Figure 5. Both ZAP-70 and Lck kinase activities are required for the association of RhoH with ZAP-70. HEK293 cells were transfected with combinations of the plasmids expressing HA-RhoH, CA-Lck, kinase-dead Lck (KD-Lck), ZAP-70 or kinase-dead ZAP-70 (KD- ZAP-70). Total lysates were submitted to immunoprecipitation (IP) of anti-ZAP-70 or anti-HA Ab and immunoprecipitates and total lysates were analyzed by immunoblotting. doi:10.1371/journal.pone.0013970.g005 ZAP-70 with the T cell membrane was enhanced by expression of Myr-ZAP-70 compared with T cells expressing EGFP alone (Fig. 7C right panel). These data demonstrate that RhoH functions as an adaptor for ZAP-70 to facilitate the translocation of Lck into the TCR complex and membrane-targeted ZAP-70 can partially correct the defective TCR signaling in Rhoh-/- T cells. Discussion RhoH is an hematopoietic-specific GTPase-deficient GTPase of the RhoE family first identified as a fusion of LAZ3/BCL6 in non Hodgkin’s Lymphoma (NHL)[1]. Subsequently, RhoH has been found to be mutated in multiple myeloma and diffuse large B cell lymphomas (DLBCL)[39,40] and in AIDS-associated NHL[41], although the pathophysiological relevance of these findings are still unknown. Due to the presence of alternative residues at the highly conserved amino acids analogous to positions 12 and 61 of the Ras RhoH as an Adaptor for ZAP-70 proteins regulating GTPase activity, RhoH remains GTP bound. Thus, the cellular activity and function of RhoH has been hypothesized to be dependent on protein levels in the cell. Previous studies have implicated RhoH in T cell development and TCR signaling, inside-out integrin signaling and adhesion in T cells and antagonism of the activity of the Rac GTPase pathways in cells of hematopoietic lineages[4,5,7,42]. Using gene- targeted mice deficient in RhoH, we have previously demonstrated defective thymocyte development, positive and negative selection and TCR signaling. Rhoh-/- mice have T lymphopenia and demonstrate reduced TCR-induced T cell proliferation in vitro. Proteomic studies demonstrated that RhoH interacts with ZAP-70 in TCR signaling, providing an initial insight into the molecular mechanism of defective T cell signaling in Rhoh-/- cells. Here we show that RhoH plays a critical role in facilitating the localization of multiple TCR signaling components-likely via the ZAP-70 interaction-to the membrane, particularly the detergent insoluble fraction of the membrane that contains lipid rafts. Lck and ZAP-70 have been shown to translocate to microclusters of receptors and signaling molecules in the peripheral regions of the T-APC interface within lipid rafts during initiation of the TCR activation signal. The clusters form a central supramolecular activation cluster (c-SMAC) of the IS [24,25,26]. In addition, ZAP-70 has been reported to promote CD3f phosphorylation by recruiting Lck to TCR complex in a kinase activity-independent manner[9]. In this regard, although Lck kinase activity appears normal in the absence of RhoH, here we demonstrate the Rhoh-/- T cells show defective ZAP-70 and Lck localization to the IS and defective downstream signaling of the TCR pathway. Our studies demonstrate protein interactions between Lck, ZAP-70 and RhoH and that the translocation of both ZAP-70 and Lck to the IS is decreased in Rhoh-/- T cells. Thus, RhoH appears to be involved in the coordinated movement of proteins in the IS. We are currently studying which domains in addition to the ITAM-like motifs of RhoH are critical for these interactions. Previous studies have implicated the translocation of Lck, ZAP- 70 and the TCR into the IS in effective TCR signaling [29,30,31]. Binding of ZAP-70 to ITAM motifs of CD3f has been suggested to induce a conformational change of ZAP-70 to facilitate the phosphorylation of Tyr315 and Tyr319 in interdomain B of ZAP- 70, which establishes and stabilizes the active conformation of ZAP-70[17,34]. We have previously shown that RhoH associates with ZAP-70 in phosphorylation-dependent manner[3]. In the studies reported here, exogenously-expressed RhoH alone did not affect the association of ZAP-70 and Lck or the phosphorylation of ZAP-70 on Tyr319 and 493 of ZAP-70 by Lck. However, expression of CA-Lck and functional ZAP-70 together enhanced the interaction of ZAP-70 with RhoH and the phosphorylation of RhoH. The motifs involved in these interactions are currently under additional investigations. RhoH-dependent localization of Lck to the IS via ZAP-70 would suggest that ZAP-70 can function both upstream and downstream of Lck in the localization of TCR signaling components and phosphorylation of CD3f. This is consistent with previous reports that TCR-induced phosphoryla- tion of CD3f is reduced in ZAP-70-/- mice, suggesting a role for ZAP-70 as an upstream facilitator of Lck localization and function[43]. Taken together, these data suggest that RhoH may act as an adaptor molecule for ZAP-70 and Lck recruitment which, in turn, phosphorylates RhoH to enhance the heterotrimeric association of these molecules. In support of this hypothesis, CA-Lck is required for the activation of ZAP-70 and co-expression of CA-Lck and ZAP-70 enhanced the phosphorylation and binding affinity of RhoH to ZAP-70. Since CA-Lck alone did not phosphorylate PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e13970