shown and Ref. 11). CMS-C also bound directly to human CAPZ {alpha}1{beta}2 heterodimer (Figs. 4C and 2B, lane 8) and not to CD2:322–339. A motif (VEALK) within the coil-coil region of CMS known to selfassociate (11) is also present in the C terminus of human CAPZ {beta}2, raising the possibility that this region mediated binding to CAPZ. However, the coiled-coil region of CMS, CMS-CC (Fig. 2, A and B, lane 5), which formed a tetramer as described for CIN85 (22) demonstrating a capability to form higher order complexes, did not bind to CAPZ (Fig. 4C, inset). CIN85-C (Fig. 2, A and B, lane 7) was also dimeric (data not shown) and bound immobilized hCAPZ with high avidity (Fig. 4D). In the reverse orientation, hCAPZ bound to immobilized CMS and CIN85, showing that binding was not an artifact of CAPZ immobilization (Fig. 4, E and F). Thus CAPZ interacts through a discrete fragment of the CMS/CIN85 C-terminal region, which is distinct from the upstream putative endophilin-binding sites (23) and three of the four putative actin binding sites in the coiled-coil region at the C terminus of CMS (11) (Fig. 5).



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FIG. 5.
CMS and CIN85 bind CAPZ, providing a link between the cell surface protein CD2 and the actin cytoskeleton.

 

Overexpression of the C-terminal Region of CD2AP Enhances Antigen-specific IL-2 Production—The functional consequences of disrupting CMS/CIN85 binding to CAPZ and potentially a link between CD2 and the actin cytoskeleton were explored. Responses of a T cell hybridoma expressing mouse CD2 to antigen presented by Chinese hamster ovary cells with major histocompatibility complex class II on their surface were measured (17). For these experiments we used a C-terminal fragment of the mouse equivalent of CMS, CD2AP, to maintain optimal interactions in the murine hybridoma. The N-terminal SH3 domains of CMS and CD2AP are identical; thus CMS-SH3d1 and CD2AP-SH3d1 are interchangeable, and for consistency in describing the functional experiments in mouse cells, we have used the nomenclature CD2AP-SH3d1. Expression of retroviral constructs encoding EGFP fusion proteins was readily observed by microscopy in the packaging cell line where fluorescence of CD2AP-C (Fig. 6A) and CMS-CC (Fig. 6B) fusion proteins was consistent with localization at the plasma membrane in contrast to EGFP (Fig. 6C), which remained cytoplasmic. Membrane localization of CMS-CC is consistent with it mediating interactions either by its capacity to form tetramers or heterodimerization (24). 2B4 T hybridoma cells expressing these constructs were produced and tested for comparable levels of EGFP (Fig. 6D) and CD3 (Fig. 6E). EGFP levels were high enough to quantitate by flow cytometry but very faint in microscopic examinations. The cells were then assayed for antigen-specific IL-2 production (Fig. 6, F and G). Antigen dose response is shown in Fig. 6F as [3H]thymidine incorporation to establish the criteria for subsequently comparing levels of IL-2 production for one antigen concentration in the linear part of the curve (Fig. 6G). Cells expressing CD2AP-C gave enhanced levels of IL-2 in response to antigen and concanavalin A in contrast to cells expressing CMS-CC or EGFP (Fig. 6, F and G). The effects of CMS-CC relative to a high level of EGFP were marginal in contrast to CD2AP-C. In subsequent experiments CMS-CC was used as the sole control vector.

Interactions of the N-terminal SH3 Domain of CD2AP Are Important in Regulating T Cell Signaling—It is the specific location of the CD2-CD2AP (or CIN85) interaction that will be important in the way CD2 utilizes this pathway in the cell. Thus it was essential to know whether disruption of binding to the N-terminal SH3 domain of CD2AP is significant. To directly compare with the dimeric CD2AP-C, we constructed a dimeric form of CD2AP-SH3d1 by fusing it to the coiled-coil region of CMS. As expected based on the behavior of CMS-CC, CD2AP-SH3d1-CC localized to the plasma membrane, as did constructs expressing CIN85-SH3d1-CC and CD2BP1-SH3d1-CC (data not shown). Antigen-specific IL-2 production was enhanced by expression of CD2AP-SH3d1-CC relative to cells expressing CMS-CC (Fig. 7A). CD2AP-SH3d1-CC had a significant effect, although with comparable levels of EGFP and CD3 (data not shown), CD2AP-C was more effective than CD2AP-SH3d1-CC in the enhancement of IL-2 production (Fig. 7A). Overexpression of truncation mutants of CIN85 potentiates signaling by inhibiting receptor-mediated endocytosis (23, 25). Cells expressing EGFP fusion proteins of CD2AP-C, CD2AP-SH3d1-CC, and, as a control, CMS-CC were compared for their effects on antigen-induced TCR down-modulation. The same trend was observed reproducibly (Fig. 7, B and C). Relative effects on down-modulation were comparable with those observed for EGF receptor internalization in similar experiments (25). CD2AP-C and CD2AP-SH3d1-CC inhibited TCR down-modulation consistent with enhanced IL-2 production being the result of prolonged signaling. As for results for IL-2 production, in cells with comparable EGFP and CD3 levels, CD2AP-C appeared more effective than CD2AP-SH3d1-CC.

