A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific, GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

Anja Troeger,Hee-Don Chae,Mumine Senturk,Jenna Wood,David A Williams

doi:10.1074/jbc.m113.505727

Abstract

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved "CAAX" box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal "insert" domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh(-/-) mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh(-/-) T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function.

Highlights

RhoH is required for T cell development and non-coding RhoH mutations are found in B-cell lymphoma
RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia
To analyze the effect of the mutated domains on protein stability, the transduced cells were treated with CHX and RhoH levels were determined over time by immunoblot. wtRhoH was characterized by a protein half-life of less than 3 h (Fig. 1, C and D), which is similar to published data on the half-lives of other atypical Rho GTPases such as RhoB [32]

Summary

Background

RhoH is required for T cell development and non-coding RhoH mutations are found in B-cell lymphoma. We have demonstrated that ex vivo and in vivo Lenalidomide treatment is associated with decreased RhoH protein levels in human CLL cells [13] These observations suggest a potential therapeutic benefit of targeting RhoH expression in B cell malignancies. Due to its constitutively active state, RhoH activity appears to be mainly determined by the protein level and posttranslational modifications [4, 7, 17, 24] In this regard, we have previously demonstrated the functional importance of phosphorylation of an immunoreceptor tyrosine-based activation motif-like sequence, unique in RhoH among all Rho GTPases, as one mechanism of regulation [8]. The LFSINE domain does not affect RhoH function in normal T and B cell development This suggests a potential drug target for modulation of RhoH protein levels in malignant cells

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION