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University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Departments of Pharmacology [P. A. S., C. J. D.] and Microbiology and Immunology [W. H., L. S.], Chapel Hill, North Carolina 27599, and Department of Pharmacology, University of Wisconsin Medical School and Comprehensive Cancer Center, Madison, Wisconsin 53706 [P. J. K.]
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and MethodsReferences |
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Rho family GTPases are mediators of diverse cellular functions:
(a) They are regulators of actin cytoskeletal organization. Whereas Cdc42 promotes filopodia formation, Rac1 promotes formation of lamellipodia or membrane ruffles, and RhoA triggers actin stress fiber and focal adhesion formation (reviewed in Refs. 1 , 2 ). In contrast, RhoE causes a disruption of stress fibers and cell rounding (6 , 7) . Consequently, Rho GTPase function can influence cell-cell and cell-substratum interactions, which in turn can modulate cell movement. For example, we showed that Rac1 and Cdc42 activation promoted the motility and invasion in vitro of T47D human breast epithelial cells (8) . Activated Rac1 promoted T-cell invasion in vitro (9) . Overexpression of RhoC has been shown to promote the metastatic properties of melanoma cells in vivo (10) . Thus, inhibitors of Rho GTPases have been considered as novel anticancer drugs (11) .
(b) Rho GTPases are regulators of gene expression and stimulate the activity of various transcription factors, including NF-
B3
(12
, 13)
, SRF (14)
, and c-Jun (15
, 16)
.
(c) Rho GTPases can regulate cell cycle progression and cellular proliferation. For example, constitutive activation of RhoA, Rac1, and Cdc42 has been shown to promote tumorigenic transformation of rodent fibroblasts, and the function of these GTPases is required for the transforming activity of Ras and other oncoproteins (reviewed in Ref. 2 ). Finally, among a diverse spectrum of other functions, we determined recently that RhoA activation antagonizes HIV-1 viral replication and gene expression (17 , 18) . In light of the diverse signaling and biological activities of RhoA, it is not surprising that a large spectrum of functionally diverse proteins have been identified as downstream effector targets of RhoA (1 , 2 , 19) .
In addition to GDP/GTP cycling, Rho GTPase function is also believed to be critically dependent on posttranslational modification by isoprenoid lipids. Similar to Ras, Rho GTPases also terminate in a COOH-terminal CAAX tetrapeptide sequence motif (where C is cysteine, followed by two aliphatic amino acids, ending in a variant amino acid; reviewed in Refs. 20 , 21 ). This sequence signals for covalent attachment of an isoprenoid lipid group to the cysteine of the CAAX sequence, followed by endoprotease removal of the -AAX residues and carboxymethylation of the now terminal prenylated cysteine. These modifications promote the association of Ras and Rho GTPases with plasma and intracellular membranes.
The CAAX motif of Ras and Rho GTPases signal for covalent modification by either of two types of isoprenoid lipids (reviewed in Refs. 20 , 21 ). The FTase enzyme catalyzes the addition of a C15 farnesyl group to a subset of CAAX sequences, when X is serine, methionine, cysteine, alanine, or glutamine. For CAAX sequences where X is leucine or isoleucine, the GGTaseI enzyme catalyzes addition of the more hydrophobic C20 geranylgeranyl moiety. All isoforms of Ras are modified by farnesylation, whereas the closely related R-Ras and TC21/R-Ras2 proteins are modified by geranylgeranylation. A majority of Rho GTPases is modified by the geranylgeranyl group (e.g., RhoA, Rac, and Cdc42), whereas some are modified by a farnesyl group (e.g., RhoE/Rnd3; Ref. 22 ), and others can be modified by both isoprenoids (e.g., RhoB; Refs. 23 , 24 ).
