To generate WASpΔGBD mice, a WASp cDNA deleted for sequences encoding amino acids 238–252 (WASpΔGBD) was introduced downstream of the CD2 promoter elements in the p29Δ2 vector and the transgene construct was then injected into CD1 embryos. Transgene+
founders were identified by Southern analysis and PCR using primer pairs: 5′-GGAGCACATCAG AAGGGCTGGCTT-3′ (forward) and 5′CGGAGGAACCAGAGGTGGACCT-3′ (reverse). Founders were then backcrossed to the C57BL/6 background and pups were screened for WASpΔGBD expression by immunoblotting analysis of lymphoid cell lysates using anti-WASp antibody. Selected WASpΔGBD transgenic lines were then mated with WASp-deficient (WAS−/−
) mice (18
) to derive animals expressing WASpΔGBD on the WAS−/−
ΔGBD). Mice expressing the OVA-specific OT-II TCR transgene on the C57BL/6 background (provided by F. Carbone, University of Melbourne, Melbourne, Australia, and T. Watts, University of Toronto, Toronto, Canada; reference 19
) were bred with WAS−/−
ΔGBD, and Fyn−/−
(Jackson ImmunoResearch Laboratories) mice to generate WAS−/−
ΔGBD/OT-II, and Fyn−/−
/OT-II animals. C57BL/6 Itk−/−
mice were provided by D. Fowell (University of Rochester Medical Center, Rochester, NY).
Reagents used for these studies included: polyclonal antibodies specific for WASp (raised against a synthetic peptide representing WASp residues 224–238), PSTPIP1 (5
), and PTP-PEST (provided by M. Tremblay, McGill University, Montreal, Canada, and A. Veillette, Clinical Research Institute of Montreal, Montreal, Canada); monoclonal antibodies recognizing human and murine CD3 (from the OKT3 and 2C11 hybridomas, respectively), human and murine CD28 (from the 9.2 and 37.51 hybridomas, respectively), and human protein kinase C (PKC)-θ (Transduction Labs); monoclonal antiphosphotyrosine (pTyr) pTyr4G10 antibody (Upstate Biotechnology); goat anti–hamster IgG (BD Biosciences); Cy5-conjugated goat anti–rabbit Ig, Cy5–sheep anti–mouse Ig, and goat anti–hamster IgG (Jackson ImmunoResearch Laboratories); anti-Fyn antibodies and purified rabbit Ig (Santa Cruz Biotechnology, Inc.); rhodamine, FITC, and Alexa 350–phalloidin (Molecular Probes); and anti–green fluorescent protein (GFP) antibody (AbCam). Expression constructs for immunofluorescence studies were derived by subcloning cDNAs encoding full-length WASp, PSTPIP1, and PTP-PEST (provided by M. Tremblay) into the pEGFP-C3, pDSRED, and pcDNA3 expression vectors (CLONTECH Laboratories, Inc.). WASp tyrosine point mutation (tyrosine→phenylalanine) GFP-tagged expression constructs were generated by PCR-mediated mutagenesis of each WASp tyrosine residue and subcloning of the mutant cDNAs into the pEGFP-C3 vector. Expression constructs (pcDNA3) for cdc42, cdc42-V12, and cdc42-N17 were obtained from G. Downey (University of Toronto, Toronto, Canada) and expression constructs for Fyn K296M were obtained by PCR-mediated mutagenesis of the Fyn cDNA and subcloning into pcDNA3 vector.
T Cell Stimulation.
For T cell activation, 2 × 107
Jurkat E6, JCam-1, or JCam-1-Lck cells (20
) cultured in RPM1 1640 plus 10% fetal bovine serum and 2 mM l
-glutamine penicillin/streptomycin (GIBCO BRL) were serum starved overnight and then stimulated for varying times with 10 μg/ml anti-CD3 and 5 μg/ml anti-CD28 antibodies. Alternatively, 2 × 107
murine thymocytes or lymph node T cells were incubated with 5 μg/ml anti-CD3 and 2 μg/ml anti-CD28 antibodies on ice for 30 min followed by cross-linking with 5 μg anti–hamster IgG for varying times at 37°C. Aliquots of cell cultures were subjected to anti-pTyr immunoblotting analysis to confirm cell activation.
