Introduction
The Wiskott-Aldrich syndrome protein (WASp) is a hemopoietic lineage-restricted cytosolic effector of T cell activation, integrally involved in the coupling of TCR engagement to transcriptional activation and cytoskeletal rearrangement (1). WASp effects on TCR signaling are mediated through its tandemly located GTPase binding domain (GBD) and proline-rich region, flanking NH2-terminal–located Ena/Vasp homology 1, and COOH-terminal verprolin homology central region-acidic region (VCA) domains. The VCA domain represents a newly identified structural motif that provides WASp and several WASp-related proteins with the capacity to bind components of the Arp2/3 complex and thereby induce Arp2/3 actin-nucleating activity (2,3). Affinity of this domain for Arp2/3 complex binding has very recently been shown to be enhanced by its constitutive serine phosphorylation (4). The other functional domains of WASp complement this activity by mediating effector interactions that enable WASp recruitment into TCR signaling pathways and the consequent linkage of TCR stimulatory signals to induction of actin polymerization. For example, TCR-stimulated binding of the WASp proline-rich region to the SH3 domain–containing proline, serine, threonine phosphatase interacting protein (PSTPIP)1 adaptor, allows WASp to be targeted to the T cell–APC interface and thereby direct the regional actin cytoskeletal rearrangements required for formation of the immunological synapse (5). Similarly, WASp proline-rich region-mediated interactions with other SH3 domain–containing adaptors, such as CrkL and intersectin 2, and WASp Ena/Vasp homology 1 domain–mediated interactions with the WASp-interacting protein, WIP, appear to enable WASp translocation to specific subcellular sites wherein its actin polymerizing properties can be deployed so as to augment cell activation (6–9). Such interactions may also account for the capacity of WASp to modulate lipid raft clustering after TCR engagement (10). By contrast, the WASp GBD mediates an interaction with the activated form of the cdc42 Rho GTPase that does not modulate WASp localization, but instead induces WASp actin polymerizing function by releasing WASp from an autoinhibitory structural configuration that constrains VCA effects on the Arp2/3 complex (11).
The structural and biochemical data showing WASp actin polymerizing properties to be constitutively, but reversibly, autoinhibited, provide a mechanical explanation for the inducibility of actin cytoskeletal change in response to cell stimulation (12, 13). However, data showing that WASp promotes clustering in the absence of cdc42 binding imply that WASp Arp2/3 stimulatory function can be evoked by mechanisms other than cdc42-GTP association with its GBD (13). This possibility is also suggested by nuclear magnetic resonance data showing that a WASp tyrosine residue (Y291) in close proximity to the WASp GBD is structurally positioned such that its phosphorylation might disrupt WASp autoinhibitory conformation and thereby evoke VCA domain activity (12). This tyrosine residue has been shown to be phosphorylated in vitro by the protein tyrosine kinases (PTKs) Btk and Hck (14, 15), and its phosphorylation has been associated with enhanced macrophage filopodium formation (15). However, although WASp has also been shown to interact with Fyn and Itk (16, 17), the regulation of its tyrosine phosphorylation and the relevance of WASp tyrosine phosphorylation to its biological functions in T cells remain unknown.
In this study, the sites, mechanisms, and biologic relevance of WASp tyrosine phosphorylation in relation to T cell activation were investigated. The data indicate inducible phosphorylation of the WASp tyrosine residue at position 291 to be essential for the coupling of TCR engagement to NFAT activation, actin polymerization, and immunological synapse formation. By contrast, WASp effects on T cell development and activation as well as its inducible tyrosine phosphorylation can occur independently of the WASp GBD motif. The data also identify Fyn as the WASp-binding PTK required for phosphorylation of Y291 after TCR stimulation and indicate that WASp association with the PSTPIP1 adaptor allows WASp to colocalize with and be dephosphorylated by the protein tyrosine phosphatase (PTP), PTP-PEST. Like WASp, both Fyn and PTP-PEST are inducibly translocated to the contact zone formed between T cells and stimulatory APCs, but although WASp and Fyn are required for immunological synapse formation, PTP-PEST overexpression essentially abrogates synapse development. Similarly, Fyn markedly enhances, whereas PTP-PEST in combination with PSTPIP1 impairs, WASp capacity to stimulate Arp2/3 actin polymerizing activity in vitro. Importantly, Fyn effects on WASp phosphorylation and its actin polymerizing role appear to be unaffected by changes in cdc42 activity and disruption of the WASp–cdc42 interaction, respectively. These findings indicate Y291 phosphorylation to be essential to the triggering of WASp effector function during T cell activation. The data also reveal that WASp tyrosine phosphorylation can occur independently of WASp–cdc42 interaction and its modulation represents a key mechanism whereby Fyn and PTP-PEST regulate the signaling events linking TCR ligation to transcriptional activation and cytoskeletal rearrangement.