SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9

Maria Simarro1,4, Arpad Lanyi1,5, Duncan Howie1, Florence Poy2, Joost Bruggeman1, Michelle Choi1, Janos Sumegi3, Michael J. Eck2 and Cox Terhorst1

1 Division of Immunology, Beth Israel Deaconess Medical Center and 2 Department of Cancer Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 3 Division of Hematology and Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA 4 Present address: Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 5 Present address: Institute of Immunology, University of Debrecen, H-4012, Hungary

Correspondence to: C. Terhorst or M. Simarro; E-mail: terhorst{at}caregroup.harvard.edu or msimarro{at}rics.bwh.harvard.edu
Transmitting editor: T. Tedder


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells. Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells. Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck. While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck. The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT. In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity. By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells. Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.

Keywords: signal transduction, Src, T-lymphocyte, X-linked lymphoproliferative disease


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SAP is a free Src homology 2 (SH2)-domain-containing protein that controls signal transduction events induced by at least six members of the SLAM family of receptors on the surface of T lymphocytes and NK cells (1). The absence or mutation of SAP causes X-linked lymphoproliferative (XLP) immunodeficiency, a disease with three major clinical phenotypes, fulminant mononucleosis, dysgammaglobulinemia and B cell lymphomas, which is often only manifested after Epstein Barr virus infections (17). Although the XLP syndrome is primarily caused by defective T cell responses, impaired NK activity has also been detected (814).

We and others have shown that SAP-deficient CD4+ and CD8+ cells demonstrate enhanced IFN-{gamma} production after infection with lymphocytic choriomeningitis virus (LCMV) (15,16). Conversely, in vitro and in vivo studies of SAP-deficient mice reveal that SAP is required for the development of T helper 2 cells (15,16). Recent studies by Ahmed and colleagues show that SAP is also required for the CD4+ T cell role in generating long-term humoral immunity after LCMV infection (17). NK cell functions are also impaired in SAP–/– mice, as they are in XLP patients (our unpublished observations).

SAP binds with high affinity to the motif TxYxxV/I which is found in the cytoplasmic tail of at least six SLAM related receptors: SLAM (CD150), CD84, 2B4 (CD244), Ly9 (CD229), NTB-A (SF2000) and CS1 (also called 19A, CRACC or novel Ly9) (2,13,1827). SLAM is one of the two measles virus receptors and also functions in T and B cell activation (2834). In T cells, anti-SLAM antibodies induce IFN-{gamma} secretion (30,32,33). CD84 is a self-ligand expressed on B and T cells (21,35,36). Similarly to SLAM, cross-linking CD84 increases IFN-{gamma} production by human T cells (36). 2B4, CS1 and NTB-A trigger activating signals in NK cells (13,26,27,37). The exact function of Ly9 remains unknown. The mechanistic principle of the interactions of SAP with these receptors is important for an understanding of the pathogenesis of XLP. Indeed, studies with a series of SAP missense mutations derived from XLP patients show that binding to the various SLAM receptors can be differentially affected by these mutations (38,39).

A number of in vitro and in vivo studies using transfection experiments in COS and Ba/F3 cells have shown that SAP prevents the binding of the SH2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, to phosphotyrosine motifs in the tail of SLAM-related receptors (2,19,22,32,40). Physico-chemical studies show that the SH2-domain of SAP has a very high affinity for the TxYxxV/I motif in the cytoplasmic tail of SLAM (2,25,39). These observations are consistent with crystallographic data and nuclear magnetic resonance analyses of the SAP/SLAM interactions, which reveal that SAP binds to SLAM using a ‘three-pronged’ modality (25,39,41). This high affinity interaction enables SAP to act as a natural inhibitor molecule, blocking recruitment of SHP-1 or SHP-2 to SLAM, 2B4, Ly9 or CD84 (2,19,22,32,40). However, SAP also appears to have an adaptor function, which implicates FynT (FynT is the Fyn isoform found in T cells) as the key kinase that phosphorylates SLAM and its downstream targets inositol 5-phosphatase SHIP, Dok1 and Dok2 (42,43). We and others recently showed that SAP binds directly to FynT, an interaction mediated by the FynT SH3 domain (40,43,44).

In this paper, we show that SAP is required for SLAM and Ly9 phosphorylation in thymocytes and peripheral T cells. In vitro and yeast two-hybrid analyses show that SAP binds directly to both FynT and Lck. However, while SAP interacts with both the SH3 domain and the kinase domain of FynT, it interacts solely with the kinase domain of Lck. Finally, in vitro kinase assays show that SAP interaction with the SH3 domain of FynT increases FynT catalytic activity.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Wild type (wt) Balb/c or C57BL/6 and Fyn–/– (129/Sv x C57BL/6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). SAP–/– mice (15) were back-crossed with wt Balb/c or C57BL/6 mice for seven generations.

