RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development Yi Gu 1,3 , Hee-Don Chae 1,3 , Jamie E Siefring 1 , Aparna C Jasti 1 , David A Hildeman 2 & David A Williams 1 RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family with unknown physiological function. Here we demonstrate that Rhoh –/– mice have impaired T cell receptor (TCR)–mediated thymocyte selection and maturation, resulting in T cell deficiency. RhoH deficiency resulted in defective CD3f phosphorylation, impaired translocation of the signaling molecule Zap70 to the immunological synapse and reduced activation of Zap70-mediated signaling in thymic and peripheral T cells. Proteomic analyses demonstrated that RhoH is a component of TCR signaling and is required for recruitment of Zap70 to the TCR through interaction with RhoH noncanonical immunoreceptor tyrosine-based activation motifs (ITAMs). In vivo reconstitution studies also demonstrated that RhoH function depends on phosphorylation of the RhoH ITAMs. These findings suggest that RhoH is a critical regulator of thymocyte development and TCR signaling by mediating recruitment and activation of Zap70. As demonstrated by studies of human immunophenotypes and a variety of gene-targeted mouse lines, functional T cell development is critical for the adaptive immune system. Maturation of T lymphocytes with the appropriate immune repertoire occurs in the thymus and involves a tightly controlled, sequential process of T cell receptor (TCR) gene rearrangement, positive and negative selection and proliferative expansion 1 . There are three critical checkpoints in the TCRab lineage maturation, comprising b-selection regulated by a pre-TCR signaling complex 2,3 and positive and negative selection controlled by signals derived from a fully rearranged TCRab antigen complex 4 . The Rho family GTPases are Ras-like molecules increasingly recog- nized as important signaling proteins involved in the pre-TCR and TCRab pathways during T cell differentiation and proliferation 5 . In general, Rho GTPases cycle between GTP-bound, active and GDP-bound, inactive states and are regulated by the activity of guanine nucleotide–exchange factors and GTPase-activating proteins. Rhoh, first identified as a hypermutable gene in non-Hodgkin lym- phoma 6,7 and a member of the RhoE subfamily, lacks intrinsic and/or agonist-induced GTPase activity and remains in the GTP-bound, active conformation 8 . Thus, regulation of the cellular activity of this GTPase remains unclear but it may be depend in part on cell-specific expression that determines the amount of intracellular protein. Indeed, RhoH is hematopoietic specific and has high expression in mouse thymus and human T cells 8,9 . Experimental alteration of RhoH expression affects the proliferation and engraftment of hematopoietic progenitor cells 9 and integrin-mediated adhesion in Jurkat cells 10 . Studies have reported that transcriptional regulation of the Rhoh gene and alternative splicing of 5¢ exons of Rhoh mRNA occur in hema- topoietic and lymphoid cells 8,11 . However, key unanswered biological issues remain, including the mechanism of physiological regulation and the function of RhoH in the hematopoietic lineages. Intrathymic selection events are dependent on TCR signal transduc- tion mediated by protein tyrosine kinases 12 . Engagement of antigen receptors by self antigens expressed on the thymic stromal cells initiates activation of the Src family kinases Lck and Fyn. Phospho- rylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of CD3z subunits by Src family kinases induces interaction of CD3z with Zap70 in the plasma membrane and cytoskeleton-enriched fractions and activation of Zap70 (refs. 13,14). Zap70 subsequently stimulates ‘downstream’ signaling cascades, including the activation (phosphorylation) of mitogen-activated protein kinases 15 . Zap70-deficient mice have developmental arrest of thymocytes at the CD4 + CD8 + double-positive (DP) stage 16 . The importance of Zap70 in human T cell differentiation and/or function has also been demonstrated in patients with loss- of-function mutations in the gene encoding Zap70 (refs. 17,18) or defective recruitment of Zap70 to the signaling competent TCR complex 19 . So far, the molecular mechanism involved in regulating the recruitment of Zap70 to the TCR subunits for signaling activation is incompletely understood, and as-yet-unidentified molecules have been linked to the process. Our studies here of gene-targeted Received 11 May; accepted 25 August; published online 8 October 2006; doi:10.1038/ni1396 1 Division of Experimental Hematology and 2 Division of Immunobiology, Cincinnati Children’s Research Foundation and Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA. 3 These authors contributed equally to this work. Correspondence should be addressed to D.A.W. (david.williams@cchmc.org) or Y.G. (yi.gu@cchmc.org). 1182 VOLUME 7 NUMBER 11 NOVEMBER 2006 NATURE IMMUNOLOGY ARTICLES © 2006 Nature Publishing Group http://www.nature.com/natureimmunology