Most of the Rho GTPases are ubiquitously expressed proteins. Our studies examined, for the first time, the expression of RhoH in normal tissues and showed that RhoH transcripts are detected only in hematopoietic tissues with the highest level of expression in the thymus. In a wide range of nonhematopoietic tissues examined, RhoH transcripts were not detected. From the representative cell lines we have examined, RhoH appears to be preferentially expressed in T and B cells. We cannot rule out the possibility that myeloid cells at later stages of differentiation, such as neutrophils, can express RhoH. However, the much lower level of RhoH mRNA in bone marrow than in the thymus and spleen suggests that RhoH is encoded by a lymphoid tissue-specific gene. Therefore, RhoH very likely has a function of particular relevance to lymphoid cells. In a Northern blot analysis, murine RNA was probed with human cDNA under high-stringency conditions and the strong signal obtained indicated a very high degree of nucleotide and amino acid similarity between humans and mice.
An extensive body of literature has addressed the involvement of Rho GTPase in the regulation of nuclear signaling, including activation of transcriptional factors involved in stress and inflammatory pathways (30
). The precise mechanism by which Rho, Rac, and CDC42 activate NF-κB has not been fully elucidated, but existing evidence suggests that these GTPases do so by inducing or enhancing the phosphorylation of IκB (46
). A nodal point at which several pathways that lead to NF-κB activation converge is the phosphorylation of IκB by the IκB kinase (IKK-α, -β, and -
) complex (4
) Phosphorylation of IκB at serine residues leads to ubiquitination of IκB and its degradation in the proteasome complex, resulting in the release of NF-κB and translocation of the p50/p65 dimers to the nuclei (5
). RhoH cannot activate NF-κB. Instead, RhoH is a potent suppressor of activation of NF-κB induced by TNF, Rac1, RhoA, and IKK in both hematopoietic and nonhematopoietic cells. Since the inhibitory action of RhoH is directed at or downstream of IKK, it is not surprising that RhoH inhibits the activation of NF-κB by both Rac1 and RhoA. Although we have not tested CDC42, it is most likely that RhoH can also inhibit CDC42-mediated activation of NF-κB. While additional work needs to be done to confirm the data by various other approaches, our results show that suppression by RhoH appears to be due not to inhibition of the phosphorylation of IκB by IKK but rather to retardation of the degradation of both total and phosphorylated IκB. There are a number of ways in which RhoH may cause this effect, including inhibition of the ubiquitination of IκB, release of ubiquitinated IκB from NF-κB dimers, and degradation of free ubiquitinated IκB by proteasomes (4
). Further work will attempt to unravel the mechanism involved. Nevertheless, our results show that RhoH acts very differently from the RhoA-Rac-CDC42 group of Rho GTPases and, together with a recent report that RhoB represses NF-κB signaling (16
), indicate that the effect of Rho GTPases on this important pathway can be either positive or negative. Since NF-κB regulates the expression of a wide range of genes, including those involved in inflammatory response and regulation of cell death, it is reasonable to construe that RhoH may be an important modulator of these responses in hematopoietic cells.
Further evidence that the inhibitory effect of RhoH on NF-κB activation is a specific function is supported by the next set of experiments examining other transcriptional pathways known to be influenced by RhoA, Rac, and CDC42, namely, the stress and inflammatory response pathways. The ERK kinases or p42/p44 proteins are activated and phosphorylated by mitogenic stimuli. In contrast to p42/p44 mitogen-activated protein kinases, JNKs and p38 are poorly activated by mitogens but strongly activated by inflammatory cytokines and cellular stress. Evidence that these stimuli exert their effects through Rho GTPases is provided by the fact that expression of activated forms of the Rho GTPases has been shown to induce activation of ERK, JNK, and p38 in various cell types (60
). The selective inhibition of the TNF-mediated activation of p38 by RhoH but not by JNK or ERK indicates that this is a specific and physiological function of RhoH. This is further supported by the ability of RhoH to suppress p38 activation by Rac1L61 and CDC42L61 equally well in hematopoietic and nonhematopoietic cells.