Antibodies
A polyclonal antibody directed at human SAP was generated by immunizing rabbits with a 25 amino acid long peptide (amino acids 104–128) (2). A polyclonal antibody to mouse SAP was obtained by immunizing rabbits with an 11 amino acid long peptide (amino acids 111–121) (45). Mouse monoclonal anti-human SLAM (2E7) was a gift from DNAX Research Institute (Palo Alto, CA). Rat anti-mouse SLAM (9D1) is specific for the extracellular region of mouse SLAM (33). A polyclonal antibody to the cytoplasmic tail of human SLAM was produced by immunizing rabbits with a 17 amino acid long peptide (amino acids 304–320) (2). Rat anti-mouse Ly9.1 monoclonal antibody (clone 30C7) was obtained from American Type Culture Collection (ATCC, Rockville, MD). Sources of commercial antibodies are as follows: hamster anti-mouse CD3{epsilon} (clone 145–2C11) and goat polyclonal anti-rat Ig G were obtained from BD Pharmingen (San Diego, CA); hamster anti-mouse CD28 (clone 37.51) was obtained from eBioscience (San Diego, CA); mouse monoclonal anti-Glutathione S-transferase (GST) (sc-138), mouse monoclonal anti-Lck (clone 3A5, sc-433) and rabbit polyclonal anti-Fyn (sc-16) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); horseradish peroxidase-conjugated anti-phosphotyrosine was obtained from Zymed (South San Francisco, CA); mouse monoclonal anti-FLAG M5 was obtained from Kodak (Rochester, NY); rabbit anti Src [pY416] or anti-Fyn [pY417] phosphospecific antibody was obtained from Biosource International (Camarillo, CA); rabbit polyclonal anti-myc-Tag and rabbit polyclonal anti-Lck were obtained from Cell Signalling Technology (Beverly, MA).

Cells
The antigen-specific mouse T cell hybridoma BI-141 (kindly provided by Dr M. C. Miceli, University of California, CA) (46) was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). 293T cells (obtained from ATCC, Rockville, MD) were grown in DMEM supplemented with 10% fetal bovine serum. Mouse CD4+ and CD8+ splenic T cells were isolated using negative selection columns (R&D Systems, Minneapolis, MN). CD4+ T cells were activated in vitro using plate bound anti-CD3{epsilon} (clone 145–2C11) at 10 µg/ml, together with anti-CD28 (clone 37.51) at 2 µg/ml, for 48 h. The cells were then cultured for an additional 24 h in a medium supplemented with recombinant mouse IL2 (20 U/ml) (BD Pharmingen, San Diego, CA).

cDNA constructs
Human wild type SAP or the SAP mutants R32Q, T53I or T68I (38) were cloned into either pGEX2T (Amersham Pharmacia Biotech, Piscataway, NJ) or pCMV2-Flag (Kodak, Rochester, NY). The human SLAM cDNA in vector pJFE14-SR{alpha} was a gift from DNAX Research Institute (Palo Alto, CA). FynT cDNA fragments corresponding to the unique domain (amino acids 1–83), the unique + SH3 domains (amino acids 1–142) and the full-length FynT were amplified from pSR{alpha}2-FynT (a gift from Dr M. Streuli, Dana Farber Cancer Institute) into PEGB vector (a gift from Dr C. Rudd, Imperial College London, London, UK). cDNAs encoding mouse SLAM, mouse wild type SAP and the mouse SAP mutant R78E were cloned into pCI (Promega, Madison, WI). The mouse SAP mutant R78E was generated by overlapping polymerase chain reactions using oligonucleotide primers containing the mutation.

Transfections
293T cells were transiently transfected with 2 µg of each construct using FuGENETM Transfection reagent (Roche, Indianapolis, IN). Forty-eight hours after transfection, cells were harvested and post-nuclear lysates were prepared and used for binding assay. BI-141 T cells were transfected by electroporation. Briefly, BI-141 T cells were suspended in RPMI-medium supplemented with 10% fetal bovine serum at a concentration of 3 x 107 cells per 250 µl per cuvette. Cells were electroporated with 100 µg of each construct at 300 V, 960 µF using a Gene Pulser (Bio-Rad, Hercules, CA). The cells were incubated at 37°C, 5% CO2 for 24 h before use.

Coprecipitation and immunoblotting
For detection of Ly9, the cell surface proteins were biotinylated with Sulfo-NHS-Biotin (Pierce, Rockford, IL). Briefly, a total of 4 x 107 cells were washed three times in phosphate buffered saline, resuspended in 800 µl saline, and surface labeled with 200 µl of Biotin 1 mg/ml for 20 min at 4°C. After washing the cells once in RPMI media with 10% fetal bovine serum and four times in phosphate buffered saline, the cells were lysed in lysis buffer containing 1% Nonidet P-40. The precleared lysates were then incubated with appropriate antibodies and protein G–Sepharose. Immunocomplexes were recovered by centrifugation and washed. Precleared lysates or immunocomplexes were subjected to SDS–PAGE and transferred to polyvinylidine difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Membranes were blocked with 10% non-fat milk or 3% bovine serum albumin, incubated with the indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies, and developed using enhanced chemiluminescence (Pierce, Rockford, IL).

