T-cell antigen receptor (TCR) engagement induces formation of multi-protein signalling complexes essential for regulating T-cell functions. Generation of a complex of SLP-76, Nck and VAV1 is crucial for regulation of the actin machinery. We define the composition, stoichiometry and specificity of interactions in the SLP-76, Nck and VAV1 complex. Our data reveal that this complex can contain one SLP-76 molecule, two Nck and two VAV1 molecules. A direct interaction between Nck and VAV1 is mediated by binding between the C-terminal SH3 domain of Nck and the VAV1 N-terminal SH3 domain. Disruption of the VAV1:Nck interaction deleteriously affected actin polymerization. These novel findings shed new light on the mechanism of actin polymerization after T-cell activation.

Following T-cell antigen receptor (TCR) engagement, multiprotein signaling complexes essential for regulating T-cell functions are formed. A multi-molecular complex consisting of SLP-76, Nck and VAV is recruited to the T-cell/antigen presenting cell (APC) site during initial T cell activation and is crucial for regulation of the actin machinery. Most of the studies performed to date have concentrated on the role of the signaling molecules that comprise this molecular complex. However, the regulation of the complex formation and the cooperation and competition among its components during formation have not yet been explored. In this study, a multidisciplinary approach including molecular imaging, biochemical and biophysical techniques was utilized in order to define the composition, stoichiometry, and specificity of the SLP-76:Nck:VAV complex. High-resolution imaging techniques, such as fluorescence resonance energy transfer (FRET) analysis, were performed in live activated T cells in order to focus on the binding properties within the trimolecular complex. Our data reveal a direct interaction between Nck and VAV which is mediated by the binding of the C-terminal SH3 domain of Nck to VAV’s N-terminal SH3 domain. Disruption of the VAV:Nck interaction significantly impaired actin polymerization. This research provides novel insights into the regulation of a signaling complex formation critical for immune response.