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RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development

Nature Immunology volume 7, pages 1182–1190 (2006)Cite this article

Abstract

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 CD3ζ 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.

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Figure 1: Substantial blockade of intrathymic T cell development in Rhoh−/− mice.
Figure 2: Defective TCR-mediated thymic positive selection in Rhoh−/− mice.
Figure 3: Transgenic expression of RhoH restores Rhoh−/− intrathymic development and T cell function.
Figure 4: RhoH is tyrosine-phosphorylated and interacts with Zap70.
Figure 5: Conserved tyrosine-phosphorylated sequence motifs in RhoH mediate the interaction with Zap70 and RhoH function in thymocyte development.
Figure 6: Rhoh−/− thymocytes have impaired translocation of Zap70 to the plasma membrane and the immunological synapse and reduced phosphorylation of Zap70, CD3ζ and p42-p44.

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Acknowledgements

We thank D. Schumann and Y. Yuan (Genome Research Institute, University of Cincinnati, Cincinnati, Ohio) for assistance with mass spectrometry analysis; A.S. Shaw (Washington University School of Medicine, St. Louis. Missouri) for the pGEM3Z-Zap70, pGEM3Z-Zap70ΔN-SH2 and pGEM3Z-Zap70ΔC-SH2 constructs; C.T. Baldari (University of Siena, Siena, Italy) for the constitutively active Lck construct; P. Marrack (National Jewish Medical and Research Center, Denver, Colorado) for breeding pairs of p14 lymphocytic choriomeningitis virus TCR–transgenic mice; S. Li, J. Bailey and V. Summer (Cincinnati Children's Hospital, Cincinnati, Ohio) for technical assistance; and Y. Zheng for discussions and critical comments on the manuscript. Supported by the National Cancer Institute (KO1 CA107110 to Y.G.) and the US National Institutes of Health (RO1 DK62757 to D.A.W. and RO1 CA113969).

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Author notes

  1. Yi Gu and Hee-Don Chae: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Experimental Hematology, Cincinnati Children's Research Foundation and Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, 45229, Ohio, USA

    Yi Gu, Hee-Don Chae, Jamie E Siefring, Aparna C Jasti & David A Williams

  2. Division of Immunobiology, Cincinnati Children's Research Foundation and Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, 45229, Ohio, USA

    David A Hildeman

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Contributions

Y.G. designed and did experiments and wrote the draft of the paper; H.-D.C. did experiments; J.E.S. and A.C.J. assisted in experiments; D.A.H. provided advice and directed some experiments; and D.A.W. provided oversight for all experiments, designed experiments and edited the draft paper.

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Correspondence to Yi Gu or David A Williams.

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Supplementary information

Supplementary Fig. 1

Gene-targeted deletion of the murine Rhoh allele. (PDF 756 kb)

Supplementary Fig. 2

Viability and proliferation of WT and Rhoh−/− thymocytes. (PDF 337 kb)

Supplementary Fig. 3

Thymic cellularity and distribution of CD4+CD8+ thymocyte subsets in p14tg/+ Rhoh−/− mice. (PDF 163 kb)

Supplementary Fig. 4

Impaired phosophorylation and recruitment of Zap70 to the immunological synapse in Rhoh−/− peripheral T cells. (PDF 968 kb)

Supplementary Table 1

T and B lymphocyte populations in Rhoh−/− mice. (PDF 84 kb)

Supplementary Table 2

Engraftment and T cell reconstitution of the RhoH-transduced Rhoh−/− BM cells in Rag2−/− recipient mice. (PDF 87 kb)

Supplementary Methods (PDF 70 kb)

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Gu, Y., Chae, HD., Siefring, J. et al. RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development. Nat Immunol 7, 1182–1190 (2006). https://doi.org/10.1038/ni1396

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