RhoH as an Adaptor for ZAP-70 Figure 1. RhoH mediates the interaction of Lck and CD3f. Thymocytes (A) and lymph node (LN)-derived T cells (B) from wild type (WT) and Rhoh-/- mice were left unstimulated or stimulated with anti-CD3/28 Abs for 2 min. Lck autophosphorylation activity was measured by immune complex kinase assay with [c-32P]ATP. Total lysates were submitted to immunoprecipitation (IP) of Lck and immunoprecipitates and total lysates were immunoblotted (IB) for CD3f, p-Tyr and Lck. Lck phosphorylation were analyzed by immunoblotting for anti-pY394-Lck (anti-pY416-Src) or Lck of Lck IP products. Data are representative of three or more experiments with similar results. doi:10.1371/journal.pone.0013970.g001 regulators of actin polymerization, cytoskeleton assembly and integrin function, we examined the effect of RhoH-deficiency on actin polymerization in the IS. Actin polymerization was normal at the T-APC contact site in RhoH-deficient CD8+ T cells (Fig. 2B), while other markers such as CD8 and the TCRb chain localize to the IS (data not shown). These data suggest that the defect in Lck and ZAP-70 translocation in RhoH-deficient T cells is not due to secondary effects on actin polymerization. Subcellular localization of Lck, CD3f, ZAP-70 and RhoH Engagement of the TCR induces the translocation of several molecules such as Lck, ZAP-70 and CD3f to the detergent-insoluble membrane fraction of T cells that contains lipid rafts[29,30,31]. For further biochemical studies of the role of RhoH in Lck/ZAP-70/ CD3f complex subcellular translocation, we expressed HA-tagged RhoH in Jurkat T cells. As measured by immunoblot, anti-CD3e antibody stimulation led to the translocation of both HA-RhoH and ZAP-70 to the detergent-insoluble membrane fraction (31.4762.7% to 46.3464.08%, RhoH, unstimulated vs stimulated p,0.01; 11.9362.29% to 2464.92%, ZAP-70, unstimulated vs stimulated p,0.02; mean6SD; N = 3) with a significant amount of ZAP-70-bound HA-RhoH (Fig. 3A). Since ZAP-70 is defective in translocation to the TCR complex and the detergent-insoluble membrane fraction in RhoH-deficient T cells[3], we next analyzed the association of ZAP-70 and Lck in different subcellular fractions by immunoprecipitation experiments. ZAP-70 co-immunoprecipitated with Lck in the detergent-soluble membrane fraction of transfected Jurkat T cells without stimulation. Treatment with anti-CD3e Ab enhanced the association of ZAP-70 with Lck in the detergent-soluble and detergent-insoluble mem- brane fraction (upper panel in Fig. 3B). As expected from previous studies most of the complex remained in the detergent-soluble membrane fraction. Since Lck phosphorylates CD3f and phospho- CD3f recruits ZAP-70 to TCR complex to activate downstream signaling molecules[32,33] we investigated the possible interactions of Lck with RhoH and CD3f. After anti-CD3e stimulation most CD3f-bound ZAP-70 was detected in the detergent-insoluble membrane (Fig. 3 B, lower panel). In these studies, fully phosphorylated CD3f (p23) localized mostly to the detergent- insoluble membrane (Fig. 3C, lower panel) and Lck co-immuno- precipitated primarily with the phosphorylated form of CD3f in anti-CD3e stimulated cells. RhoH together with CD3f was associated with Lck and ZAP-70 as determined by co-immunopre- cipitation analysis after anti-CD3e stimulation. Lck also co- immunoprecipitated with HA-RhoH in the detergent-soluble membrane fraction after anti-CD3e Ab stimulation (Fig. 3B & C). Enhancement of ZAP-70/RhoH complex formation by Lck Binding of ZAP-70 to the ITAM motifs of CD3f has been suggested to induce a conformational change of ZAP-70 that facilitates the phosphorylation of Tyr315 and Tyr319 in inter- domain B of ZAP-70[17,34]. These phosphorylation events establish the active conformation of ZAP70. We have previously shown that RhoH associates with ZAP-70 in a phosphorylation- dependent manner[3]. To further study the potential formation of multi-protein complexes containing RhoH, we transfected HEK293 cells with RhoH, ZAP-70 and a constitutive active mutant of Lck (CA-Lck). Exogenously-expressed RhoH did not affect the association of ZAP-70 and Lck (Fig. 4A, upper panel, lane 4 vs lane 5 and summarized in C) or the phosphorylation on Tyr319 and PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13970