We did not observe a reproducible CD2-dependent effect in IL-2 production or TCR down-modulation between CD2+ and CD2– T cell hybridoma cells (data not shown). Comparisons were made using antigen presenting Chinese hamster ovary cells, which did not express CD48 to avoid the complication of the dominant effects of the CD2-CD48 interaction. The responses were enhanced only in CD2+ cells when CD48+ antigen presenting Chinese hamster ovary cells were used (data not shown). Similar effects of CD2AP-SH3d1-CC expression on IL-2 production and TCR down-modulation were observed in CD2+ and CD2– hybridoma cells (data not shown). The dominant effects of adhesion between CD2 and CD48 reduced the relative effects of the EGFP fusion proteins in CD2+ cells and did not assist in assessing a role for the CD2 cytoplasmic region.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biochemical data presented here provide proof that there is a direct link between the cell surface receptor, CD2, and the cytoskeleton via CAPZ, and this is shown schematically in Fig. 5. CMS and CIN85 are widely expressed, and linkage to CAPZ has implications for a fundamental role in regulation of the cytoskeleton in many cell types not just in T cell triggering (11, 12, 26). Direct interaction between CMS/CIN85 and CAPZ provides a molecular basis for interpreting other biochemical data (24) and observations made on the localization of components of the actin cytoskeleton in cells (27). CAPZ colocalizes with actin and the Arp2/3 complex in leading edges and in small spots of actin assembly in the lamella of motile cells (27). A similar distribution of CMS (11) and CD2AP (30) has been demonstrated in transfected and normal cells, respectively. Unpublished data describing CAPZ and CD2AP colocalization are referred to in Ref. 27. The interaction with CAPZ is common to CMS and CIN85 in contrast to claims of direct binding of actin by CMS (28) and not CIN85 (29).

Comparable affinities of two structurally related proteins for the highly conserved region of the CD2 cytoplasmic tail support a model for building complex networks as against linear signaling in response to a single input signal (31) if CMS and CIN85 coupled to different downstream effectors. The affinities of CMS-SH3d1 and CIN85-SH3d1 for CD2:322–339 peptide at a KD level of ~100 µM are at the lower end of the range reported for other synthetic peptides binding to SH3 domains (21, 32). A low affinity may be an advantage in responding quickly to changes in the micro-environment (31). Given the broad distribution of CMS and CIN85 (11, 12, 26), specificity of any in vivo interaction with CD2 appears to be determined by the distribution of CD2.

The N-terminal domains of CMS and CIN85 clearly have a greater affinity for CD2 than other candidates (3, 6, 7, 29). Other candidates include CD2BP1, which is restricted to hematopoietic tissue, in particular T and NK cells, which express CD2 (7). We showed that the SH3 domain of CD2BP1 does have some capacity to bind CD2:322–339 but that it has a much weaker affinity for the peptide than CMS and CIN85, it showed less specificity, and its significance is debatable (33). An interaction between the region of CD2 encompassed by CD2:322–339 and other SH3 domains has been previously reported (3, 6), and although monomeric affinities were not measured at 37 °C, relative levels of binding with GST fusion proteins indicated that CD2:322–339 had a marked preference for CD2AP (3, 6). Ligands for the N-terminal SH3 domain of CMS and CIN85 have not been identified in cells lacking CD2, but their existence is implied from functional data discussed below. CD2AP binds the cytoplasmic region of the cell surface receptor, nephrin through a different mechanism involving the C-terminal region of CD2AP (34). The second N-terminal SH3 domains of CMS and CIN85 are crucial for binding c-Cbl (12, 35).

CAPZ is involved in control of actin polymerization and associated functions and for instance is essential for Listeria movement (36). A reduction in CAPZ concentration will reduce the rate of treadmilling and thus slow the rate of actin movement (36). However, filament length is not accounted for by a simple treadmilling model leading to speculation that CAPZ is regulated in vivo (3739). CMS and CIN85 have been reported to bind various other intracellular proteins in a manner that can be regulated (11, 12, 22, 35, 40), thus through interaction with the multiple protein binding motifs and domains in CMS and CIN85 there are multiple possibilities for regulating CAPZ activity. The phenotype of the CD2AP (mouse CMS)-deficient mouse is consistent with a role of CD2AP in regulating movement because podocytes do not form properly in the knockout mouse (26).

Disruption of interactions, which include the link with the actin cytoskeleton through the C-terminal regions of these adaptor proteins leads to loss of regulation of receptor signaling. Antigen-specific responses in a T cell hybridoma were enhanced by expression of the C-terminal region or N-terminal SH3 domain of CD2AP. A corresponding reduction in TCR down-modulation indicates a mechanism reported recently for receptors on other cell types (23, 25). A similar approach to perturbing CIN85 function resulted in potentiated signaling caused by a loss of receptor-mediated endocytosis (23, 25). The role ascribed to CIN85 in receptor-mediated endocytosis is attributed to its association with endophilin (23, 25). Interestingly the EGFP-CD2AP-C protein did not contain the putative endophilin site that is conserved between CD2AP (CMS) and CIN85 (23); thus interactions of the more distal region of these proteins are important in receptor internalization.

Interfering with binding to the N-terminal SH3 domain had a marked effect on adaptor-regulated receptor internalization (Fig. 7 and Ref. 25). However, a role for CD2 in the pathways initiated by CMS/CIN85 and CAPZ in regulating TCR signaling has not yet been defined. Hyper-responsiveness has been observed in CD2-deficient mice where thymocytes are more readily positively selected (42, 43), which could also be explained by a failure to down-regulate TCR. Failure to down-regulate TCR has indeed been observed in T cells from CD2-deficient mice (2). The difficulty in interpreting these experiments in models using CD2-deficient mice is distinguishing between the effects on extracellular and intracellular interactions of CD2. This is avoided in the model of transgenic mice expressing human CD2, which is not engaged by endogenous CD48 so that effects can be directly attributed to the cytoplasmic region (44). However, comparisons between TCR down-modulation in TCR transgenic mice null, heterozygous, or homozygous for a single copy of human CD2 (44) did not reveal a consistent trend.2 Discerning a role for CD2 in the pathways initiated by CMS/CIN85 and CAPZ will require a more refined assay system.

The key to understanding why CD2 has hijacked this general cell biological mechanism in a T cell will be its effect on localization of CMS (CD2AP) and CIN85. Concentration of CD2 at the contact site between a T cell and an antigen presenting cell is initially dependent on the optimal size of the complex it forms with its cell surface ligand, thus aiding in recruiting CD2AP (41). This supports a model in which CD2 influences the organization of the actin cytoskeleton. The CD2AP-CD2 interaction is enhanced on cell activation (3), providing a molecular explanation for earlier observations where cross-linking CD2 with mAbs led to immobilization of CD2 indicative of an interaction with the cytoskeleton (45). Cross-linking with CD2 mAbs provided evidence that perturbation of CD2 can affect the activity of actin-associated proteins as it resulted in cofilin dephosphorylation and translocation (46). However, the effects of cross-linking CD2 by mAbs are dependent on engagement of a classical signaling receptor (47). Thus the dependence of CD2 reorganization on engagement of TCR by antigen can be interpreted as regulation of CD2 distribution by the cytoskeleton (3). This latter scenario is more in keeping with the model proposed for CD43 where there are clear data showing cytoskeleton-dependent relocation of CD43 (48). Either way, whether a cell surface receptor regulates the cytoskeleton or it is the other way around, a link between CD2 and actin via CMS/CIN85 and CAPZ provides a firm molecular basis for a mechanism.


    FOOTNOTES
 
* This work was supported by the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Everest Biotech. Ltd, Littlemore Park, Oxford OX4 4SS, UK. Back

|| Present address: Dept. of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0446. Back

** To whom correspondence should be addressed: Sir William Dunn School of Pathology, South Parks Rd., Oxford OX1 3RE, UK. Tel.: 01865-275595; Fax: 01865-275591; E-mail: Marion.Brown{at}path.ox.ac.uk.

1 The abbreviations used are: mAb, monoclonal antibody; CIN85-SH3, CIN85 SH3 domains 1–3; CD2BP1-SH3, CD2BP1 SH3 domain; -SH3d1, SH3 domain 1; -C, C-terminal region; -CC, coiled-coil region; pMAL, maltose binding protein; RU, response units; IL, interleukin; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; EGFP, enhanced green fluorescence protein; TCR, T cell receptor. Back

2 N. Clarkson, M. H. Brown, A. Filby, and R. Zamoyska, unpublished data. Back


    ACKNOWLEDGMENTS
 
We thank Philip Khulman for generously providing purified human CAPZ, Despoina Voulgaraki for data on a pull-down experiment in the presence of cations, Kathryn Kirsch and Sachiko Kajigaya for CMS and CIN85 cDNA, Takashi Obinata for the chicken CAPZ expression vector, Mike Dustin for anti-CD2AP sera, John Cooper and Emily Barron-Casella for a helpful introduction to CAPZ, and Anton van der Merwe, Gillian Griffiths, and Lisa Durrant for critical review of the manuscript.



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