RhoH is an hematopoietic-specific GTPase-deficient GTPase of the RhoE family first identified as a fusion of LAZ3/BCL6 in non Hodgkin's Lymphoma (NHL)
. Subsequently, RhoH has been found to be mutated in multiple myeloma and diffuse large B cell lymphomas (DLBCL)
and in AIDS-associated NHL
, although the pathophysiological relevance of these findings are still unknown. Due to the presence of alternative residues at the highly conserved amino acids analogous to positions 12 and 61 of the Ras proteins regulating GTPase activity, RhoH remains GTP bound. Thus, the cellular activity and function of RhoH has been hypothesized to be dependent on protein levels in the cell.
Previous studies have implicated RhoH in T cell development and TCR signaling, inside-out integrin signaling and adhesion in T cells and antagonism of the activity of the Rac GTPase pathways in cells of hematopoietic lineages
. Using gene-targeted mice deficient in RhoH, we have previously demonstrated defective thymocyte development, positive and negative selection and TCR signaling. Rhoh-/-
mice have T lymphopenia and demonstrate reduced TCR-induced T cell proliferation in vitro
. Proteomic studies demonstrated that RhoH interacts with ZAP-70 in TCR signaling, providing an initial insight into the molecular mechanism of defective T cell signaling in Rhoh-/-
Here we show that RhoH plays a critical role in facilitating the localization of multiple TCR signaling components-likely via the ZAP-70 interaction-to the membrane, particularly the detergent insoluble fraction of the membrane that contains lipid rafts. Lck and ZAP-70 have been shown to translocate to microclusters of receptors and signaling molecules in the peripheral regions of the T-APC interface within lipid rafts during initiation of the TCR activation signal. The clusters form a central supramolecular activation cluster (c-SMAC) of the IS 
. In addition, ZAP-70 has been reported to promote CD3ζ phosphorylation by recruiting Lck to TCR complex in a kinase activity-independent manner
. In this regard, although Lck kinase activity appears normal in the absence of RhoH, here we demonstrate the Rhoh-/-
T cells show defective ZAP-70 and Lck localization to the IS and defective downstream signaling of the TCR pathway. Our studies demonstrate protein interactions between Lck, ZAP-70 and RhoH and that the translocation of both ZAP-70 and Lck to the IS is decreased in Rhoh-/-
T cells. Thus, RhoH appears to be involved in the coordinated movement of proteins in the IS. We are currently studying which domains in addition to the ITAM-like motifs of RhoH are critical for these interactions.
Previous studies have implicated the translocation of Lck, ZAP-70 and the TCR into the IS in effective TCR signaling 
. Binding of ZAP-70 to ITAM motifs of CD3ζ has been suggested to induce a conformational change of ZAP-70 to facilitate the phosphorylation of Tyr315 and Tyr319 in interdomain B of ZAP-70, which establishes and stabilizes the active conformation of ZAP-70
. We have previously shown that RhoH associates with ZAP-70 in phosphorylation-dependent manner
. In the studies reported here, exogenously-expressed RhoH alone did not affect the association of ZAP-70 and Lck or the phosphorylation of ZAP-70 on Tyr319 and 493 of ZAP-70 by Lck. However, expression of CA-Lck and functional ZAP-70 together enhanced the interaction of ZAP-70 with RhoH and the phosphorylation of RhoH. The motifs involved in these interactions are currently under additional investigations. RhoH-dependent localization of Lck to the IS via ZAP-70 would suggest that ZAP-70 can function both upstream and downstream of Lck in the localization of TCR signaling components and phosphorylation of CD3ζ. This is consistent with previous reports that TCR-induced phosphorylation of CD3ζ is reduced in ZAP-70-/-
mice, suggesting a role for ZAP-70 as an upstream facilitator of Lck localization and function
Taken together, these data suggest that RhoH may act as an adaptor molecule for ZAP-70 and Lck recruitment which, in turn, phosphorylates RhoH to enhance the heterotrimeric association of these molecules. In support of this hypothesis, CA-Lck is required for the activation of ZAP-70 and co-expression of CA-Lck and ZAP-70 enhanced the phosphorylation and binding affinity of RhoH to ZAP-70. Since CA-Lck alone did not phosphorylate RhoH efficiently, we also investigated the potential role of ZAP-70 in RhoH phosphorylation. Co-expression of kinase-dead Lck and ZAP-70 did not result in measurable phosphorylation of RhoH. However, a constitutive active mutant of Lck induced phosphorylation of RhoH and enhanced interaction among ZAP-70/Lck/RhoH in Jurkat T cells, whereas kinase-dead Lck inhibited the phosphorylation of RhoH and CD3ζ. Constitutive active ZAP-70 also enhanced ZAP-70/RhoH complex formation through Lck activation because AA-ZAP-70 showed enhanced CD3ζ phosphorylation and association with Lck in Jurkat T cells. Co-expression of kinase-dead ZAP-70 with CA-Lck resulted in reduced phosphorylation of RhoH, and the KA-ZAP-70 did not co-immunoprecipitate with RhoH and showed lower binding affinity to Lck. These data suggest that functional ZAP-70 is required for the Lck-mediated RhoH phosphorylation and optimal association of RhoH with ZAP-70.
