RhoH as an Adaptor for ZAP-70 Figure 2. Impaired recruitment of Lck to the immunological synapse in Rhoh-/- T cells. (A) CD8+ T cells from WT or Rhoh-/- p14 TCR transgenic mice were conjugated with gp33 peptide-preloaded APC cells (CH.B2 cells) for 5 min. Cells were fixed and stained with anti-ZAP-70 (green) and anti-Lck (red). The localization of the immune synapse is indicated with a white arrow. Bars, 3 mm. (B) T cell-APC conjugates were fixed and stained with TRITC-Phalloidin for detection of F-actin. Differential interference contrast (DIC) images show the antigen-specific T cell-APC conjugates. Bars, 3 mm. At least 100 cell conjugates were examined per condition. doi:10.1371/journal.pone.0013970.g002 Tyr493 of ZAP-70 by CA-Lck (Fig. 4B, lane 4 vs lane 5). CA-Lck alone did not phosphorylate RhoH to the same level as co- expression of CA-Lck and ZAP-70(Fig. 4A, lower panel, lane 2 vs lane 4 and C). Expression of CA-Lck, ZAP-70 and RhoH together enhanced the interaction of ZAP-70 with RhoH and the phosphorylation of RhoH (Fig. 4A, upper panel, lane 4 and lower panel and C). These data and the fact that RhoH did not co- immunoprecipitate with Lck in the absence of ZAP-70 suggest that Lck does not interact directly with RhoH in the absence of ZAP-70. Since CA-Lck alone did not phosphorylate RhoH efficiently, we next investigated whether ZAP-70 could phosphorylate RhoH with or without expression of Lck. As noted above, co-expression of CA-Lck, ZAP-70 and RhoH led to increased phosphorylation of RhoH which is immunoprecipitated with ZAP-70 (Fig. 5, lane 3). A co-expressed kinase-dead mutant of Lck (KD-Lck) and ZAP-70 did not complex with RhoH and was not associated with phosphorylation of RhoH (Fig. 5, lane 4). While co-expression of a kinase-dead ZAP-70 mutant (KA-ZAP-70) with CA-Lck could still lead to increased phosphorylation of RhoH, CA-Lck did not enhance the association of ZAP-70 with RhoH and KA-ZAP-70 also showed lower affinity to CA-Lck. (Fig. 5, lane 5). These data suggest that the kinase activities of both ZAP-70 and Lck are required for the association of RhoH with ZAP-70 and efficient phosphorylation of RhoH. We next investigated whether Lck and ZAP-70 activity is required for the association of ZAP-70 with RhoH and phosphorylation of RhoH in T cells (Fig. 6). Expression of CA- Lck in Jurkat T cells induced the phosphorylation of ZAP-70, CD3f, and RhoH. CA-Lck also enhanced the association of ZAP- 70 with RhoH (Fig. 6, lane 5 vs. lane 1). In contrast, KD-Lck inhibited the phosphorylation of CD3f, ZAP-70, and RhoH (Fig. 6, lane 3) and reduced the association of RhoH with ZAP70 (Fig.6, lane 3 vs lane 6). Jurkat T cells expressing a constitutive active ZAP-70 mutant (AA-ZAP-70) showed enhanced association PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13970