Interaction of the Wiskott-Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells

Abstract

The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9DeltaPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation.

Fig. 1.

SNX9 inducibly binds and colocalizes with WASp. (A) Schematic showing the domain organization of SNX9 and the SNX9ΔSH3 and ΔPX3 constructs. (B) Lysates prepared from anti-CD3 and anti-CD28 antibody-stimulated BI-141 cells were immunoprecipitated with anti-WASp antibody, subjected to SDS/PAGE and sequentially immunoblotted with anti-SNX9 and anti-WASp antibodies. (C) BI-141 T cell lysates were incubated with GST or indicated fusion proteins immobilized on glutathione Sepharose beads, resolved by SDS/PAGE, and immunoblotted by using anti-WASp and anti-GST antibodies. (D) Cos-7 cells were transfected transiently with dsRED WASp and/or pEGFP-SNX9 and analyzed by immunofluorescent microscopy. Actin and clathrin were visualized with rhodamine phalloidin and anti-clathrin/Cy3-secondary antibody, respectively. Images are representative of three independent experiments.

Fig. 2.

SNX9 associates with PI(3,4,5)P₃ and forms a multimeric complex with WASp, p85, and CD28. (A) GSTSNX9-PX or GST fusion proteins were used to probe membranes containing the indicated PtdIns and bound proteins detected by immunoblotting with anti-GST antibody. (B) Lysates prepared from anti-CD3 and anti-CD28 antibody-stimulated BI-141 cells were immunoprecipitated with anti-p85 antibody and sequentially immunoblotted with anti-SNX9, anti-WASp, anti-CD28, and anti-p85 antibodies. (C) BI-141 T cell lysates were incubated with GST or indicated fusion proteins bound to glutathione Sepharose beads, the complexes resolved by SDS/PAGE and immunoblotted by using anti-p85 and then anti-GST antibodies.

Fig. 3.

CD28 endocytosis is clathrin-mediated, PI3K-dependent, and SNX9-regulated. (A) BI-141 cells were transfected with pEGFP or pEGFP-Eps15DN. GFP-positive cells were incubated with anti-CD28 and then biotinylated goat anti-hamster antibody. Cells were washed, warmed to 37°C, and aliquots were removed at 0, 5, 10, or 15 min and treated with 0.1% NaN₃. Cells were stained with PE streptavidin, fixed, and analyzed for surface CD28 expression by flow cytometry. Results are the percentage change in the geometric mean of PE-streptavidin fluorescence between time 0 and indicated times. Each value represents the mean (± SEM) of three determinations, and data represent four independent experiments. (B) BI-141 T cells were cultured for 15 min with indicated Wortmannin concentrations and CD28 internalization assessed as in A. Each value represents the means (±SEM) of three determinations, and data represent three independent experiments. (C) BI-141 T cells were stimulated for 2, 5, 10, or 30 min with anti-CD28 antibody followed by immunogold-anti-hamster IgG. Ultrathin sections were prepared and cells were visualized by electron microscopy. Arrows indicate gold-labeled CD28 in membrane invaginations at 1 min and then in vesicular structures. (D) BI-141 T cells were transfected with pEGFP, pEGFP SNX9, or pEGFP SNX9ΔPX, and CD28 internalization was assessed as in A. Values represent means (±SEM) of three assays and are representative of three independent experiments.

Fig. 4.

WASp colocalizes with CD28 and modulates CD28 endocytosis and CD28-induced actin polymerization. (A) Thymocytes from wild-type (WT) and WAS^−/− mice were incubated with anti-CD28 and then biotinylated goat anti-hamster antibodies, warmed to 37°C, and CD28 internalization was assessed as in Fig. 3. Results represent the mean (± SEM) of three assays and are from four independent experiments. (B) pEGFP-SNX9 or WASp BI-141 transfectants stimulated with anti-CD3 and anti-CD28 antibodies were stained with antibodies to clathrin, p85 or WASp, and appropriate secondary antibodies and analyzed by immunofluorescent microscopy. Images represent three independent experiments. (C) WT and WAS^−/− thymocytes were stimulated with anti-CD28 antibody for 30 min, followed by secondary antibody cross-linking. Cells were fixed and F-actin content was quantified by flow cytometry of FITC phalloidin-stained resting (black) and stimulated (white) cells. Data are representative of three independent experiments.

Fig. 5.

SNX9 regulates induction of NFAT activation. (A) BI-141 T cells were cotransfected with an NFAT luciferase reporter vector and with either pEGFP-SNX9, pEGFP-SNX9ΔPX, pEGFP, or pEGFP-Eps15DN. Transfected cells were stimulated for 8 h with anti-CD28 antibody, lysed, and assayed by luminometry for luciferase expression. Values represent the means (± SEM) of three assays and represent six independent experiments.