RhoH Functions as an Adaptor Molecule for ZAP-70 in TCR Signaling Pathway.

Abstract

RhoH is a hematopoietic-specific and GTPase-deficient Rho GTPase of the RhoE family. In a previous report, we showed that RhoH plays a critical role in T cell receptor (TCR) signaling via recruitment of ZAP-70 in T cell development (Gu et al., Nat. Immunol., 2006). RhoH-deficient T cells show impaired CD3ζ phosphorylation which is critical for the recruitment of ZAP-70 to the TCR complex. In this study, we investigated whether RhoH acts as an adaptor molecule for ZAP-70 membrane targeting or a regulator of the Lck activity. Although Lck activity was not significantly decreased in RhoH-deficient T cells as determined by the level of Lck phosphorylation and in vitro kinase assay, Lck protein did not translocate to the immunological synapse in these cells and the association between Lck and CD3ζ was impaired. ZAP-70 has been reported to mediate the recruitment of Lck into TCR complex with subsequent CD3ζ phosphorylation. In RhoH-deficient T cells, ZAP-70 did not translocate to TCR complex in the plasma membrane, especially the detergent-insoluble (cytoskeletal) fraction. To further determine if RhoH mediated membrane localization of ZAP-70 and if this localization was responsible for normal T cell differentiation, we generated a myristoylation sequence-fused ZAP-70 (Myr-ZAP-70). Myr-ZAP-70 localized to the membrane fraction and resulted in the partial rescue of RhoH−/ − T cell phenotype as measured by T cell reconstitution in Rag2−/ − recipient mice (Table 1). These results suggest that RhoH plays a key role in TCR signaling as an adaptor for ZAP-70 plasma membrane localization.Table 1:Engraftment and T cell reconstitution of the transduced RhoH−/ − BM cells in Rag2−/ − recipient miceDonor cells (retroviral vector transduction)CD4+ CD8− cells (x 10e5)CD4− CD8+ cells (x 10e5)SpleenPB (ml)SpleenPB (ml)RhoH−/− (empty vector)3.330.91.20.36RhoH−/− (ZAP-70)16.71.23.10.6RhoH−/−(Myr-ZAP-70)35.23.39.12.2RhoH−/− (HA-RhoH)65.03.913.61.3