Prenylation may critically influence subcellular location of a protein as well as regulate its ability to interact with other proteins. The contribution of CAAX-signaled processing to protein function has been best studied with Ras proteins (25, 26, 27) . Mutation of the CAAX motif of Ras proteins prevents prenylation and abolishes plasma membrane association, causing a complete loss of oncogenic Ras transforming activity. This information has led to the development of FTIs to block Ras processing and function, and hence, are being developed as anticancer drugs to treat ras mutation-positive human cancers. To address the importance of specific isoprenoid modification for Ras function, CAAX mutants of Ras that undergo modification by geranylgeranylation were generated. Although the subcellular location of these variants was altered, with localization to intramembrane compartments, geranylgeranylated versions of oncogenic Ras retained potent transforming activity (28 , 29) . Therefore, it appears that oncogenic Ras function can be facilitated by modification with either isoprenoid group.
The importance of protein prenylation for Rho GTPase function has been best evaluated with RhoB. Activated RhoB can promote growth transformation, and a nonprenylated version of activated RhoB showed a loss of transforming activity (30 , 31) . However, it retained the ability to stimulate SRF activation. Thus, some but not all RhoB function is dependent on prenylation. However, in contrast to the observations with Ras, RhoB function appears to be critically dependent on specific isoprenoid function. RhoB appears to be modified primarily by farnesylation in vivo. However, FTI treatment caused an accumulation of geranylgeranylated RhoB (32) . Prendergast and colleagues (33 , 34) showed that geranylgeranylated RhoB inhibited the growth of rodent and human cells. Thus, whereas farnesylated RhoB is a growth-promoting protein, geranylgeranylated RhoB is an apoptosis-inducing protein. Consistent with these opposing functions, farnesylated RhoB function was shown to be required for Ras transformation (30) , whereas geranylgeranylated RhoB blocked Ras transforming activity (33) . Consequently, the FTI-induced formation of inhibitory geranylgeranylated RhoB has been proposed as the basis for the antitumor activity of FTIs.
RhoA and RhoB share significant sequence identity (>85%). For example, they share complete identity in core effector domain sequences (residues 32–40) required for interaction with downstream effectors. RhoA and RhoB exhibit greatest sequence divergence in residues immediately NH2-terminal to the CAAX motif. Because these sequences contain elements that influence the targeting of Ras and Rho GTPases to specific membrane compartments, it is not surprising that RhoA and RhoB exhibit different subcellular locations. One study found that only a minor fraction of RhoA was associated with the plasma membrane, with the majority of the protein located in the cytosol (35) . In contrast, RhoB was associated with early endosomes and a prelysosomal compartment. However, a recent analysis of GFP-tagged proteins in live cells determined that RhoB was located predominantly at the plasma membrane, and it was suggested that the earlier localization of RhoB to endosomes may be an artifact of fixation (36) . In contrast, RhoA showed a predominantly cytosolic location.
RhoA is modified exclusively by geranylgeranylation. Whether all RhoA function is critically dependent on prenylation and whether modification by farnesylation can support RhoA function has not been determined. Additionally, pharmacological inhibitors of GGTaseI, the enzyme that modifies RhoA and other Rho GTPases, have been shown to possess antitumor activity, possibly by blocking the function of RhoA (37, 38, 39, 40) . RhoA function is mediated by interaction with a multitude of downstream effectors, and the precise location of RhoA may critically influence proper interaction with effectors (1 , 2 , 19) . In the present study, we determined the requirement for modification by geranylgeranylation for RhoA function, whether modification by farnesylation could support the diverse biological functions RhoA, and whether RhoA is the critical target for GGTIs. We determined that RhoA function was dependent on prenylation, but that a farnesylated form of RhoA was unchanged in function and subcellular location. Thus, our observations contrast with those of RhoB and show that RhoA function is not dependent on modification by a specific isoprenoid. Finally, cells expressing farnesylated RhoA were still sensitive to growth inhibition by GGTI treatment, indicating that RhoA is not the only critical target for GGTIs.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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(a) The WT CAAX sequence (CLVL) was mutated to the CAAX sequence of the farnesylated H-Ras protein [CVLS; designated RhoA(63L)-CVLS]. This CAAX motif has been determined to be specific to signal for farnesylation as opposed to geranylgeranylation (21 , 41) .
