WASP Recruitment to the T Cell:APC Contact Site Occurs Independently of Cdc42 Activation

Abstract

Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.

SLP-76 Coordinates Nck-Dependent Wiskott-Aldrich Syndrome Protein Recruitment with Vav-1/Cdc42-Dependent Wiskott-Aldrich Syndrome Protein Activation at the T Cell-APC Contact Site

“…We found that Cdc42-GTP accumulates at the site of T cell-APC interactions (22). Like Cdc42-GTP, WASP accumulates at the T cell-APC contact site (16,22).…”

“…Secondary Abs conjugated to fluorochromes were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). GFP-WASP-GTPase binding protein (GBD) was prepared and used as described previously (22). WASP mAb 26E6, which specifically recognizes the open active conformation of WASP, will be described elsewhere (S. D. Leung, C. Labno, D. You, J. Burkhardt, and M. Rosen, manuscript in preparation).…”

“…FLAG-tagged WASP cDNA was described previously (22). GFP-tagged clones expressing wild-type and mutant Nck and Grb2 were described elsewhere (39).…”

“…Transient transfection was performed as described previously (22). Briefly, 20 ϫ 10 6 T cells in log-phase were combined with 20 g of DNA and electroporated using a Genepulser II (Bio-Rad, Hercules, CA) at 0.24 kV and 1500 F. Dead cells were removed by passage over Ficoll-Paque (Amersham Pharmacia, Piscataway, NJ).…”

“…Cell conjugations and microscopy were performed as described previously (22). Briefly, Nalm-6 B cells were dyed with 7-amino-4-cholormethylcoumarin and incubated with or without 2 g/ml SEE.…”