SLAM and Ly9 crosslinking experiments
Thymocytes and CD4+ or CD8+ T cells from different mouse strains (5 x 107 per lane) were incubated on ice with either rat-anti mouse Ly9.1 (clone 30C7) or rat-anti mouse SLAM monoclonal antibody (clone 9D1) (1 µg per 1 x 107 cells). After 30 min, goat polyclonal anti-rat Ig G (1 µg per 2 x 107 cells) was added for the indicated time periods at 37°C. Following crosslinking, the reactions were stopped by the addition of ice-cold PBS and cells were lysed in lysis buffer containing 1% Nonidet P-40 before anti-SLAM or anti-Ly9 immunoprecipitation and Western blotting.

Yeast two-hybrid analysis
For yeast two-hybrid assays the following constructs were used: the full-length human FynT, and FynT fragments containing the unique domain (amino acids 1–83), the SH3 domain (amino acids 84–142), the SH2 domain (amino acids 143–248), the kinase domain (amino acids 253–534), the C-terminal regulatory tail (amino acids 523–534), as well as the fragments containing the SH3 domain with either the N-terminal unique domain or/and the C-terminal SH2 domain, were amplified from pSR{alpha}2-FynT (a gift from Dr M. Streuli, Dana Farber Cancer Institute) and cloned into pYESTrp (Invitrogen, Carlsbad, CA) in-frame with the B42 activation domain. The full-length human Lck, the Lck fragment containing the unique domain, the SH3 and SH2 domains (amino acids 1–228) and the Lck kinase domain (amino acids 229–509) were amplified from pSR{alpha}2-Lck and cloned into pYESTrp.

Interaction analysis between human SAP and FynT or Lck was performed by first transforming the yeast strain EGY48 p8op lacZ with the vector PEG202 (a gift from Dr V. K. Kuchroo, Harvard Medical School) containing the LexA binding domain fused to full-length human SAP. The resultant bait yeast strain was transformed with the vector pYESTrp containing various FynT or Lck fragments fused to the B42 activation domain. The transformants were plated on synthetic UTH medium (lacking uracil, tryptophan and histidine), to maintain selection for the prey and bait plasmids. To test protein–protein interactions, colonies from UTH plates were plated on UTHL/Gal/Raf induction medium (lacking uracil, triptophane, histidine, leucine and containing galactose and raffinose), and assayed for ß-galactosidase activity by a qualitative in vivo plate assay, as described in the Clontech yeast protocols. The following plasmid constructs were used as controls: pYESTrp-Jun (negative control for prey) (Invitrogen, Carlsbad, CA), and PEG202-Lamin (negative control for bait plasmid).

In vitro protein binding assays
[35S]-labeled full-length FynT and Lck were prepared using a coupled transcription/translation system (TNTR T7 Quick for PCR DNA, Promega, Madison, WI). Briefly, the coding regions of FynT or Lck were amplified by polymerase chain reaction using gene specific primers. In addition, the 5' primer contained the Kozak sequence and the T7 promoter. In vitro translation was performed in the presence of 20 µCi of [35S]methionine (Amersham Pharmacia Biotech, Piscataway, NJ) as suggested by the manufacturer. Translation mixtures were diluted to 500 µl final with lysis buffer containing 0.5% Triton X-100. Equimolar amounts of bacterially expressed GST, GST–wild type SAP and GST–SAP mutants were added to the translation mixtures followed by precipitation with glutathione–Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ). After protein complexes were subjected to SDS–PAGE, gels were treated with Amplify Fluorographic Reagent (Amersham Pharmacia Biotech, Piscataway, NJ), and subjected to autoradiography.

In vitro kinase assay
Human wild type SAP (residues 1–104) and human SAP mutant R78E proteins were expressed and purified as previously described (25,44). In brief, bacterial lysates were fractionated by cation exchange chromatography (HiTrap S HP, Amersham Pharmacia Biotech, Piscataway, NJ) and fractions containing SAP were pooled and further purified by phosphotyrosine affinity chromatography (47). Human CD3{zeta} chain (amino acids 52–163) was expressed and purified as previously described (48) with some modifications. After elution from Ni+2–Sepharose (metal chelating Sepharose FF, Amersham Pharmacia Biotech, Piscataway, NJ), the N-terminus His6 tag was removed by TEV protease; CD3{zeta} chain was then further purified by cation exchange chromatography. Human FynT (residues 83–534) was expressed with an N-terminus His6 tag from a T7-based expression plasmid (Novagen, Madison, WI) in Escherichia coli strain BL21(DE3); the soluble fraction of FynT was purified by Ni+2–Sepharose, anion exchange (HiTrap Q HP, Amersham Pharmacia Biotech, Piscataway, NJ) and gel filtration (Superdex75 prep grade column, Amersham Pharmacia Biotech, Piscataway, NJ). Wild type SAP, SAP mutant R78E and FynT proteins were stored at –80°C in 20 mM Tris pH 8, 150 mM NaCl and 5 mM DTT; CD3{zeta} was stored at 4°C in 20 mM Tris pH 8, 150 mM NaCl.

