RhoH as an Adaptor for ZAP-70 Figure 6. Constitutive active Lck enhances the phosphorylation and ZAP-70 association of RhoH in Jurkat T cells. Jurkat T cells transduced with a retroviral vector expressing HA-RhoH were transfected with combinations of the plasmids expressing EGFP, CA-Lck, kinase-dead Lck (KD-Lck), constitutive active ZAP-70 (AA-ZAP-70) or kinase-dead ZAP-70 (KA-ZAP-70). Total lysates were submitted to immunoprecipitation (IP) of anti-ZAP-70, anti-Lck or anti-HA Ab and immunoprecipitates and total lysates were analyzed by immunoblotting. doi:10.1371/journal.pone.0013970.g006 RhoH efficiently, we also investigated the potential role of ZAP-70 in RhoH phosphorylation. Co-expression of kinase-dead Lck and ZAP-70 did not result in measurable phosphorylation of RhoH. However, a constitutive active mutant of Lck induced phosphor- ylation of RhoH and enhanced interaction among ZAP-70/Lck/ RhoH in Jurkat T cells, whereas kinase-dead Lck inhibited the phosphorylation of RhoH and CD3f. Constitutive active ZAP-70 also enhanced ZAP-70/RhoH complex formation through Lck activation because AA-ZAP-70 showed enhanced CD3f phos- phorylation and association with Lck in Jurkat T cells. Co- expression of kinase-dead ZAP-70 with CA-Lck resulted in reduced phosphorylation of RhoH, and the KA-ZAP-70 did not co-immunoprecipitate with RhoH and showed lower binding affinity to Lck. These data suggest that functional ZAP-70 is required for the Lck-mediated RhoH phosphorylation and optimal association of RhoH with ZAP-70. Supporting a critical role for RhoH in functionally important localization of ZAP-70 to the membrane, anti-CD3/28 Abs- induced phosphorylation of CD3f and LAT was partially restored in Rhoh-/- thymocytes expressing Myr-ZAP-70. As expected, a higher proportion of Myr-ZAP-70 compared with wild-type ZAP- 70 showed association with the cell membrane in T cells. Myr- ZAP-70 partially rescued in thymocyte development and TCR signaling in the absence of RhoH. Membrane-bound ZAP-70 can activate TCR minimally without stimuli, but still requires functional Lck activity [35,36]. Since Myr-ZAP-70 can partially rescue the thymocyte development and TCR signaling in Rhoh-/- cells, we suggest that RhoH function is required for the membrane localization of ZAP-70, particularly to the IS. In the experiments reported here, there could be two reasons why Myr-ZAP-70 did not completely restore the TCR signaling in Rhoh-/- cells completely. First, the sustained TCR activation by membrane- bound ZAP-70 might induce a refractory state that reduces TCR signaling efficiency as previously reported[35]. Second, src myristoylation sequence broadly localizes ZAP-70 to the mem- brane, and not to specific TCR signaling regions (Fig. S1)[35]. In Rhoh-/- T cells expressing Myr-ZAP-70, Lck was polarized towards the interface of cells and microbeads coated with anti-CD3e and anti-CD28 antibodies, while Lck remained dispersed over the entire membrane in Rhoh-/- T cells expressing only EGFP. These data strongly suggest that RhoH functions with ZAP-70 to also facilitate the translocation of Lck into the TCR complex. Taken together with the data showing partial rescue of T cell development after Rhoh-/- cells expressing Myr-ZAP-70 are transferred into Rag2-/- mice, these data suggest that this function of RhoH is physiologically important. Other investigators have confirmed that RhoH promotes the ZAP70-dependent phosphorylation of the LAT signalosome[7]. Although they did not show defective localization and activation of ZAP-70, there was complete loss of CD3f phosphorylation[44]. One possibility to explain the difference in ZAP-70 phosphory- lation in these studies is that this group stimulated T cells using CD3 and CD4 co-crosslinking, which could lead to the direct recruitment of Lck. Altogether, these data suggest that Lck/ZAP-70 complex may facilitate the phosphorylation of RhoH, which then enhances the PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e13970