(b) We also generated a nonprenylated version by mutating the isoprenoid-accepting cysteine into a serine [SLVL; designated RhoA(63L)-SLVL]. We verified that the two mutant proteins were stable and could be expressed at levels comparable with that of the WT protein. Transiently or stably transfected NIH 3T3 cells showed that RhoA(63L) and RhoA(63L)-CVLS were expressed at comparable levels (Fig. 1A)
. Interestingly, in both transiently and stably transfected cells, we observed that the RhoA(63L)-SLVL was expressed at higher levels than RhoA(63L).
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. Thus, modification by a different isoprenoid did not cause a detectable change in subcellular location.
Because previous studies of RhoB localization found differences when evaluated in live cells when compared with fixed cells (36
, 42)
, we also evaluated the location of HA epitope-tagged versions of these two proteins in fixed cells (Fig. 1C)
. Essentially similar results were seen, where both WT and CVLS versions of RhoA(63L) showed punctate, perinuclear staining patterns. In contrast, the nonprenylated RhoA(63L)-SLVL mutant showed a diffuse cytoplasmic localization. We also evaluated subcellular distribution by high-speed fractionation into cytosolic S100 soluble and membrane-containing P100 particulate fractions. Both RhoA(63L)-WT and RhoA(63L)-CVLS were found predominantly in the P100 fraction, although RhoA(63L)-CVLS showed a reproducibly greater percentage of protein in the S100 fraction (data not shown). Thus, in contrast to what has been described for RhoB, mutation of the CAAX motif to alter the specific isoprenoid modification did not cause a significant change in subcellular location.
We then determined whether the distribution of the CVLS mutant was sensitive to inhibition by a FTI. As expected, treatment with the FTI-2153 inhibitor caused a redistribution of RhoA(63L)-CVLS to a diffuse cytoplasmic and nuclear location, whereas the distribution of RhoA(63L)-WT was unchanged (Fig. 1B)
. Conversely, treatment with the GGTI-2166 GGTaseI inhibitor resulted in a diffuse distribution of RhoA(63L)-WT but did not alter the perinuclear distribution of RhoA(63L)-CVLS. Similar results were also seen with the HA epitope-tagged proteins, where RhoA(63L)-CVLS, but not RhoA(63L)-WT, was sensitive to FTI treatment, as measured by a change in mobility in SDS-PAGE (data not shown). We conclude that RhoA(63L)-CVLS is modified by farnesylation, but this alternative prenylation did not result in the significant change in subcellular location that has been described for the different prenylated forms of RhoB (32)
.
RhoA-mediated Transcriptional Activation of SRF, NF-B, and the Cyclin D1 Promoter Is Dependent on Prenylation.
Previously, it has been shown that RhoA can activate several transcription factors, such as SRF (14)
and NF-B (12)
, and can stimulate transcription from the cyclin D1 promoter (43)
. Therefore, to determine whether RhoA requires the modification by a specific prenyl group for activation of these factors, transient expression analyses were done using reporter plasmids, where expression of the luciferase gene is regulated by minimal promoters that contain SRF- or NF-B-responsive elements or by the human cyclin D1 promoter. We found that RhoA(63L)-WT and RhoA(63L)-CVLS expression caused comparable activation of SRF (
10-fold), NF-B (11-fold), and cyclin D1 (3-fold; Fig. 2
). In contrast, the RhoA(63L)-SLVL mutant failed to cause significant stimulation in any assay. Thus, although prenylation is essential for the activation of these pathways, farnesylated and geranylgeranylated RhoA activate similar signaling pathways.
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; Ref. 47
). The farnesylated RhoA(63L)-CVLS mutant showed a comparable level of focus-forming activity, whereas the unprocessed RhoA(63L)-SLVL mutant showed greatly impaired focus-forming activity.
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and retained a refractile morphology and formed multilayered cultures (Fig. 4B)
. In contrast, both the empty vector- and RhoA(63L)-SLVL-transfected cells had become quiescent and formed a monolayer of nonrefractile cells. These results demonstrate that prenylation is crucial for the growth-promoting property of RhoA(63L), but either isoprenoid modification could support this activity.