The effect of SAP on FynT kinase activity was measured by a spectrophometric assay that couples the utilization of ATP to the oxidation of NADH via phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase (49). Reaction mixtures (80 µl total volume) contained 100 mM Tris pH 8, 10 mM MgCl2, 1 mM phosphoenolpyruvate, 0.28 mM NADH, 89 U/ml pyruvate kinase, 124 U/ml lactate dehydrogenase, 1 µM FynT, 20 µM CD3 {zeta} chain (120 µM phosphorylation sites), and 0–200 µM wild type SAP or SAP R78E. The reactions were initiated by the addition of 0.5 mM ATP and carried out at 30°C; the decrease in absorbance at 340 nm was monitored by a microplate spectrophotometer (SPECTRAmax Plus plate reader, Molecular Devices, Sunnyvale, CA). Rates were determined by a linear least squares fit of the absorbance versus time data and Va (the velocity measured in the presence of ligand minus the velocity measured in its absence) was plotted against ligand concentration.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SAP enhances tyrosine phosphorylation of SLAM
Our previous experiments using an overexpression system demonstrated that FynT is able to phosphorylate SLAM. Moreover, the presence of SAP greatly increased FynT-mediated phosphorylation of SLAM (2,18). Here we extend these studies to a second T cell src kinase: Lck. Specifically, 293T cells were transiently transfected with a combination of plasmids encoding SAP, SLAM and FynT or Lck. The experiments (Fig. 1A and B) demonstrate that both FynT and Lck phosphorylate the cytoplasmic region of SLAM after coexpression in 293T cells. The mere presence of SAP induces tyrosine phosphorylation of SLAM by endogenous protein tyrosine kinases and augments tyrosine phosphorylation of SLAM induced by FynT and Lck. These observations support the notion that SAP acts as an adaptor, which enhances phosphorylation of SLAM by FynT and Lck.



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Fig. 1. The presence of SAP enhances tyrosine phosphorylation of SLAM by Src-like PTKs. Tyrosine phosphorylation of human SLAM (CD150) was examined after transfection of cDNAs encoding human SLAM, SAP and FynT (A) or SLAM, SAP and Lck (B) into 293T cells. Anti-human SLAM immunoprecipitates (IP:{alpha}-hSLAM) were analyzed by SDS–PAGE and western blotting (WB) with antibodies against phosphotyrosine ({alpha}-PY), human-SLAM ({alpha}-hSLAM) and human-SAP ({alpha}-hSAP).

 
Tyrosine phosphorylation of SLAM and Ly9 in T lymphocytes requires SAP
Recent findings (43,44) were indicative that phosphorylation of the receptor SLAM in thymocytes required the presence of the adaptor SAP. Here we compare the role of SAP in phosphorylation of the receptors SLAM and Ly9 in both thymocytes and peripheral T cells.

Anti-SLAM mediated crosslinking induced rapid tyrosine phosphorylation of SLAM in thymocytes from wt mice (Fig. 2A). A significant basal level of SLAM phosphorylation was also detected in unstimulated thymocytes. By contrast, SLAM tyrosine phosphorylation was completely absent in SAP–/– thymocytes. Tyrosine phosphorylation of SLAM was reduced in thymocytes from Fyn–/– mice. In three independent experiments, SLAM phosphorylation was reduced by ~70% in thymocytes from Fyn–/– mice. The remaining phosphorylation can be attributed to other protein tyrosine kinases, i.e. Lck.



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Fig. 2. Phosphorylation of SLAM and Ly9 is absent in SAP-deficient T cells and thymocytes, and is reduced in Fyn-deficient thymocytes. Phosphorylation of SLAM or Ly9 was induced by incubation of cells of T lineage for various lengths of time, with either rat anti-mouse-SLAM ({alpha}-SLAM) or anti-Ly9 ({alpha}-Ly9), together with a secondary antibody (goat anti-rat IgG). Cell lysates were subjected to immunoprecipitation with monoclonal antibodies recognizing mouse SLAM or mouse Ly9. Samples were analyzed by SDS–PAGE and western blotting with anti-phosphotyrosine antibody ({alpha}-PY), anti-mouse SAP antibody ({alpha}-mSAP), anti-mouse SLAM antibody ({alpha}-mSLAM) and horseradish peroxidase-conjugated avidin (Av-HRP), as indicated to the right of each panel. (A) Anti-SLAM induced phosphorylation of SLAM in freshly isolated thymocytes from wt, Fyn-deficient (Fyn–/–) and SAP-deficient (SAP–/–) C57BL/6 mice. (B) Anti-SLAM induced phosphorylation of SLAM in activated CD4+ T cells isolated from wt and SAP-deficient (SAP–/–) C57BL/6 mice. Prior to SLAM ligation with rat anti-mouse SLAM antibody, CD4+ T cells were stimulated with plate-bound anti-CD3{epsilon} (10 µg/ml) plus anti-CD28 (2 µg/ml) for 48 h, followed by a rest period of 24 h in the presence of recombinant IL2 (20 U/ml). (C) Anti-Ly9 induced phosphorylation of Ly9 in freshly isolated thymocytes from wt and SAP-deficient BALB/c mice. Cell surface proteins were biotinylated prior to lysis and the immunoprecipitated Ly9 was detected using horseradish peroxidase-conjugated avidin (Av-HRP). (D) Anti-Ly9 induced phosphorylation of Ly9 in freshly isolated CD4+ T cells from SAP-deficient BALB/c mice. Cell surface proteins were biotinylated prior to lysis and the immunoprecipitated Ly9 was detected using horseradish peroxidase-conjugated avidin (Av-HRP). (E) Anti-Ly9 induced phosphorylation of Ly9 does not take place in freshly isolated CD8+ T cells from SAP-deficient BALB/c mice. Cell surface proteins were biotinylated prior to lysis and the immunoprecipitated Ly9 was detected using horseradish peroxidase-conjugated avidin (Av-HRP).

