Functional Characterization of the RhoH c-Terminus and Insert Domain As Potential Drug Targets

Abstract 1781

RhoH is a hematopoietic-specific, ‘GTPase-deficient’ member of the RhoE/Rnd3 subfamily that remains in a GTP-bound, constitutively active state. Thus, RhoH effects are thought to be regulated at the level of expression or posttranslational modification. RhoH functions as an adaptor molecule facilitating the colocalization of LCK and ZAP70 to the immunological synapse after TCR activation (Chae et al., 2010). Rhoh^−/− mice exhibit a profound T cell defect (Gu et al., 2006; Dorn et al., 2007). Furthermore RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia (CLL) and disease progression is attenuated in a Rhoh^−/− mouse model of CLL (Sanchez-Aguilera et al., 2009). These data suggest that RhoH represents a new therapeutic target in CLL and detailed understanding of the regulation of RhoH activity is critical for development of new treatment strategies. Similar to other Rho GTPase family proteins, RhoH has a polybasic domain and prenylation site that regulate its membrane localization and function (Chae et al., 2008) but also an unique insert domain (LFSINE) whose function is unknown. We therefore investigated the impact of the RhoH c-terminus on T cell development, ZAP70 interaction, subcellular localization and protein levels. EGFP-fused or HA-tagged WT and c-terminal deleted RhoH mutants (Table 1) were cloned into retroviral expression vectors and expressed in murine bone marrow (BM) and Jurkat T cells. Given the critical role of RhoH and ZAP70 interaction in TCR signaling and T cell function, we evaluated the relevance of the c-terminal RhoH domains for in vivo T cell development using Rhoh^−/− mouse BM cells in transplantation rescue experiments. Rhoh^−/− BM cells were transduced with EGFP-RhoH mutants, transplanted into lethally irradiated recipients and 2 months later T cell reconstitution (EGFP⁺/CD3⁺ cells) was assessed in the peripheral blood. Mice transplanted with empty vector or RhoH^δCT transduced Rhoh^−/− BM remained T cell deficient (0.1+0.02 and 0.1+0.01 EGFP⁺ T cells (K/μl), respectively; n=4-5; mean +/− SEM), re-expression of WT RhoH or RhoH^δLFSINE in Rhoh^−/− BM rescued the T cell defect to near normal levels compared to WT BM (0.6+0.1 and 0.5+0.1 vs. 0.9+0.1 EGFP⁺ T cells (K/μl), respectively). We next studied the interaction of RhoH mutant proteins with ZAP70 in transduced Jurkat cells by immunoprecipitation. WT and particularly RhoH^δLFSINE immunoprecipitated with ZAP70, RhoH^δCT and RhoH^δPR did not. Cell fractionation experiments revealed disruption of membrane localization in prenylation site-deleted RhoH^δPR and RhoH^δCT. RhoH^δLFSINE and WT protein localized to the membrane fraction. RhoH^δLFSINE also accumulated more than WT protein in the cytosolic fraction. To determine the basis for the excessive accumulation of RhoH^δLFSINE studies on protein stability were undertaken. Loss of the prenylation site reduced RhoH protein half life, while deletion of the insert domain substantially enhanced protein stability (Table1). Treatment with proteosome inhibitors enhanced RhoH^δCT and RhoH^δPR protein levels, but had no effect on WT RhoH or RhoH^δLFSINE. Treatment with a HSP90 inhibitor (a component of the chaperone-mediated autophagy (CMA) pathway) accelerated the degradation of WT RhoH and RhoH^δCT with less effect on RhoH^δLFSINE levels. Lysosomal inhibition increased WT RhoH protein levels but had no effect on RhoH^δLFSINE. Taken together, these findings imply that membrane localization and ZAP70 interaction both rely on the function of prenylation sites but are not affected by loss of the insert domain. Post-translational degradation of WT RhoH is regulated via CMA. The significantly enhanced protein stability of RhoH^δLFSINE suggests that LFSINE serves as unique lysosomal recognition sequence regulating lysosomal RhoH uptake. Overall, the c-terminal protein domains are critical for RhoH protein stability and activity and could serve as future target sequences to treat disorders associated with altered RhoH expression.