The mechanism by which RhoH inhibits Rac and CDC42 activation of p38 remains unresolved. Although we have tested only one exchange factor, TIAM-1, it is quite unlikely that RhoH inhibits other Rho GTPases by sequestering their exchange factors. The identity of the downstream effectors that link the Rho GTPases to activation of p38 is not fully understood. An increasing number of the serine/threonine kinases known as PAKs (3
) have also been identified as immediate downstream effectors because they bind to Rho GTPase in a GTP-dependent manner and become activated upon binding (3
). Further downstream, various kinases, such as the dual-specificity kinase SEK1/MEKK4, activate JNK and p38, and MKK3 and MKK6 specifically activate p38 (48
). Here we have shown that RhoH is also able to inhibit MKK6glu activation of p38, indicating that the inhibitory effect of RhoH is likely to be at or downstream of MKK6. Whether RhoH also inhibits other effectors, such as MKK3, remains to be systematically tested.
It was surprising that overexpression of wild-type RhoH was sufficient to induce a potent and specific effect on transcription. In most functional experiments with Rho GTPases, this effect has been best demonstrated by using dominant active or negative mutant proteins since overexpression of the wild-type form alone exerts a weaker or minimal effect. Amino acid substitution of valine for glycine at codon 12 or of leucine for glutamine at codon 61 (Rac1 numbering) has been extensively used to generate constitutively active Rho GTPases (13
), and crystal structure studies have confirmed an essential role for both residues in GTP hydrolysis (25
). Either mutation alone is sufficient to prevent intrinsic and GAP-induced GTP hydrolysis.
Both residues are replaced in wild-type RhoH, and the vitro GTPase and nucleotide dissociation assays showed that wild-type RhoH is GTPase deficient and exists only in the GTP-bound form. This result explains why the overexpression of wild-type RhoH alone is sufficient to exert the powerful inhibitory effect we had observed in various biochemical studies.
A few small GTPases containing substitutions at one or two of these positions have been found to maintain and exhibit GTPase activity in spite of the difference (37
). This implies that a certain degree of structural change within the catalytic region is allowed.
In this regard, RhoH resembles RhoE, which has also been shown to be GTPase deficient. (15
). In RhoE, amino acid substitutions exist in three highly conserved positions that are critical for normal GTPase activity and, similar to RhoH, RhoE appears to exist only in the GTP-bound state. Interestingly, it has been shown by microinjection of MDCK cells that RhoE induces complete disappearance of stress fibers, together with cell spreading (19
). It was postulated that RhoE inhibits signaling downstream of RhoA. We did not observe any dramatic effect of RhoH on the cytoskeleton or cell morphology in 3T3 and MDCK cells. This may not be universal because the effect of Rho GTPases on actin reorganization can be cell dependent and we cannot entirely rule out the possibility that RhoH has a definitive role in cytoskeletal organization in other cell types. Therefore, whether RhoH has a role in cytoskeletal changes in hematopoietic cells needs to be further studied.
Another Rho GTPase, RhoD, has been shown to cause disassembly of stress fibers and inhibition of cell motility (58
). Introduction of the constitutively active form of RhoD (G26V) into fibroblasts by microinjection or transfection resulted in disassembly of actin stress fibers and focal adhesions. Furthermore, stress fiber enhancement by RhoA or RhoA-activating lysophosphatidic acid was reversed by the transfection of RhoD cDNA. Thus, RhoD, -E, and -H constitute a category of Rho GTPases that either have no effect on actin polymerization or are antagonistic to the effect of RhoA, Rac, or CDC42 on actin.
The C-terminal residues of RhoH, CKIF, represent a typical CAAX motive present in the entire ras superfamily of small GTP-binding proteins. Depending on the identity of the carboxyl-terminal amino acid (X), proteins will be geranylated if X is leucine or phenylalanine. This posttranslational modification is crucial for the localization of both Ras and Rho proteins to the plasma membrane and for their biological activities. Many of the Rho proteins identified thus far end with leucine or phenylalanine and are geranylated (1
). Therefore, RhoH is most likely geranylated and, like other Rho GTPases, would have a certain fraction of the protein associating with membrane fractions during biological activities. However, confocal images of immunostained RhoH-transfected cells showed that the protein is diffusely distributed in the cytoplasm. We could not detect any definitive plasma membrane staining. We cannot, however, rule out the possibility that, inside the cell, RhoH is localized to certain membrane compartments. Furthermore, it may be informative to formally demonstrate if RhoH is geranylated.
Analogous to other GTP-binding proteins, the GTP-bound state of RhoH is most likely the active state. The cycling of Rho GTPases between the GDP-bound and GTP-bound states is controlled by three regulatory factors. GDP exchange factors (GEFs) catalyze the release of GDP and replacement with cytosolic GTP (9
). Since RhoH exists only in GTP-bound form, GEFs are not likely to be relevant to its function. The down regulation of active Rho GTPases is achieved mainly by specific GAPs that strongly enhance their intrinsic GTPase activities. RhoH is not responsive to Rho GAP p50, which is a GAP for Rho, Rac, and CDC42. About 20 GAPs for Rho GTPases have been identified to date (8
). The human genome data have shown that chromosome 2 alone potentially encodes eight GAPs. It is possible that, in some physiological context, RhoH is stimulated to hydrolyze GTP by some GAP. While further tests with other GAPs or with cellular lysates would be useful in resolving this issue, we would argue that since RhoH shows little or no intrinsic GTPase activity, it is likely that RhoH is not regulated by some specific cellular Rho GAP.
A third regulatory protein that is involved in GTPase activity is the GDI (2
). The exact in vivo function of Rho GDIs remains unclear. In vitro assays have shown that GDIs inhibit GDP dissociation and compete with GEF for activation of Rho GTPases (2
). They also bind to Rho GTPases strongly enough that that they are capable of displacing them from membranes (33
). In one of the proposed models for the regulation of GTPases, the GDIs extract the GDP-bound form of GTPases from the membrane after inactivation by hydrolysis and return the GTPases to the cytosolic compartment in a GDI-Rho GTPase complex. Another proposal is that Rho GDIs act as shuttles carrying and targeting Rho GTPases to their site of activity. Finally, Rho GDI-α or Rho GDI-1 has also been shown to be capable of inhibiting GAP activity (22
). Of the various functions attributed to the GDIs, the shuttling of GTPases to sites of activity may be relevant to a naturally GTPase-deficient protein like RhoH. Our results showed that after cotransfection of GDIs with RhoH, immunoprecipitation of the GDIs consistently coprecipitated RhoH, indicating that, in vivo, RhoH interacts avidly with the GDIs. Thus, it is possible that the GDIs play a role in the regulation of the cellular function of RhoH.
Most significantly, the selective inhibitory function of RhoH raises the question of how such a protein may compete with other GTPases in the same cell. The fact that RhoH is GTPase deficient and exists only in the GTP-bound form implies that the regulation of the protein must follow a mechanism other than nucleotide cycling. Possible mechanisms for regulating RhoH activity include phosphorylation states, mRNA expression levels, and protein level changes by various proteolysis mechanisms.
We have focused on searching for evidences that expression of RhoH is regulated. Our finding that the RhoH transcript is rapidly down regulated after treatment with a physiological dose of PMA provides clear evidence that RhoH expression is transcriptionally responsive to cell stimulation. The rapid down regulation of the RhoH transcript within 60 min also suggest that active degradation of RhoH mRNA is involved. Further experiments comparing this with the natural half-life of RhoH mRNA should resolve this issue.
A clue to what may be the physiological conditions under which differential expression of RhoH occurs was obtained from our analysis of Th1/Th2 cell differentiation.
Li et al. (34
) have shown that Rac2 is preferentially expressed in the Th1 subset of T cells. The authors also showed that Rac2 is required for the production and release of IFN-γ and that dominant negative Rac1N17 almost completely suppresses cytokine release by activated Th1 cells. Significantly, it was shown that this function of Rac2 depends on its ability to activate NF-κB and p38 and that the activation of each alone is not sufficient to induce IFN-γ release. Given our observation that RhoH appears to be expressed only in lymphoid cells and that the biochemical effect of RhoH is the opposite of that of Rac2, we hypothesized that RhoH may be differentially expressed in Th1 and Th2 cells.
The expression of RhoH is clearly differentially regulated between Th1 and Th2 cells. The significantly higher level of RhoH expression in Th1 cells suggests that RhoH has a role in the regulation of the difference in function between Th1 and Th2 cells.
Since RhoH exerts an inhibitory effect on transcription factors in T cells that are activated by Rac2, we postulate that RhoH functionally competes with Rho GTPases such as Rac2 and that, together, the two GTPases modulate T-cell functions such as the secretion of cytokines. Furthermore, the lowering of RhoH mRNA in Th1 cells after restimulation with anti-CD3 indicates that in certain cells, signaling through the T-cell receptor is linked to transcriptional alteration of RhoH.
Here we have obtained the first evidence of how important the transcriptional response of RhoH may be by showing that a reduction of endogenous RhoH in lymphocytes by αs-RhoH overexpression resulted in dose-dependent enhancement of a Rac-induced inflammatory response.
It is possible that a signal that activates Rac proteins is linked to a signal that down regulates RhoH expression in order to remove the inhibitory effect of RhoH. However, the kinetics of activation of Rac is faster than the transcriptional down regulation of RhoH. Therefore, the reduction of RhoH inhibition of Rac would not be synchronous with the activation of Rac, implying that RhoH targets are not immediate Rac effectors but, rather, are further downstream in pathways affected by Rac activation. Whether there is an additional mechanism for the rapid degradation of RhoH protein is an interesting possibility that remains to be tested.
One possibility is that, rather than being linked directly to the rapid on-off activation of other Rho GTPases, RhoH functions as a “thermostat” that sets the levels of response to activation of other Rho GTPases. Another potential hypothesis recognizes the fact that Th1 CD4+ T cells produce large amounts of inflammatory cytokines, including TNF-α, LTα (TNF-β), and IFN-γ. As we have shown, increased expression of RhoH in these cells would reduce the activation of signaling pathways downstream of the TNFs and thus render these cells resistant to the toxic effects of the cytokines they produce.
The possibility that regulation of RhoH does not occur by nucleotide cycling also raises the relevance of binding to GTP. In the context of RhoE, another GTPase-deficient Rho GTPase, it has been suggested that in these noncycling GTP-binding proteins, GTP plays only a structural role, such as coordination of the correct conformation of the protein (15
). Whatever the answer, the properties of RhoH serve to further support the notion that nucleotide exchange is not absolutely necessary for the activation of all GTP-binding proteins.
Finally, RhoH and at least three other known Rho GTPases, RhoB (16
), RhoD, and RhoE, constitute a category of Rho GTPases having functional effects opposite or antagonistic to those of other Rho GTPases, such as RhoA, Rac, and CDC42. This suggests that another level of regulation of Rho GTPase activity that has to be considered is one in which Rho GTPases with opposing modes of function compete or work together to modulate the final outcome of particular Rho GTPase-activated pathways (Fig. ). Further work to elucidate the mechanism of RhoH function and additional evidence from other Rho GTPases would help to resolve the validity of such a model involving competing Rho GTPases. Recent work showing the competition between RhoA and RhoE in cell transformation supports such a model of disease development (21
). In this regard, it is pertinent to note that mutations of RhoH have recently been found in as many as 46% of diffuse large-cell lymphomas (45
). Most interestingly, all of the mutations are in the first intron of the gene and do not involve the protein coding exon. Whether this translates into abnormal alterations of RhoH expression is currently under investigation.
Model of competing Rho GTPases. Two groups of Rho GTPases that have opposite functions are shown as having a positive or negative effect. The model suggests that some cellular end responses may be modulated by competition between these Rho GTPases.