 
We next examined whether SAP is required for SLAM phosphorylation in activated peripheral T cells, which are known to express high levels of SLAM (32,33). SLAM is only weakly tyrosine phosphorylated either in the absence or in the presence of anti-SLAM, even though large amounts of SLAM were detectable by western blotting (Fig. 2B). Nonetheless, SLAM was never phosphorylated in T cells from SAP–/– mice.

Next, the phosphorylation of Ly9, a second SAP binding cell surface receptor, was examined in a similar fashion (19,50). Crosslinking with anti-Ly9 antibodies led to a strong and transient tyrosine phosphorylation of Ly9 in thymocytes from wt mice, while only a very low level of Ly9 phosphorylation was detected in SAP–/– thymocytes (Fig. 2C). Crosslinking with anti-Ly9 induced a rapid and transient phosphorylation of Ly9 in both resting CD4+ and CD8+ T cells. Whereas no Ly9 phosphorylation occurred in SAP–/– CD4+ T cells (Fig. 2D), some Ly9 phosphorylation was detectable in SAP–/– CD8+ T cells (Fig. 2E). Thus, SAP appears to control phosphorylation of Ly9 in a similar fashion to SLAM in thymocytes and peripheral T lymphocytes.

Taken together, these data demonstrate that phosphorylation of SLAM and Ly9 in T lymphocytes is dependent upon the presence of SAP.

SAP is essential for the association of SLAM with FynT
Whereas SAP is known to participate in the formation of a SLAM/SAP/FynT complex (43,44), a SLAM/SAP/Lck complex has not yet been described. This Lck-containing ‘triple complex’ would account for the residual phosphorylation of SLAM in Fyn–/– thymocytes. To determine the existence of a SLAM/SAP/Lck complex, thymocytes from wt mice and SAP–/– mice were incubated with anti-SLAM antibody for short periods of time and upon precipitation with anti-SLAM antibody, western blotting was done with antibodies directed at phosphotyrosine, the auto-phosphorylation site of FynT [pY417] and Lck. In unstimulated thymocytes, a small amount of FynT was found associated with SLAM (Fig. 3); upon engagement by anti-SLAM, the rapid increase in tyrosine phosphorylation of SLAM was matched by an increased association of FynT. By contrast, SLAM did not associate with FynT in thymocytes from SAP–/– mice. No Lck could be detected in anti-SLAM immunoprecipitates with three different anti-Lck antibodies.



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Fig. 3. SAP is required for SLAM binding to FynT. Thymocytes from wt and SAP-deficient C57BL/6 mice were stimulated with rat anti-mouse SLAM antibody ({alpha}-mSLAM) and goat anti-rat IgG for the indicated time points. Cell lysates were subjected to immuno precipitation (300 x 106 cells/immunoprecipitation) using anti-mouse SLAM antibody. The precipitates were separated on a 4–15% SDS–polyacrylamide gel followed by western blotting with antibodies directed at phosphotyrosine ({alpha}-PY), phosphorylated Y417 of FynT ({alpha}-FynT[pY417]), Lck (clone 3A5, {alpha}-Lck), mouse SAP ({alpha}–mSAP) and mouse SLAM ({alpha}–mSLAM).

 
Thus, our results demonstrate that SAP is indispensable for the interaction of SLAM with FynT and for the phosphorylation of SLAM. Under similar experimental conditions, Lck does not form a stable ternary complex with SLAM and SAP.

SAP binds directly to the tyrosine kinases FynT and Lck
Next, we further explored whether SAP binds directly to both FynT and Lck, using in vitro binding assays. This was done using recombinant SAP and in vitro translated FynT and Lck (Fig. 4A). Bacterially expressed GST-tagged SAP, either wt SAP or SAP mutants (R32Q, T53I and T68I), were used. All three SAP mutants were used because each has a mutation that affects the interaction with SLAM family receptors in a distinct fashion (38,39). As shown in Figure 4(A), both [35S]-labeled FynT and Lck bound to wild type SAP. Interestingly, all three SAP mutants, i.e. R32Q, T53I and T68I, bound to FynT in a fashion similar to that observed for wild type SAP. This strongly suggested that the mode of binding of SAP to FynT was different from that involved in the SLAM–SAP interactions.