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. This enhancement was not seen with cells expressing the unprenylated SLVL mutant. Therefore, similar to our observations with the NIH 3T3 transformation assays, activated RhoA(63L)-stimulated migration of T47D cells was also dependent on prenylation but could be facilitated by either farnesylation or geranylgeranylation.
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(a) We observed previously that the RhoA(63L) mutant inhibited HIV-1 replication, as measured by the production of infectious virus and of virion-associated reverse transcriptase activity (80 and 50% reduction, respectively). A similar level of inhibition was seen with the RhoA(63L)-CVLS mutant in both assays (80 and 60% reduction), whereas RhoA(63L)-SLVL was impaired significantly (30% reduction) in this ability (Fig. 6A)
.
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. This suggests the importance of RhoA prenylation, but not the specific isoprenoid modification, in mediating inhibition of HIV-1 function.
Farnesylated RhoA Does Not Protect Cells from Growth Inhibition Caused by Inhibition of GGTaseI.
Pharmacological inhibition of GGTaseI has been shown to inhibit normal and tumor cell proliferation, in part by blocking progression through G1 of the cell cycle (50)
. Rho GTPases are substrates for GGTaseI and have been shown to be required for proliferation through G1 (51
, 52)
. Hence, geranylgeranylated Rho GTPases constitute logical targets for the inhibitory action of GGTIs. One approach for evaluating this possibility is to determine whether variants of Rho GTPases that are insensitive to GGTIs can protect cells from GGTI growth inhibition. In this study, we have shown that farnesylation can support the diverse signaling and biological functions of RhoA. Thus, this farnesylated variant of RhoA can serve as a useful reagent to evaluate whether RhoA is a critical target of GGTIs.
To determine whether RhoA is an important target for the inhibitory activity of GGTIs, we compared the sensitivity of NIH 3T3 cells stably expressing RhoA(63L)-WT and RhoA(63L)-CVLS. Similar to what has been described previously, treatment of cells expressing RhoA(63L)-WT with GGTI-2166 caused a dose-dependent inhibition of proliferation. RhoA(63L)-WT expressing cells cultured in growth medium supplemented with 3 µM GGTI-2166 showed an 30% reduction in cell number and an 80% reduction at 10 µM GGTI-2166 (Fig. 7)
. Essentially identical levels of growth inhibition was seen with RhoA(63L)-CVLS-expressing cells. Thus, the growth-inhibitory activity caused by GGTI treatment is not attributable solely, if at all, to the loss of RhoA function.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Our analyses of the role of isoprenoid modification on RhoA function extend on previous studies that address whether specific isoprenoid modification is critical for protein function. Approximately 0.5% of mammalian proteins undergo posttranslational modification by isoprenoid lipids, and of the two types of prenylation, geranylgeranylation is the predominant modification (21
, 25)
. Interestingly, proteins that are both structurally and functionally related can be modified by different isoprenoid moieties. For example, the three Ras isoforms (H-Ras, K-Ras, and N-Ras) are all modified by farnesylation, whereas the closely related R-Ras1 and R-Ras2/TC21 proteins are modified by geranylgeranylation. However, despite differences in isoprenoid modification, constitutively activated mutants of Ras and R-Ras isoforms all cause growth transformation. Similarly, whereas RhoA is modified by geranylgeranylation, the related RhoE protein is modified by farnesylation (22)
. In this case, though, these proteins have opposing functions, with RhoA promoting actin stress fiber formation and RhoE causing a disruption of stress fibers. Whether the different prenylated nature of these two proteins accounts for their opposing functions has not been determined. The 
subunits of heterotrimeric G proteins are also modified by prenylation. Among the 11 known subunits, 1, 8, and 11 are farnesylated, whereas the remaining subunits are modified by geranylgeranylation (53)
. When analyzed, no striking isoprenoid-specific differences in subunit function has been found. Thus, to date, studies evaluating a role for specific isoprenoid modification in protein function have determined surprisingly subtle or no differences in isoprenoid regulation of protein function (29
, 54
, 55)
.
