The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation

Abstract

Background: The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated.

Results: By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release.

Conclusions: These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Figure 1

The WAVE2 Complex Localizes to the IS (A) Jurkat T cells were stimulated for indicated time points (above lanes). After stimulation, cells were lysed, clarified, and subsequently immunoprecipitated with control IgG antibody or antibody against WAVE2. IPs were separated by SDS-PAGE, transferred, and subsequently blotted for PIR121, HEM-1, Abi-1/2, and WAVE2. (B) Primary CD4⁺ T cells were isolated, lysed, immunoprecipitated, and subsequently blotted as in (A). (C) Ovalbumin peptide-pulsed A20 B cells (blue) were incubated with secondary DO11.10 transgenic T cells (top). Conjugated cells were bound to poly-L-lysine coated coverslips, fixed, and stained with antibody against WAVE2 (green) and rhodamine-phalloidin (red). Also shown are primary CD4⁺ T cells conjugated to superantigen cocktail (SAC) pulsed Raji B cells (bottom).

Figure 2

Depletion of WAVE2 Leads to Subsequent Degradation of Other Members of the Complex (A) Three subsequent IPs for WAVE2 were performed on 0.5 mg of Jurkat T cell lysate (WAVE2#1-3, lanes 1–3). After preclearing the lysate of any residual WAVE2 antibody (bead clear, lane 4), an IP for HEM-1 and PIR121 was performed (PIR121/HEM-1, lane 5). HEM-1 and PIR121 was also directly IP’ed from 0.5 mg of Jurkat T cell lysate (PIR121/HEM-1, lane 6). Bound proteins from each IP were separated by SDS-PAGE, transferred, and subsequently blotted for WAVE2, HEM-1, and PIR121. (B) Jurkat T cells were transfected with a control vector (vector) or one of two shWAVE2 suppression vectors (shWAVE2-A/-B). Cells were lysed at the 24, 48, and 72 hr time points. Equal amounts of protein were separated by SDS-PAGE, transferred, and blotted for WAVE2 and ZAP-70. (C) Cells were transfected as in (B) with the addition of two suppression vectors against HEM-1 (shHEM-1-A/-B). After 72 hr, cells were harvested as in (B) and blotted for PIR121, HEM-1, Abi-2, WAVE2, and ZAP-70.

Figure 3

Suppression of WAVE2 Decreases Conjugate Formation and Actin Accumulation at the IS (A) Cells were transfected with either vector control or suppression vectors targeting either WAVE2 or HEM-1. After 72 hr, transfected cells were incubated with NALM6 B cells stained red with ethidium bromide and pulsed with SEE (+SEE) or left unpulsed (−SEE). Using GFP expression as a marker for transfected cells, conjugate formation was determined using two-color flow cytometry. Shown is a representative example of three independent experiments. (B) Jurkat T cells were transfected as in (A) and were analyzed for their ability to bind to fibronectin following no stimulation (unstimulated) or stimulation of the TCR (OKT3) for 10 min at 37ºC.After Following washing, adherent cells were collected and the adhesion of GFP-low, intermediate, and high cells in each sample was determined by flow cytometry. (C) Cells were transfected as in (A) and after 72 hr were incubated with SEE-pulsed NALM6 B cells (blue). Conjugates were bound to poly-L-lysine coated coverslips, fixed, and stained with rhodamine-phalloidin (red) to visualize the accumulation of F-actin. (D) Conjugates formed in (C) and Figure S4 were quantified on their ability to form F-actin at the IS. Shown is the average of three independent experiments.

Figure 4

WAVE2-Suppressed T Cells Fail to Spread Properly in Response to TCR Ligation Jurkat T cells stably expressing GFP-actin were transfected with (A) control shRNA-cherry vector or (B) cherry-shWAVE2-B. After 48 hr, the cells were placed onto OKT3 mAb-coated coverslips and activation events were recorded by taking images every 5 s, as explained in the Experimental Procedures. Shown are representative time-lapsed images occurring after initial contact with the OKT3-coated coverslips. More than 100 cherry-expressing cells were analyzed for each transfection. Images were taken at the same magnification. Movies are available online with the Supplemental Data.

Figure 5

WAVE2 Regulates T Cell Activation Independently of PLCγ1, Erk, and Jnk Activation (A) Jurkat T cells were transfected with IL-2p luciferase reporter along with either a control vector or one of two shWAVE2 suppression vectors (shWAVE2-A/-B). After 72 hr, cells were left unstimulated (Nil) or stimulated with OKT3 and anti-CD28 for 5 hr. Luciferase activity was measured and normalized to TK-Renilla readings. (B) Jurkat T cells were transfected with vector control or shWAVE2 suppression vector. After 72 hr, cells were stimulated with OKT3 and goat anti-mouse antibody for the indicated time points. Whole-cell extracts were prepared and proteins were separated by SDS-PAGE, transferred, and analyzed for phosphorylation of PLCγ1 (Tyr 783) by immunoblot. (C and D) Same as in (B) but blotted for phosphorylation of Erk1 and Erk2 and Jnk1 and Jnk2. (E and F) Cells were transfected as in (A), but with a reporter construct for (E) AP-1 or (F) NFκB. Cells were stimulated for 2, 3, 4, or 5 hr, and luciferase activity was measured and normalized to TK-Renilla readings. Data shown are representative examples of three independent experiments for each.

Figure 6

WAVE2 Regulates CRAC Channel Activation Leading to NFAT-Mediated Gene Transcription (A) Jurkat T cells were transfected with vector control or shWAVE2 suppression vector. After 72 hr, calcium mobilization in response to OKT3/ goat anti-mouse was measured using Indo-1 staining and flow cytometry. (B) Jurkat T cells were transfected as in (A). After 72 hr, calcium measurements were performed using single cell fluorescence ratio (Fura-2 imaging) of GFP positive control and WAVE2 suppressed Jurkat cells. In calcium-free bath solution, increases in Fura-2 ratio reflect Ca²⁺ release from intracellular stores. Extracellular Ca²⁺ entry via activated CRAC channels was subsequently assessed by reintroduction of extracellular calcium (2 mM Ca²⁺). Shown are representative examples of three independent experiments for each condition and each trace represents the average response of at least 100 cells in the recording chamber. (C and D) Cells were prepared and analyzed as in (B) and stimulated as indicated above the graphs. (E and F) Jurkat T cells were transfected with vector control or shWAVE2 suppression vector and either a (E) NFAT/AP-1 or (F) NFAT luciferase reporter. 72 hr post transfection, cells were stimulated with OKT3 and anti-CD28 for 2, 3, 4, and 5 hr. Luciferase activity was measured at each time point and normalized to TK-Renilla readings. Shown are representative examples of three independent experiments for each.