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Fig. 4. SAP binds directly to FynT and Lck, but uses the SH3 domain of FynT exclusively. (A) In vitro binding of SAP to FynT and Lck occurs even if SAP mutants with impaired binding to the SLAM family of receptors are used. In vitro translated [35S]-labeled FynT (left panel) or Lck (right panel) were incubated with the GST proteins indicated at the top of the figure (GST alone, GST–SAP wild type (wt), GST–SAP R32Q, GST–SAP T53I and GST–SAP T68I). The proteins were then bound to glutathione–Sepharose beads and were analyzed by SDS–PAGE and autoradiography. Equal amounts of GST proteins were used, as assessed by Coomassie staining. The positions of FynT, Lck, GST and GST–SAP are indicated with arrowheads. (B) SAP binds to the N-terminal and to the C-terminal segment of FynT, but only to the C-terminal (kinase) part of Lck. Binding between human SAP and FynT or Lck was assayed by in vivo plate (qualitative) ß-galactosidase assays in Saccharomyces cerevisiae strain EGY48. Full length SAP was fused to the LexA binding domain. Full length FynT, N-terminal segment of FynT (FynT-N, unique+SH3+SH2 domains, amino acids 1–248), C-terminal segment of FynT (FynT-C, kinase domain, amino acids 253–534), Full length Lck, N-terminal segment of Lck (Lck-N, unique+SH3+ SH2 domains, amino acids 1–228) and C-terminal segment of Lck (Lck-C, kinase domain, amino acids 229–509) were fused to B42 activation domain. Jun served as prey negative control. 4C, SAP binds mostly to the SH3 domain of FynT as judged by yeast two hybrid analysis. Binding of SAP to full length FynT and its subdomains were assayed by in vivo plate (qualitative) ß-galactosidase assays in S. cerevisiae strain EGY48. Jun served as prey negative control. 1 = full-length; 2 = unique domain; 3 = SH3 domain; 4 = SH2 domain; 5 = unique+SH3 domains; 6 = SH3+SH2 domains; 7 = unique+SH3+SH2 domains; 8 = kinase domain; 9 = C-terminal tail.

 
Evidence for a direct interaction between SAP and FynT was also obtained using a yeast two-hybrid system. SAP was used as bait and the full-length form of FynT and Lck as preys. To this end, SAP was fused to the LexA binding domain and both FynT and Lck were fused to the B42 activation domain. In control experiments, lamin was used as bait. We induced the expression of FynT and Lck at the end of the assay to overcome the toxicity of Src-like PTKs in yeast (see Methods). As judged by the ß-galactosidase assay, SAP interacts with both FynT and Lck (Fig. 4B). Interestingly, the outcomes of the colorimetric assays suggest that SAP binding to full-length FynT was significantly stronger than the binding to Lck (Fig. 4B, left panels). Furthermore, SAP binds to both the N-terminal and C-terminal segment of FynT (Fig. 4B, FynT-N and FynT-C), yet only to the C-terminal segment of Lck (Fig. 4B, Lck-C).

Taken together, the two-hybrid analyses and in vitro binding assays demonstrate direct interactions between SAP and FynT and between SAP and Lck and show that the binding of SAP to FynT is distinct from the SAP/Lck interaction.

SAP binds to both the SH3 domain and to the kinase domain of FynT, but only to the kinase domain of Lck
To further dissect the SAP/FynT interaction, yeast two-hybrid analysis was employed. We constructed yeast expression vectors encoding a fusion of either full-length FynT or Lck, or fragments of FynT or Lck, in-frame with the B42 activation domain to assess interaction with SAP. As shown in Figure 4(C), the unique domain of FynT, the C-terminal regulatory tail and the SH2 domain of FynT failed to show a substantial ß-galactosidase activity. However, any fragment that contained the FynT SH3 domain interacted strongly with SAP, as assessed by ß-galactosidase activity (Fig. 4B and C). A weak interaction between the FynT kinase domain and SAP was also observed. In contrast to the SAP–FynT interactions, SAP interacted solely with the kinase domain of Lck. Notably, no binding to the Lck unique SH3–SH2 fragment was observed (Fig. 4B). These results suggest that the kinase domain of Lck is sufficient for interaction with SAP. Nevertheless, based on this analysis, binding to the kinase domain of Lck was not nearly as effective as binding to full-length Lck.

Because the two-hybrid analyses suggested a relatively high affinity of SAP for the FynT SH3 domain, we examined whether SLAM/SAP/FynT or SLAM/SAP/FynT SH3 domain ternary complexes could be formed in mammalian cells. To this end, 293T cells were transfected with cDNAs encoding combinations of SLAM, SAP, GST–FynT SH3 domain and GST–FynT. As shown in Figure 5, SLAM coprecipitated with the FynT SH3 domain or with FynT only when SAP was coexpressed. No interaction with other segments of FynT could be detected (data not shown).



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Fig. 5. Ternary complexes comprising either SLAM/SAP/FynT SH3 or SLAM/SAP/FynT in 293T transfectant cells. 293T cells were transfected with combinations of cDNAs, encoding GST, GST–FynT SH3 domain (GST-FynT SH3), GST–FynT full-length (GST-FynT) and human SLAM and human SAP, and assessed for complex formation. Human SLAM immunoprecipitates (top panel, {alpha}-hSLAM) and whole cell lysates (bottom panel) were resolved on SDS–PAGE and western blotted with anti-human SLAM ({alpha}-hSLAM), anti-GST ({alpha}-GST) and anti-human SAP ({alpha}-hSAP) antibodies. The position of immunoglobulin heavy chains is indicated with asterisks.