Our observations that RhoA transforming activity could be mediated by geranylgeranylation as well as farnesylation contrasts sharply with the observations made with RhoB. Whereas farnesylated RhoB was found to exhibit a growth-promoting activity that was required for Ras transformation, geranylgeranylated RhoB instead showed a proapoptotic function that antagonized Ras transformation (33 , 34) . These strikingly different consequences of different isoprenoid modifications on RhoA and RhoB function are unexpected in light of their strong structural (>85% amino acid sequence identity) and functional (e.g., promotion of actin stress fibers) similarities. However, RhoA and RhoB diverge significantly in their COOH-terminal sequences and consequently do localize to very distinct regions in the cell. This may account for the different consequences of isoprenoid modification on RhoA and RhoB biological activity.
Our observations with RhoA transformation are similar to previous observations that geranylgeranylated versions of oncogenic H-Ras proteins also retained the ability to cause transformation (29) . However, we also found that a geranylgeranylated mutant of WT H-Ras was growth inhibitory. This result suggests that specific isoprenoid modification is important for the normal function of H-Ras. Whether normal RhoA function is also dependent of specific isoprenoid modification remains to be determined. Finally, it will be important to determine whether the function of other Rho GTPases will be dependent (i.e., RhoB) or independent (i.e., RhoA) of specific isoprenoid modification.
RhoA function is mediated by interaction with a large number of functionally diverse effectors. The precise effector(s) important for mediating the ability of RhoA to cause growth transformation of NIH 3T3 cells, motility of T47D cells, and to block HIV-1 function remain to be determined. However, our analyses of a panel of effector domain mutants of activated RhoA(63L),5 which are impaired differentially in effector interactions, indicate that these three RhoA functions are initiated through the use of different effectors. Our observation that nonprenylated RhoA(63L) was deficient in all three activities indicates that membrane association is essential for RhoA interaction with the effectors involved in these processes. Finally, we found that RhoA activation of SRF was also dependent on prenylation. This result contrasts with the observation that nonprenylated RhoB can still activate SRF and further suggests that the distinct subcellular locations of RhoA and RhoB must greatly influence their functions (31) .
During the course of our studies, Allal et al. (56) published a study showing that the function of activated RhoA was also independent of the type of isoprenoid modification. They determined that farnesylated RhoA(14V) was unchanged in subcellular location, interaction with RhoGDI, actin reorganization, growth transformation of NIH 3T3 cells, suppression of p21CIP1 gene expression, and stimulation of SRF. Thus, our observations are in agreement with and extend their observations to other biological functions of RhoA. We showed additionally that farnesylation can support two other diverse RhoA-mediated functions, induction of breast tumor cell migration in vitro and RhoA inhibition of HIV-1 function. Hence, it appears that the majority of RhoA effector function can be supported by either isoprenoid modification. Because a goal of our studies was to use farnesylated RhoA to evaluate the mechanism of GGTI-mediated growth inhibition, a complete evaluation of whether diverse functions of RhoA can be supported by alternative prenylation is critical. Additionally, because there are currently conflicting observations regarding whether RhoB function is dependent on a specific isoprenoid (33 , 57) , independent studies that show that RhoA function can be supported by modification by either isoprenoid is important.
Pharmacological inhibitors of GGTaseI have been developed as potential anticancer drugs (11) and have shown potent antitumor activity in cell culture and animal preclinical studies. The mechanism of growth inhibition of GGTIs has been ascribed, in part, to inhibition of progression through G1 of the cell cycle (50) . Because RhoA, a GGTaseI substrate, has been shown to be critical for G1 progression (51 , 52) , it represents a possible target for GGTI-induced growth inhibition. In the present study, we established that farnesylated RhoA retained all signaling and biological functions of the authentic geranylgeranylated RhoA protein. This provided important validation that farnesylated RhoA will be a useful RhoA variant to determine whether RhoA is indeed the critical target for GGTIs. However, we found that NIH 3T3 cells stably expressing farnesylated RhoA showed the same sensitivity to GGTI-induced growth inhibition. Although this result does not eliminate RhoA as a critical target, it does indicate that other GGTaseI substrates must also be involved in GGTI growth inhibition. Logical candidates include two other Rho GTPases, Rac1 and Cdc42, that are substrates for GGTaseI and have also been shown to be required for G1 progression. Because loss of function of RhoA, Rac1, or Cdc42 alone was sufficient to block G1 progression, perhaps GGTI-mediated loss of function of all three Rho GTPases is responsible for GGTI growth inhibition. To evaluate this possibility, it must first be determined whether farnesylated versions of Rac1 and Cdc42 also retain the same functions as their authentic geranylgeranylated counterparts. If so, then it will be important to determine whether coexpression of farnesylated RhoA, Rac1, and Cdc42 can reduce the sensitivity of cells to GGTI-induced growth inhibition. If not, then other GGTaseI substrates must be targeted by GGTIs to cause growth inhibition. The identity of these geranylgeranylated targets will be important for the further development of GGTIs as anticancer drugs.
