Studies of novel interactions between Nck and VAV SH3 domains

Following T-cell antigen receptor (TCR) engagement, a multi-molecular complex consisting of SLP-76, Nck, and VAV1 is formed and recruited to the T-cell antigen-presenting-cell (APC) interaction site. This complex is crucial for the regulation of the actin machinery. The molecules Nck (an adaptor) and VAV1 (a GEF for small G-proteins) were previously shown to bind SLP-76. Using high-resolution imaging techniques, together with gene silencing and biochemical analysis, we studied the dynamics of this signaling complex formation. We recently showed that VAV1 and Nck can bind each other independently of SLP-76. This direct interaction is mediated by the binding of the Nck C-terminal SH3 domain and the VAV1 N-terminal SH3 domain. This interaction contributes to the cooperative nature of the complex formation. This observation was confirmed in functional studies: disruption of the Nck-VAV1 interaction strongly inhibited actin polymerization. Here, we show that Nck-VAV1 interaction is not required for Ca2+ mobilization, since a point mutation in the VAV1 N-terminal SH3 domain, which prevent the direct interaction between Nck and VAV1, has no effect on Ca2+ flux and minimal effects on ZAP-70, LAT, or PLCγ1 phosphorylation.