 
Thus, SAP binds both to the SH3 domain and to the kinase domain of FynT, but solely to the kinase domain of Lck. Furthermore, the SH3 domain of FynT is sufficient in itself to form a ternary complex with SLAM and SAP.

SAP binding the FynT SH3 domain leads to a marked increase in the tyrosine kinase activity of FynT
By which mechanism does SAP augment phosphorylation of SLAM and Ly9? One possibility is that SAP recruits an active pool of FynT to the receptor tail. Alternatively, binding of SAP to FynT may effect both recruitment and catalytic activation. In order to differentiate between these possibilities, we asked whether addition of SAP to autoinhibited FynT in vitro could activate the enzyme. This system also allows for an analysis of the functional importance of the interaction between SAP and the FynT SH3 domain by using the SAP mutant R78E, which is unable to bind to the FynT SH3 domain in vitro (44). As shown in Fig. 6(A), wild type SAP induced an increase in FynT kinase activity in a dose dependent fashion. By contrast, the SAP mutant R78E did not activate FynT (Fig. 6B). We conclude therefore, that SAP can activate FynT by SH3 displacement.



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Fig. 6. The interaction between the FynT SH3 domain and SAP is critical for activation of the FynT kinase. (A) In vitro activation of the autoinhibited FynT kinase by SAP. Increasing concentrations of bacterially expressed wild type SAP or SAP mutant R78E (0–200 µM) were added to 1 µM FynT and to 20 µM CD3{zeta} in the presence of 0.5 mM ATP. The amount of phosphate transferred to substrate by FynT was monitored by a spectrophometric assay for 1 h at 30°C, with measurements taken every 15 sec. Top panel, trendlines represent the average of three experiments for each wild type SAP concentration. Trendlines for FynT assayed in the presence of SAP R78E mutant (not shown) show no significant difference than FynT alone. Bottom panel, Va (the velocity measured in the presence of ligand minus the velocity measured in its absence) is plotted against ligand concentration. (B) Reduced SLAM tyrosine phosphorylation when the SAP/FynT SH3 domain interaction is impaired. BI-141 T cells were transfected with mouse SLAM, mouse SAP or mouse SAP R78E. Cell transfectants were stimulated with rat anti-mouse SLAM antibody ({alpha}-mSLAM) and goat anti-rat IgG for the indicated time points. Subsequently, cell lysates were immunoprecipitated with anti-mouse SLAM antibody ({alpha}-mSLAM, 300 x 106 cells/immunoprecipitation). The immune complexes were separated on a 4–15% SDS–polyacrylamide gel followed by Western blotting with antibodies directed at phosphotyrosine ({alpha}-PY), phosphorylated Y417 of FynT ({alpha}-FynT [pY417], Lck (clone 3A5, {alpha}-Lck), mouse SAP ({alpha}-mSAP) and mouse SLAM ({alpha}-mSLAM). Detergent lysates of each cell were blotted with anti-mouse SAP antibody ({alpha}-mSAP).

 
We next wanted to examine whether the absence of the SAP/FynT SH3 domain interaction would diminish SAP-dependent SLAM phosphorylation in vivo. For these experiments, we used BI-141 T cell line, which expresses neither SAP nor SLAM (42,46). These transfection experiments demonstrated that the SAP/FynT interaction via the FynT SH3 domain was critical for optimal SLAM phosphorylation, because transfection of the SAP mutant R78E, compared to wild type SAP, resulted in almost no phosphorylation of SLAM (Fig. 6B). Crosslinking with anti-SLAM had no effect on the phosphorylation level of SLAM in the presence of either form of SAP. These in vivo results confirm previous data by Latour et al. (43).

Thus, the result of this series of experiments was consistent with the in vitro experiment, in which the SAP/FynT SH3 domain interaction was essential for the increase in FynT kinase activity, and consequently for the phosphorylation of SLAM.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SAP, a single SH2 domain-containing protein that regulates the signaling of the SLAM receptors in T and NK cells, acts both as a natural inhibitor of SHP-2 and SHP-1-mediated interactions as well as an adaptor by recruiting the Src-like PTK FynT (1,51). Here we show that SAP is indispensable for the phosphorylation of SLAM and its related receptor Ly9 by using thymocytes, peripheral T cells, transfected 293T cells and BI-141 T cell line. In vitro studies and yeast two-hybrid assays demonstrated that SAP binds directly to the Src-like PTKs FynT and Lck. However, while SAP interacts with both the SH3 domain and the kinase domain of FynT, it interacts solely with the kinase domain of Lck. Additionally, in vitro enzyme assays demonstrated that SAP increased FynT kinase activity, which is dependent upon the SAP/FynT SH3 domain interaction.