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Materials and Methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Cell Culture, Transfection, and Transformation Assays.
NIH 3T3 mouse fibroblasts were maintained in DMEM supplemented with 10% calf serum (Hyclone). Human embryonic kidney 293T cells and the HeLa-MAGI human cervical carcinoma cells were maintained in DMEM supplemented with 10% FCS (Life Technologies, Inc.). T47D human breast epithelial cells were grown in RPMI supplemented with 10% FCS and 0.2 units/ml insulin. For cooperation focus formation assays with Raf(340D), NIH 3T3 cells were transfected by the calcium phosphate precipitation along with a glycerol shock as described previously (47)
. For these transfections, 1 µg of the pZIP-rhoA and 1 µg of pZIP-raf(340D) plasmid DNA were cotransfected into NIH 3T3 cells. Twenty-one days after transfection, the dishes were stained with crystal violet, and the appearance of foci of transformed cells was quantitated by visual inspection.
To establish cell lines stably expressing WT or mutant RhoA(63L) protein, NIH 3T3 were transfected by calcium phosphate precipitation as above with 500 ng of the pZIP-NeoSV(x)1 retrovirus expression vector constructs. Three days after transfection, the transfected cultures were subcultured at a 1:5 split ratio into growth medium supplemented with 400 µg/ml of G418. Mass populations of G418-resistant colonies were pooled together to establish cell lines that were then used for GGTI sensitivity experiments, growth transformation assays, and immunofluorescence analysis for determination of protein location. Western blot analyses with anti-HA antibody (Covance) were done to verify that there was comparable expression of each RhoA(63L) protein in the established cell lines.
To evaluate the ability of each cell line to proliferate in growth medium supplemented with low serum, three dishes were plated with 103 cells/60-mm dish and were allowed to attach in growth medium. After 16 h, the growth medium was replaced with DMEM supplemented with 0.5, 2, or 10% calf serum. The dishes were cultured for 21 days, and the appearance of colonies of proliferating cells was visualized by staining with 0.4% crystal violet.
To evaluate the ability of each cell line to proliferate in growth medium supplemented with GGTI-2166 GGTI (FTI-277, FTI-2153, GGTI-298, and GGTI-2166 provided by Said Sebti and Andrew Hamilton; Ref. 59 ), NIH 3T3 cells stably expressing the WT and CVLS versions of RhoA(63L) proteins were plated at 103 cells/35-mm dishes in growth medium. After 18 h, normal growth medium was replaced with growth medium supplemented with either vehicle (DMSO), 3, or 10 µM GGTI-2166. Every 24 h, the medium was replaced with fresh vehicle/GGTI-containing medium. On days 3, 5, 6, 8, 10, and 12, the cells from two dishes were trypsinized and were counted on a hemacytometer for each condition.
Transient Expression Reporter Gene Assays.
