RhoH Regulates Subcellular Localization of ZAP-70 and Lck in T Cell Receptor Signaling Hee-Don Chae1¤a, Jamie E. Siefring 1 , David A. Hildeman2, Yi Gu1¤b, David A. Williams1*¤c 1 Division of Experimental Hematology, Cincinnati Children’s Research Foundation, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 2 Division of Immunobiology, Cincinnati Children’s Research Foundation, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America Abstract RhoH is an hematopoietic-specific, GTPase-deficient Rho GTPase that plays a role in T development. We investigated the mechanisms of RhoH function in TCR signaling. We found that the association between Lck and CD3f was impaired in RhoH-deficient T cells, due to defective translocation of both Lck and ZAP-70 to the immunological synapse. RhoH with Lck and ZAP-70 localizes in the detergent-soluble membrane fraction where the complex is associated with CD3f phosphorylation. To determine if impaired translocation of ZAP-70 was a major determinant of defective T cell development, Rhoh-/- bone marrow cells were transduced with a chimeric myristoylation-tagged ZAP-70. Myr-ZAP-70 transduced cells partially reversed the in vivo defects of RhoH-associated thymic development and TCR signaling. Together, our results suggest that RhoH regulates TCR signaling via recruitment of ZAP-70 and Lck to CD3f in the immunological synapse. Thus, we define a new function for a RhoH GTPase as an adaptor molecule in TCR signaling pathway. Citation: Chae H-D, Siefring JE, Hildeman DA, Gu Y, Williams DA (2010) RhoH Regulates Subcellular Localization of ZAP-70 and Lck in T Cell Receptor Signaling. PLoS ONE 5(11): e13970. doi:10.1371/journal.pone.0013970 Editor: Derya Unutmaz, New York University, United States of America Received March 12, 2010; Accepted October 19, 2010; Published November 12, 2010 Copyright: 2010 Chae et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work in this manuscript was supported by grant R01 CA113969 from the National Institutes of Health to David Williams. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: DAWilliams@childrens.harvard.edu ¤a Current address: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America ¤b Current address: Innovation Center China, AstraZeneca, Shanghai, China ¤c Current address: Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and Harvard Stem Cell Institute, Boston, Massachusetts, United States of America Introduction RhoH, an hematopoietic-specific Rho GTPase,was first iden- tified as a fusion transcript with Bcl-6 in B-cell diffuse large cell lymphoma[1]. RhoH is a member of RhoE/Rnd3 subfamily that has no intrinsic GTPase activity and remains in a GTP-bound and constitutively active state. RhoH cellular function has been reported to be controlled at both the transcriptional and post- translational levels[2,3,4]. For example, RhoH mRNA is down- regulated after phorbol myristate acetate treatment in Jurkat T cells and after stimulation of T cell receptor (TCR) in Th1 cells[4]. RhoH has also been proposed to function as a negative regulator of other Rho GTPases, particularly Rac. For instance, RhoH is a potent inhibitor of the activation of NFkB and p38 kinase by other Rho GTPases in Jurkat T cells[4]. Overexpression of RhoH impairs the activation of Rac GTPases and negatively regulates the Rac-mediated cortical F-actin assembly and chemotaxis in hematopoietic progenitor cells (HPCs)[5,6]. The level of activated Rac GTPases is elevated in Rhoh-/- HPCs[5]. We previously demonstrated that Rhoh-/- mice have impaired TCR signaling, resulting in defective thymocyte selection and blocked maturation of T cells[3]. CD3f phosphorylation and translocation of ZAP-70 to the immunological synapse are impaired in RhoH-deficient T cells, which results in the defective activation of components of ZAP-70-mediated signaling pathway including Linker for Activation of T cells (LAT) and p44/42 MAP kinase[3,7]. Upon TCR stimulation, RhoH is phosphorylated on tyrosine residues within an immunoreceptor tyrosine-based activation motif (ITAM)-like motif which leads to enhanced interaction of RhoH and ZAP-70[3]. Thus, RhoH plays a critical role in TCR signaling and activation of ZAP-70 and is essential for T cell development and TCR-mediated function. TCR signaling is controlled by complex cross-regulation of several key downstream signaling components. ZAP-70 has been reported to promote the phosphorylation of CD3f and the association of CD3f with Lck, as ZAP-70 deficiency impairs the TCR-induced CD3f phosphorylation[8,9,10]. Although RhoH- deficient T cells also have defective recruitment of ZAP-70 to TCR and CD3f phosphorylation is impaired in Rhoh-/- T cells[3], the molecular underpinnings of how RhoH regulates Lck- mediated CD3f phosphorylation via ZAP-70 in TCR signaling remain unclear. Here, we investigated further the role of RhoH in localizing ZAP-70 to the TCR and the potential role of RhoH in Lck-mediated CD3f activation. In Rhoh-/- T cells, Lck showed impaired association with CD3f and decreased localization to the immunologic synapse. This was not associated with defects in its kinase activity as measured by in vitro kinase assay. Further biochemical data suggest that RhoH regulates CD3f phosphor- ylation by recruiting ZAP-70 and Lck to the cell membrance fraction to form the TCR complex for TCR signaling activation. In these studies, we found that membrane-targeted ZAP-70 could partially rescue the defective TCR signaling in RhoH-deficient T PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13970