Abstract
T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins.
Highlights
The regulatory mechanisms and potential redundancy of the structurally related actin nucleation promoting factors, WASp family verprolin-homologous protein-2 (WAVE2) and Wiskott-Aldrich syndrome protein (WASp), are poorly understood
Characterization of the Proteins That Form a Complex with WAVE2 upon T cell antigen receptor (TCR) Ligation—Several studies defined the molecular mechanism controlling the recruitment of WASp to the immunological synapse (IS) [20, 21, 32, 33], whereas others demonstrated the importance of WASp and WAVE2 in the regulation of actin polymerization in T cells [8, 10, 20]
We found that WAVE2 co-immunoprecipitated with phosphoproteins with molecular weights of ϳ36, 70, 76, 100, and 120 kDa; these sizes correspond to LAT, ZAP-70, SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), Vav1, and c-Cbl, respectively
Summary
The regulatory mechanisms and potential redundancy of the structurally related actin nucleation promoting factors, WAVE2 and WASp, are poorly understood. WASp and WAVE2 share several structural features and are recruited to the IS, the precise regulatory mechanisms, localization, and redundancy between them have not been fully characterized Both were shown to be involved in the formation of actin-rich lamellipodia-like (sheet-like) structures, they might differ in other functions, such as vesicular trafficking (19 –21) and integrin activation [22]. This raises the question of how WASp and WAVE2 control both similar and diverse actin-dependent cellular functions. Do they share regulatory molecular mechanisms for some functions (i.e. membrane protrusion), with distinct pathways in others (i.e. integrin activation)? Do they share regulatory molecular mechanisms for some functions (i.e. membrane protrusion), with distinct pathways in others (i.e. integrin activation)? In this study, we identified the molecular mechanism that regulates the recruitment of WAVE2 to the IS and compared it with the mechanisms governing WASp [20]
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