For the transcriptional activation assay, 293T cells were transfected by calcium phosphate coprecipitation as described above except that the glycerol shock was excluded. Five hundred ng of the pcDNA-rhoA plasmid DNA or pcDNA3 empty vector were cotransfected together with 2 µg of the luciferase gene reporter plasmids, where expression is controlled by a minimal promoter from the c-fos gene that contains multiple SRF (43)
or NF-B (60)
responsive DNA elements. The cyclin D1-Luc plasmid contains the luciferase gene under the control of the human cyclin D1 promoter (61)
. Twenty-four h after transfection, the growth medium was replaced with DMEM supplemented with 0.5% FCS. After 16–20 h, the cells were lysed and then analyzed for luciferase activity using enhanced chemiluminescence and a Monolight 2010 luminometer (Analytical Luminescence).
Cell Migration Assays.
Migration assays were performed as described previously (48)
. T47D cells were stably transfected with pZIP-NeoSV(x)1 plasmid DNA encoding the parental RhoA(63L) protein and the two CAAX mutants and selected in growth medium supplemented with 300 µg/ml G418. For the migration assay, transwell dishes (12-well cluster, 12 mm diameter, 12 µm pore size; Costar) were coated from the underside with 3 µg/ml collagen I in water (Collaborative Biomedical Products), and serum-free RPMI was placed in the lower chamber. Motility was assessed by adding 104 cells in the upper chamber of the transwell in RPMI supplemented with 5 mg/ml BSA. The transwells were incubated in a humidified CO2 incubator (10%) at 37°C for 16 h. The nonmotile cells were then removed from the upper chamber, and the transwell filter was fixed and stained with Wright-Giemsa stain (Diffquick; Baxter) and then removed and mounted on a microscope slide with Cytoseal (Baxter) solution and a coverslip. The number of remaining cells, which represent the motile cells, were counted on a Nikon microscope.
Localization Analyses.
The subcellular locations of CAAX mutants of RhoA were determined in both live and fixed cells. For live cell analyses, NIH 3T3 cells plated on coverslips in 6-well dishes were transiently transfected (Lipofectamine-Plus; Life Technologies, Inc.) with expression vectors encoding GFP-tagged RhoA(63L) proteins. Twenty-four h after transfection, the coverslips were washed twice with PBS and inverted onto microscope slides. Live cells were examined with a Axioskop 2 fluorescence microscope (Zeiss) and Openlab digital imaging software (Improvision). To evaluate the sensitivity of prenylation-dependent subcellular localization, cells expressing RhoA(63L)-WT and RhoA(63L)-CVLS proteins were treated with FTase and GGTase inhibitors. For these analyses, cells transiently expressing the GFP-tagged proteins were incubated for 24 h with growth medium supplemented with either vehicle (DMSO), 10 µM FTI-2153, or 10 µM GGTI-2166.
For our second approach to evaluate subcellular location of the RhoA mutant proteins, NIH 3T3 cells stably expressing HA epitope-tagged WT and mutated RhoA(63L) proteins were plated onto coverslips and incubated overnight in growth medium. The cells were then fixed in 3.7% formaldehyde in PBS for 10 min and incubated for 30 min in PBS supplemented with 0.1% Triton X-100 and 5 mg/ml BSA. The HA epitope-tagged RhoA(63L) proteins were visualized by indirect immunofluorescence using an anti-HA antibody, followed by a FITC-conjugated goat antimouse secondary antibody (Jackson Immunoresearch Laboratories).
Analysis of HIV-1 Replication and Gene Expression.
Inhibition of HIV-1 replication was assessed by cotransfection of the HIV-1 proviral genome pNL4-3 with each pcDNA-rhoA plasmid DNA into 293T cells using Effectene (Qiagen) as reported previously (18)
. Infectious units and virion-associated reverse transcriptase activity in culture supernatant were determined by titering on HeLa-MAGI cells at 48 h after transfection as described previously (62)
. A pAX142-lacZ reporter plasmid, where the lacZ gene is expressed constitutively from the EF1
promoter, was also cotransfected, and ß-galactosidase activity was measured to confirm similar transfection efficiency. Inhibition of the HIV-1 gene expression in human T cells (Jurkat) was analyzed by cotransfecting (Superfect; Qiagen) pcDNA-rhoA plasmid DNA together with pNL4-Luc (49)
, where the luciferase gene was under control of the HIV-1 long terminal repeat promoter. Forty-eight h after transfection, cells were lysed, and luciferase activity was determined as described (18)
.
