RhoH as an Adaptor for ZAP-70 Figure 3. Lck, ZAP-70, CD3f and RhoH form multi-protein complexes in different subcellular fractions. Jurkat T cells transduced with a retroviral vector expressing HA-RhoH were stimulated or not with anti-CD3e antibody before subcellular fractionation into cytosol (C), detergent- soluble (SM) and detergent-insoluble (IM) membrane fractions. Equal volumes of each fraction were immunoprecipitated using anti-ZAP-70, anti-Lck or anti-CD3f antibodies. (A) Interaction of RhoH with ZAP-70 in different subcellular fractions. IP products and total cell lysates as loading controls were immunoblotted for HA-RhoH (anti-HA antibody), ZAP-70 and b-actin as a loading control. (B) Interaction of ZAP-70, CD3f and Lck in different subcellular fractions. IP products were analyzed by immunoblotting for ZAP-70, Lck and CD3f. (C) Association of Lck with phospho-CD3f and RhoH in different subcellular fractions. IP products and total lysates were analyzed by immunoblotting for Lck, CD3f and HA-RhoH (anti-HA antibody). Figure shows data from one of at least three independent experiments. doi:10.1371/journal.pone.0013970.g003 between RhoH and ZAP-70, but not phosphorylation of RhoH (Fig. 6, lane 4 vs lane 1). AA-ZAP-70 induced the CD3f phosphorylation and ZAP-70/Lck complex formation. (KA- ZAP-70) did not alter the phosphorylation of RhoH and CD3f (Fig. 6). As noted in HEK293 cells, both CA-Lck and AA-ZAP-70 enhanced the co-association of ZAP-70/RhoH/CD3f complex formation, but KA-ZAP-70 inhibited ZAP-70/Lck association in Jurkat T cells as well as HEK293 cells (Fig. 5 & 6). These data suggest that Lck phosphorylates RhoH and CD3f through the interaction with functional ZAP-70. Myristoylation sequence tagged ZAP-70 can partially rescue the defective TCR signaling and thymic development in Rhoh-/- thymocytes Transient or stable membrane association of ZAP-70 mediated by the membrane-permeable synthetic ligand FK1012A or by a myristoylation sequence -tagged ZAP70 (Myr-ZAP-70) can activate downstream signaling of TCR[35,36]. Since ZAP-70 does not translocate to the TCR complex in Rho-deficient T cells, to determine if defective T cell development in Rhoh-/- mice was due to defective ZAP-70 membrane localization, we generated a retroviral vector expressing Myr-ZAP-70 or control vector and utilized these vectors to transduce Rhoh-/- low density bone marrow cells (LDBM). First to determine the functional consequences of Myr-ZAP-70, we transduced Ba/F3 cells. Expression of the Myr-ZAP-70 in Ba/F3 cells led to an increase in the proportion of ZAP-70 protein in the detergent- soluble (27% to 40%) and detergent-insoluble (6% to 22%) membrane fractions of these cells (Fig. S1A). We subsequently compared the subcellular localization of Myr-ZAP-70 with WT- ZAP-70 in the transduced LDBM cells. Most Myr-ZAP-70 was localized to the plasma membrane in these transduced cells (Fig. S1B). PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e13970