RhoH as an Adaptor for ZAP-70 Figure 7. Membrane targeting of ZAP-70 partially rescues defective Rhoh-/- thymic development. Rhoh-/- LDBM cells were transduced with a retroviral vector co-expressing Myr-ZAP-70, HA-RhoH and EGFP or EGFP alone as a control. EGFP+-sorted cells were injected intravenously into sub-lethally irradiated Rag2-/- recipient mice. Thymocytes from Rag2-/- recipient mice were analyzed 8 weeks after transplantation by flow cytometry (A) and immunoblotting (B). (A) Number of CD8 single positive cells in the thymus of Rag2-/- recipient mice. Thymocytes from the recipient mice were analyzed for CD4/CD8 subsets by flow cytometry. CD8 SP thymocytes were calculated by (frequency) x (cellularity). Data represent the mean +/- SD. N = 3 mice per transplant group. (B) Phosphorylation of CD3f, LAT and p44/42 in Myr-ZAP-70-expressing Rhoh-/- thymocytes. Thymocytes from Rag2-/- recipient mice were left unstimulated or stimulated anti-CD3e and CD28 mAbs for 2 min. Total lysates were separated on a SDS-polyacrylamide gel then transferred to PVDF membranes and immunoblotted for CD3f, p-Tyr, p-p44/42, p44/42 and p-LAT. (C) Subcellular localization of Lck in Rhoh-/- T cells expressing Myr-ZAP-70. Rhoh-/- T cells were transduced with retroviruses co-expressing Myr-ZAP-70 and EGFP or EGFP alone. EGFP+ T cells were incubated with biotin-labeled anti-CD3 and CD28 Abs, and then conjugated with Dynabeads Biotin Binder for 5 min. Cells were fixed and stained with anti-ZAP-70 or anti-Lck Ab. DIC images show the T cell-microbead conjugates. Bars, 3 mm. At least 100 cell conjugates were examined per condition. doi:10.1371/journal.pone.0013970.g007 interaction between ZAP-70 and RhoH. The enhanced association between RhoH and ZAP-70 may then facilitate the localization of Lck/ZAP-70 to the TCR complex leading to the known role of these proteins in phosphorylation of CD3f. After this, ZAP-70 bound to CD3f, ZAP-70/CD3f or ZAP-70/RhoH moves to the detergent-insoluble fraction or IS region. Overall, while RhoH appears to regulate Rac activity in some hematopoietic cell lines and in primary hematopoietic cells, the data presented here and the differences in T cell phenotypes of Rhoh-/-, Rac1-/-;Rac2-/-, ZAP-70-/- and Vav1-/- (reviewed in Wang and Zheng[45]) mice suggest that RhoH functioning in TCR signaling and T cell development is more complicated and in part related to its function as an adaptor molecule that affects localization of both ZAP-70 and Lck in the IS. This defines a novel function of Rho GTPases. Supporting Information Figure S1 Membrane localization of Myr-ZAP-70. (A) Lysates of Ba/F3 cells transduced with retroviral vectors expressing ZAP- 70 or Myristoylated ZAP-70 (Myr-ZAP-70) were separated by centrifugation into cytosol (C), detergent-soluble (SM) and detergent-insoluble (IM) membrane fractions. Equal volumes of each fractions were immunoblotted for ZAP-70 and b-actin as a loading control. (B) LDBM cells from wild type mice were infected with retroviral vectors expressing ZAP-70, Myr-ZAP-70 or EGFP alone. The transduced cells were fixed and stained with anti-ZAP- 70 (red). Bars, 3 mm. Found at: doi:10.1371/journal.pone.0013970.s001 (4.38 MB TIF) Acknowledgments We thank A. Weiss (University of California, San Francisco) for the pCDNA3-KA-ZAP-70 and pCDNA3-AA-ZAP-70; M. J. Bijlmakers (King’s College London School of Medicine, London, UK) for the pSM- CA-Lck and pSM-KD-Lck; C.T. Baldari (University of Siena, Siena, Italy) for the pRC-CMV-CA-Lck. We thank Shelli Homan and Christina Sexton for assistance with mice, and Jeff Bailey and Victoria Summey-Harner for technical assistance. We thank Sung-Yun Pai and Luigi Notarangelo for helpful discussions. Author Contributions Conceived and designed the experiments: HDC. Performed the experi- ments: HDC JES. Wrote the paper: HDC. Advised on experimental design: DW DAH YG. Reviewed and edited the manuscript: DW DAH YG. Reviewed data: DAH YG. PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13970