Supporting a critical role for RhoH in functionally important localization of ZAP-70 to the membrane, anti-CD3/28 Abs-induced phosphorylation of CD3ζ and LAT was partially restored in Rhoh-/-
thymocytes expressing Myr-ZAP-70. As expected, a higher proportion of Myr-ZAP-70 compared with wild-type ZAP-70 showed association with the cell membrane in T cells. Myr-ZAP-70 partially rescued in thymocyte development and TCR signaling in the absence of RhoH. Membrane-bound ZAP-70 can activate TCR minimally without stimuli, but still requires functional Lck activity 
. Since Myr-ZAP-70 can partially rescue the thymocyte development and TCR signaling in Rhoh-/-
cells, we suggest that RhoH function is required for the membrane localization of ZAP-70, particularly to the IS. In the experiments reported here, there could be two reasons why Myr-ZAP-70 did not completely restore the TCR signaling in Rhoh-/-
cells completely. First, the sustained TCR activation by membrane-bound ZAP-70 might induce a refractory state that reduces TCR signaling efficiency as previously reported
. Second, src myristoylation sequence broadly localizes ZAP-70 to the membrane, and not to specific TCR signaling regions (Fig. S1
. In Rhoh-/-
T cells expressing Myr-ZAP-70, Lck was polarized towards the interface of cells and microbeads coated with anti-CD3ε and anti-CD28 antibodies, while Lck remained dispersed over the entire membrane in Rhoh-/-
T cells expressing only EGFP. These data strongly suggest that RhoH functions with ZAP-70 to also facilitate the translocation of Lck into the TCR complex. Taken together with the data showing partial rescue of T cell development after Rhoh-/-
cells expressing Myr-ZAP-70 are transferred into Rag2-/-
mice, these data suggest that this function of RhoH is physiologically important.
Other investigators have confirmed that RhoH promotes the ZAP70-dependent phosphorylation of the LAT signalosome
. Although they did not show defective localization and activation of ZAP-70, there was complete loss of CD3ζ phosphorylation
. One possibility to explain the difference in ZAP-70 phosphorylation in these studies is that this group stimulated T cells using CD3 and CD4 co-crosslinking, which could lead to the direct recruitment of Lck.
Altogether, these data suggest that Lck/ZAP-70 complex may facilitate the phosphorylation of RhoH, which then enhances the interaction between ZAP-70 and RhoH. The enhanced association between RhoH and ZAP-70 may then facilitate the localization of Lck/ZAP-70 to the TCR complex leading to the known role of these proteins in phosphorylation of CD3ζ. After this, ZAP-70 bound to CD3ζ, ZAP-70/CD3ζ or ZAP-70/RhoH moves to the detergent-insoluble fraction or IS region. Overall, while RhoH appears to regulate Rac activity in some hematopoietic cell lines and in primary hematopoietic cells, the data presented here and the differences in T cell phenotypes of Rhoh-/-, Rac1-/-;Rac2-/-, ZAP-70-/-
(reviewed in Wang and Zheng
) mice suggest that RhoH functioning in TCR signaling and T cell development is more complicated and in part related to its function as an adaptor molecule that affects localization of both ZAP-70 and Lck in the IS. This defines a novel function of Rho GTPases.