Immunoprecipitation and Immunoblotting.
To prepare cell lysates, 2 × 107 Jurkat cells, thymocytes, or lymph node T cells were suspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4) and 1 μg/ml each aprotinin, leupeptin, and pepstatin (Amersham Biosciences). After 30 min of incubation on ice, unlysed cells were removed by centrifugation and 100–250 μg of the lysate proteins were then incubated with Protein A sepharose 6B beads (Amersham Biosciences) for 30 min at 4°C followed by 2 h of incubation with specific antibody or rabbit preimmune serum. The immune complexes were then collected over Protein A sepharose and eluted from the beads by boiling in Laemmli buffer. For immunoblotting analyses, immunoprecipitated proteins were suspended in loading buffer, electrophoresed through 10% polyacrylamide gels, and transferred to nitrocellulose membrane. After blocking with 5% skim milk powder in 1× TBST (20 mM Tris-HCl, pH 7.5, 0.9% NaCl, 0.1% Tween 20), membranes were incubated for 1 h with the primary antibody followed by a 1-h incubation with a 1/3,000 diluted horseradish peroxidase–conjugated secondary antibody (Bio-Rad Laboratories) and for 1 min with ECL substrate (Amersham Biosciences) followed by 1–10 min of exposure to Eastman Kodak Co. X-ray film.
Cell Proliferation Assay.
Single cell suspensions prepared from wild-type WAS−/−
ΔGBD thymi and lymph nodes were subjected to erythrocyte lysis in ammonium chloride buffer and then cultured in 96-well plates (2 × 106
cells/ml) for 48 h in culture medium alone or with 0–25 μg/ml plate-bound anti-CD3
antibody with or without 0.2 μg/ml anti-CD28 antibody. Cultured cells were pulsed with 1 μCi/well [3
H]thymidine for 18 h and incorporated radioactivity was measured using an automated β scintillation counter.
Generation of Fusion Proteins and In Vitro Binding Assays.
Fusion proteins were derived by transforming Escherichia coli with pGEX2T vectors containing full-length PSTPIP1, PTP-PEST, WASp, or cdc42 cDNAs, or PCR-amplified fragments representing the PSTPIP1 coiled coil (amino acids 120–358; PSTPIPCOIL) or SH3 (amino acids 365–415; PSTPIPSH3) domains, or pQE-30 vectors (QIAGEN) containing the Fyn, PST-PEST, or PSTPIP1 cDNAs. Fusion proteins were purified from isopropyl-1-thio-β-D-galactopyranoside–induced bacteria using glutathione-coupled sepharose 4B or Ni-NTA agarose beads (QIAGEN), and the amount of bound protein was estimated by Coomassie staining. For binding studies, 5 μg immobilized gluthathione S-transferase (GST) fusion proteins were incubated for 2 h at 4°C with either 250 μM purified 6XHis-tagged protein or lysates from stimulated Jurkat E6 cells. The immune complexes were resolved by SDS-PAGE and immunoblotting analysis was performed with the indicated antibodies. To assay WASp-cdc42 binding, 100 μg GST-cdc42 or GST alone immobilized on glutathione sepharose 4B beads was incubated for 10 min at 30°C with 50 μl GTPγS loading buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM GTPγS) followed by the addition of 10 mM MgCl2. Beads were then incubated in 5 mM MgCl2 for 2 h at 4°C with 0.5 mg lysates obtained from Cos-7 cells 36 h after Lipofectamine-mediated transfection of pcDNA3.1 vectors encoding either WASp or WASpΔGBD. Complexes were washed and resolved by SDS-PAGE and sequential immunoblotting analysis was performed with anti-WASp and anti-cdc42 antibodies.
In Vitro Kinase Assay.
Fyn immunoprecipitates obtained from TCR-stimulated Jurkat cells were washed in kinase buffer (20 mM Hepes, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 5 mM MnCl2, 0.25 mM Na3VO4, 0.5% Nonidet P-40, 0.1 mM 2-β-mercaptoethanol) and the complexes were incubated for 30 min at 30°C in kinase buffer containing 10 μCi [γ-32P]ATP (NEN Life Science Products) with 5 μg GST, GST-WASp, GST-WASpY291F, or GST-WASpΔPro fusion proteins. Samples were resuspended in Laemmli buffer and resolved in 10% SDS-PAGE gels. After electrotransfer to nitrocellulose, phosphorylated WASp was visualized by autoradiography.
NFAT Luciferase Reporter Assay.
Murine WAS−/− thymocytes (2 × 107 cells) were isolated and transfected with 50 μg of an NFAT luciferase reporter gene plus 50 μg pEGFP-C3 vectors encoding WASp-GFP, WASp Y→F GFP mutants, or GFP alone. After 4 h, viable cells were separated using Lympholyte M (Cedarlane) and the GFP+ cells were purified over a MoFlo® cell sorter (DakoCytomation) and cultured for 8 h in uncoated 96-well plates or in plates coated with 10 μg/ml anti-CD3 or 5 μg/ml anti-CD3 plus anti-CD28 antibody. Cells were lysed and luciferase activity was assayed using the luciferase assay system (Promega) and a luminometer.
LB27.4 B cells (H-2d/b–restricted B cell hybrid; American Type Culture Collection) were incubated with OVA peptide (OVA329–339) for 4 h at 37°C followed by centrifugation at 200 g for 5 min with an equal number of lymph node T cells from transgenic mice or from WAS−/−/OT-II lymphocytes transfected with pEGFP-C3 or pDSRED expression constructs. Samples were incubated at room temperature for 10 min and the cells were resuspended and plated onto poly-l-lysine–coated coverslips (Biocoat; Becton Dickinson) before fixation in 3% paraformaldehyde. Synapse formation was scored as the percent conjugates (T cell in physical contact with an APC) showing actin accumulation at the T cell–APC interface.
Transfection and Immunofluorescence Assays.
Plasmid DNA for expression constructs containing PSTPIP1, PTP-PEST, WASp, WASpY291F, and WASpY102F cDNAs were purified using CLONTECH Laboratories, Inc. Maxi-Prep kit. 5 × 104 Cos-7 cells maintained in DMEM supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin, were seeded onto glass coverslips and transfected with selected plasmid DNA using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, cells were washed and fixed with 3% ice-cold paraformaldehyde in PBS. Alternatively, unstimulated or stimulated OT-II T cells were transfected by electroporation (1 pulse, 360 mV) using a BTX electroporator and subjected to fixation at 2.5 h after transfection. After fixation, cells were blocked with 2% BSA/PBS for 10 min immediately or for intracellular staining, cells were first permeabilized with 0.1% Triton X-100/PBS. Cells were then incubated with primary and the appropriate fluorescently conjugated secondary antibodies and the stained samples were mounted in anti-fade mounting media (DakoCytomation). Images were analyzed using the Olympus 1X-70 inverted microscope equipped with fluorescence optics and Deltavision Deconvolution Software (Applied Precision).
In Vitro Actin Polymerization Assay.
Actin polymerization was evaluated by assaying increase in fluorescence of pyrene-labeled actin using the actin polymerization kit from Cytoskeleton, Inc. For these assays, cdc42-V12, PTP-PEST, and PSTPIP1 were purified as GST fusion proteins and these proteins or Fyn (Upstate Biotechnology) were added alone or in combination with 1.5× actin polymerization kit buffer containing 20 nM Arp2/3 complex, 100 nM GST-WASp or WASpΔGBD fusion protein, and 100 μl monomer pyrene actin stock in G buffer (5mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.5 mM DTT, and 0.2 mM ATP), with the final concentration of G actin being 2.8 μM. Fluorescence changes were monitored every minute for over 1 h at room temperature using a fluorometer (Photon Technology International) with filters for excitation at 365 nm and emission at 407 nm.
In Vivo Actin Polymerization Assays.
1.5 × 106 thymocytes obtained from WAS−/−ΔGBD mice or by transfection of WAS−/− thymic cells with the pEGFP-C3 WASp Y→F mutant constructs were either unstimulated or stimulated with anti-CD3 and anti-CD28 antibodies for 30 min on ice followed by cross-linking with 5 μg mouse anti–hamster IgG for 5 min at 37°C. Activation was terminated and the cells were fixed in 5% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min on ice, washed and stained with 5 μg/ml FITC-conjugated phalloidin, and analyzed using a FACSCalibur™.