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Acknowledgments |
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Footnotes |
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1 Supported by NIH Grants CA42978, CA55008, and CA63071 (to C. J. D.) and AI48407 (to L. S.). 
2 To whom requests for reprints should be addressed, at University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, CB# 7295, Chapel Hill, NC 27599-7295. Phone: (919) 966-5634; Fax: (919) 966-0162; E-mail: cjder{at}med.unc.edu
3 The abbreviations used are: NF-B, nuclear factor-B; SRF, serum response factor; FTase, farnesyltransferase; GGTaseI, geranylgeranyltransferase I; FTI, farnesyltransferase inhibitor; GGTI, geranylgeranyltransferase I inhibitor; HA, hemagglutinin; GFP, green fluorescent protein; SRF, serum response factor; WT, wild type.
4 P. J. Keely, manuscript in preparation.
5 P. A. Solski and C. J. Der, manuscript in preparation.
Received for publication 4/ 5/02. Revision received 6/20/02. Accepted for publication 6/24/02.
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References |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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 A. C. Berzat, J. E. Buss, E. J. Chenette, C. A. Weinbaum, A. Shutes, C. J. Der, A. Minden, and A. D. Cox Transforming Activity of the Rho Family GTPase, Wrch-1, a Wnt-regulated Cdc42 Homolog, Is Dependent on a Novel Carboxyl-terminal Palmitoylation Motif J. Biol. Chem., September 23, 2005; 280(38): 33055 - 33065. [Abstract] [Full Text] [PDF]
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E. J. Chenette, A. Abo, and C. J. Der Critical and Distinct Roles of Amino- and Carboxyl-terminal Sequences in Regulation of the Biological Activity of the Chp Atypical Rho GTPase J. Biol. Chem., April 8, 2005; 280(14): 13784 - 13792. [Abstract] [Full Text] [PDF]
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P. A. Solski, R. S. Wilder, K. L. Rossman, J. Sondek, A. D. Cox, S. L. Campbell, and C. J. Der Requirement For C-terminal Sequences in Regulation of Ect2 Guanine Nucleotide Exchange Specificity and Transformation J. Biol. Chem., June 11, 2004; 279(24): 25226 - 25233. [Abstract] [Full Text] [PDF]
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N. Y. Mitin, M. B. Ramocki, A. J. Zullo, C. J. Der, S. F. Konieczny, and E. J. Taparowsky Identification and Characterization of Rain, a Novel Ras-interacting Protein with a Unique Subcellular Localization J. Biol. Chem., May 21, 2004; 279(21): 22353 - 22361. [Abstract] [Full Text] [PDF]
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 K. Wennerberg and C. J. Der Rho-family GTPases: it's not only Rac and Rho (and I like it) J. Cell Sci., March 15, 2004; 117(8): 1301 - 1312. [Abstract] [Full Text] [PDF]
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 P. L. Joyce and A. D. Cox Rac1 and Rac3 Are Targets for Geranylgeranyltransferase I Inhibitor-Mediated Inhibition of Signaling, Transformation, and Membrane Ruffling Cancer Res., November 15, 2003; 63(22): 7959 - 7967. [Abstract] [Full Text] [PDF]
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J. J. Fiordalisi, R. L. Johnson II, C. A. Weinbaum, K. Sakabe, Z. Chen, P. J. Casey, and A. D. Cox High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors J. Biol. Chem., October 24, 2003; 278(43): 41718 - 41727. [Abstract] [Full Text] [PDF]
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) or by quantification of infectious units (
). The relative HIV-1 replication is presented as a percentage of vector controls (100%). B, the ability of RhoA(63L) protein to inhibit gene expression was assessed by cotransfecting pNL4-3-Luciferase plasmid with pcDNA3 expression plasmids encoding the RhoA proteins in Jurkat T cells. The level of luciferase expression (relative light units) was measured 48 h after transfection. Data points are the means of three independent measurements; bars, SE. Data shown are representative of two independent experiments.