In this study, we show that HS1 is required for the stabilization of F-actin filaments after TCR engagement, for maximal Ca2+ influx, and for NFAT and NFκB-mediated gene transcription. In addition, we find that tyrosine phosphorylation is necessary for HS1 recruitment to the IS and regulates its interaction with Lck, PLCγ1, and Vav1. These findings identify HS1 as an actin-regulatory adaptor protein essential for T cell activation.
HS1-deficient T cells show unique defects in actin polymerization at the IS; actin-rich structures are formed initially but are unstable and erratic. This phenotype is distinct from that of cells lacking WASp or WAVE2, both of which also bind Arp2/3 complex. Cells lacking WASp spread essentially normally, while cells lacking WAVE2 fail to spread altogether (Nolz et al., 2006
). The phenotype of HS1-deficient T cells resembles that of cortactin-deficient fibroblasts, which display disorganized lamellipod formation (Bryce et al., 2005
; Kempiak et al., 2005
). These phenotypes are consistent with the idea that by binding F-actin as well as Arp2/3 complex, these proteins inhibit debranching and stabilize cortical actin (Uruno et al., 2003b
). An unresolved question is whether HS1 also acts via direct interactions with WASp and WIP, as reported for cortactin (Kempiak et al., 2005
; Kinley et al., 2003
; Uruno et al., 2003a
; Weaver et al., 2001
). Though we can readily detect binding of HS1 to the WIP/WASp complex in vitro, we have so far failed to verify binding in T cell lysates. We show that HS1 is not required for recruitment of WASp or WIP to the IS. Indeed, their continued presence likely contributes to the residual actin dynamics in HS1-deficient cells.
We mapped the major sites of HS1 tyrosine phosphorylation in activated T cells to amino acids 378 and 397, and our data suggest that ZAP-70 is responsible for phosphorylating these sites. Y378 and Y397 are critical for several aspects of HS1 function. Mutation of these residues leads to defective HS1 targeting to the IS, abrogates binding to Vav1, and perturbs actin responses. Finally, these residues are required for overexpression-induced inhibition of IL-2 promoter activation. Phosphorylation-dependent membrane targeting may be a general feature of HS1 function; in B cells, tyrosine phosphorylation of HS1 mediates its recruitment to lipid rafts (Hao et al., 2004
). The protein(s) directly responsible for recruitment of HS1 remain to be identified. Our data rule out Vav1, because Vav1-deficient cells recruit HS1 efficiently. While it is possible that the functional defects observed in T cells expressing HS1 2YF are solely attributable to the aberrant localization of this mutant, it seems more likely that phosphorylation regulates multiple aspects of HS1 function. In addition to mediating binding to other signaling molecules (discussed below), phosphorylation may induce conformational changes that influence HS1 function, as already shown for cortactin (Huang et al., 1997
; Martinez-Quiles et al., 2004
One clear role of HS1 phosphorylation is to mediate binding to SH2 domain-containing proteins, including Lck, Vav1, and PLCγ1. The functional significance of Vav1 binding is underscored by the findings that HS1-suppressed T cells progressively lose Vav1 from the IS and that expression of a membrane-targeted Vav1 rescues the actin defect in HS1-suppressed cells. Interestingly, the loss of Vav1 from the IS in HS1-suppressed cells parallels the loss of F-actin at this site. This, and finding that the kinetics of Vav1/F-actin loss differ from those of HS1 phosphorylation, suggest that HS1 stabilizes Vav1 at the IS via a complex mechanism. Since Vav1 recruitment to the IS is dependent on interactions with SLP-76 and Itk (Dombroski et al., 2005
; Zeng et al., 2003
), we propose that SLP-76, Itk, and HS1 coordinately recruit Vav1 to the IS at early time points. Once this complex is localized to the IS, Vav1 initiates actin polymerization through activation of Cdc42 and Rac. HS1 participates in forming and/or stabilizing F-actin at later times, and this feeds back to stabilize Vav1 interactions with the SLP-76 complex. Stabilization could occur through direct interactions or via the F-actin scaffold. The fact that HS1 binds directly to Vav1 suggests that these important actin regulatory proteins function coordinately. It will be interesting to ask whether this interaction modifies the activity of either protein.
Phosphorylation also mediates HS1 interaction with PLCγ1, and studies are underway to probe this interaction further. Defects in signaling through PLCγ1 could account for the observed defects in Ca2+ influx in HS1-deficient T cells. However, phosphorylation of PLCγ1 at Y783 is unimpaired in HS1-suppressed cells (data not shown). Regulated interactions with PLCγ1 and the p85α subunit of PI-3K may also play a role in HS1-mediated actin regulation via effects on inositol phospholipids.
Initial analysis of HS1−/−
mice demonstrated defects in T cell proliferation and negative selection (Taniuchi et al., 1995
) but did not address the molecular basis of these defects. We show that HS1−/−
T cells have defective actin and Ca2+
responses, as well as defective IL-2 production associated with defects in activation of NFAT and NFκB transcriptional elements. It remains to be determined how alterations in HS1 function result in the observed changes in IL-2 promoter activation. Perturbations in signaling through Vav1 and/or PLCγ1 may be involved, since both proteins are required for activation of NFAT and NFκB (Cao et al., 2002
; Costello et al., 1999
; Dolmetsch et al., 1998
; Irvin et al., 2000
). Alternatively, as we recently reported for WAVE2 (Nolz et al., 2006
), HS1-dependent actin polymerization may be required to regulate CRAC activity. Studies aimed at distinguishing between these possibilities and additional analysis of HS1 function in TCR transgenic models are underway.