Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp Ϫ / Ϫ mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASpmediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.

The Wiskott-Aldrich syndrome protein (WASp) couples actin cytoskeletal rearrangement to T cell activation, but the mechanisms involved are unknown. Here, we show that antigen-induced formation of T cell:APC conjugates and synapses is abrogated in WASp-deficient T cells and that CD2 engagement evokes interactions between the proline-rich region required for WASp translocation to the synapse and the PSTPIP1 adaptor SH3 domain and between the PSTPIp1 coiled-coil domain and both CD2 and another CD2-binding adaptor, CD2AP. The induced colocalization of these proteins at the synapse is disrupted by expression of coiled-coil domain-deleted PSTPIP1. These data, together with the impairment in CD2-induced actin polymerization observed in WASp-deficient cells, suggest that PSTPIP1 acts downstream of CD2/CD2AP to link CD2 engagement to the WASp-evoked actin polymerization required for synapse formation and T cell activation.