Recently we, and others, reported that SAP is required for the tyrosine phosphorylation of SLAM in thymocytes and for the formation of the SLAM/FynT complex (43,44). Here we provide evidence that the presence of SAP is also critical for SLAM phosphorylation in peripheral T cells. In contrast to what we observed in thymocytes, SLAM triggering did not induce an increase in tyrosine phosphorylation of SLAM in CD4+ T cells. As CD4+ cells were activated to upregulate the expression of SLAM, this pre-activation state of the CD4+ T cells might have contributed to the low levels of SLAM phosphorylation after anti-SLAM treatment. On the other hand, the reported decrease of SAP expression after activation of T cells (45) might have contributed to the low levels of SLAM phosphorylation. The SLAM phosphorylation measured 2–5 min after anti-SLAM antibody treatment thus appears not to be predictive of cell activation as measured by IFN-{gamma} production and cell proliferation (30,32,33).

Ly9 is another member of the SLAM family (19,50) whose phosphorylation is controlled by SAP. The phosphorylation of Ly9 was not completely inhibited in SAP–/– thymocytes, which indicates that either Ly9 can interact directly with Src-like PTKs or that there are adaptor molecules other than SAP that can mediate Ly9 phosphorylation. Unfortunately, we could not assess FynT contribution to Ly9 phosphorylation because the available antibody against Ly9 (Ly9.1 alloantigen) does not react with the Ly9.2 alloantigen expressed on C57BL Fyn-deficient mice. However, since SAP binds preferentially to FynT, and SAP augments Ly9 phosphorylation, it appears likely that FynT plays a major role in Ly9 phosphorylation. There are two SAP binding sites in Ly9: tyrosines 558 and 581 (19). Future studies will be required to determine if one or both of these sites within Ly9 bind to SAP-FynT complexes.

Although FynT is predominantly responsible for the phosphorylation of SLAM in thymocytes, phosphorylation of SLAM can readily be detected in Fyn–/– thymocytes (Fig. 2). Thus, kinases other than FynT are involved in SLAM phosphorylation; the most likely candidate being the Src-like PTK Lck, which is expressed at high levels in thymocytes (52). Indeed, Lck was able to phosphorylate SLAM in 293T cells (Fig. 1). Interestingly, Lck has been reported to directly bind and phosphorylate 2B4 (12). Lck-mediated phosphorylation of SLAM may occur via direct binding of Lck to SLAM as occurs with 2B4, or via binding of Lck with SAP. Indeed, SAP binds to Lck, as judged by both yeast two-hybrid studies and in vitro binding experiments. Nonetheless, a SLAM/SAP/Lck ternary complex could not be detected in either thymocytes or in T cells. This may be due to the lack of adequate anti-Lck antibodies and/or the weak affinity of the interaction between SAP and Lck. The latter notion is supported by the observation that the binding of SAP to Lck is distinct from its binding to FynT. Whereas SAP binds to both the kinase domain and to the SH3 domain of FynT (40,43,44), SAP binds solely to the kinase domain of Lck, potentially reducing the overall affinity of SAP for Lck.

Previous reports suggested that formation of the SLAM/SAP/FynT complex may activate the kinase activity of FynT, because SLAM crosslinking induced phosphorylation of SHIP, Dok1 and Dok2 (42,43). Furthermore, SAP/FynT SH3 domain interaction was shown to be crucial for the role of SAP in controlling IFN-{gamma} and interleukin-4 production (43). Structural considerations (44) indicate that the FynT SH3 surface to which SAP binds is only accessible in the derepressed conformation of the kinase. Thus, SAP must either recruit previously activated FynT, or bind with sufficient affinity to compete the autoinhibitory interactions and activate the kinase. The experiments presented here not only demonstrate that the unique interaction between SAP and FynT SH3 domain increases the enzymatic activity of FynT in vitro, they also demonstrate that these interactions occur and are critical in vivo. As predicted by the structural analyses of the SAP/FynT interaction (44), arginine 78 of the adaptor SAP was indispensable for this FynT activation. This important finding represents the first evidence that a non-canonical SH3 ligand can activate Src-like kinases and also provides the mechanistic basis of the adaptor role of SAP. Thus, the adaptor function of SAP allows it to recruit and activate FynT at the cell surface receptors SLAM and Ly9. As SLAM functions as a co-stimulator of T cells, further studies are required to determine where and when during T cell activation the ternary complex between SLAM, SAP and activated FynT operates.


    Acknowledgements
 
The authors thank Drs J. Griesbach, A. C. Abadia-Molina and S. Calpe-Flores for help with the experiments, Drs Mara Ayodele and S. D. Wax for a critical review of the manuscript and Dr M. A. De la Fuente for editorial assistance. This work was supported in part by the National Institutes of Health Grants AI-15066 and AI-035714 (to C.T.). M.S. was a recipient of a Fellowship from Ministerio de Ciencia y Tecnologia (PF/99/0033960854). D.H. was a recipient of a Fellowship from the Leukemia and Lymphoma Society.


    Abbreviations
 
GST—glutathione S-transferase

IP—immunoprecipitate

LCMV—lymphocytic choriomeningitis virus

PTK—protein tyrosine kinase

SH2—Src homology 2

SHP—SH2-containing protein-tyrosine phosphatase

XLP—X-linked lymphoproliferative disease


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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