Deregulation and Mislocalization of the Cytokinesis Regulator ECT2 Activate the Rho Signaling Pathways Leading to Malignant Transformation*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 8, Issue of February 20, pp , 2004 Printed in U.S.A. Deregulation and Mislocalization of the Cytokinesis Regulator ECT2 Activate the Rho Signaling Pathways Leading to Malignant Transformation* Received for publication, June 25, 2003, and in revised form, November 17, 2003 Published, JBC Papers in Press, November 25, 2003, DOI /jbc.M Shin ichi Saito, Xiu-Fen Liu, Keiju Kamijo, Razi Raziuddin, Takashi Tatsumoto, Isamu Okamoto, Xiaoyan Chen, Chong-Chou Lee, Matthew V. Lorenzi, Naoya Ohara**, and Toru Miki From the Molecular Tumor Biology Section, Basic Research Laboratory, NCI, National Institutes of Health, Bethesda, Maryland The human ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho GTPases and regulates cytokinesis. Although the oncogenic form of ECT2 contains an N-terminal truncation, it is not clear how the structural abnormality of ECT2 causes malignant transformation. Here we show that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of ECT2. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant negative Rho GTPases, suggesting the involvement of Rho GTPases in ECT2 transformation. Although deletion of the N-terminal cell cycle regulator-related domain (N) of ECT2 did not activate its transforming activity, removal of the small central domain (S), which contains two nuclear localization signals (NLSs), significantly induced the activity. The ECT2 N domain interacted with the catalytic domain and significantly inhibited the focus formation by oncogenic ECT2. Interestingly, the introduction of the NLS mutations in the S domain of N-terminally truncated ECT2 dramatically induced the transforming activity of this otherwise nononcogenic derivative. Among the known Rho GTPases expressed in NIH 3T3 cells, RhoA was predominantly activated by oncogenic ECT2 in vivo. Therefore, the mislocalization of structurally altered ECT2 might cause the untimely activation of cytoplasmic Rho GTPases leading to the malignant transformation. The ECT2 oncogene has been isolated in a search for mitogenic signal transducers in epithelial cells, where a murine * This work was supported in part by a Breast Cancer Think Tank Award from National Institutes of Health (NIH). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported by Japan Society of Promotion of Sciences fellowships for Biomedical and Behavioral Researchers in NIH. Present address: Laboratory of Cell Biology, NCI, NIH, 37 Convent Dr. MSC 4255, Bethesda, MD Present address: Fukuoka Teishin Hospital, Yakuin, Chuoku, Fukuoka , Japan. Present address: Oncology Drug Discovery, Pharmaceutical Research Institute Bristol-Myers Squibb, P. O. Box 4000, Princeton, NJ ** Present address: Dept. of Bacteriology, Faculty of Dentistry, Nagasaki University, Nagasaki , Japan. To whom correspondence should be addressed: Molecular Tumor Biology Section, Basic Research Laboratory, NCI, NIH, 37 Convent Dr. MSC 4255, Bethesda, MD Tel.: ; Fax: ; toru@helix.nih.gov. This paper is available on line at keratinocyte expression cdna library was introduced into fibroblasts to induce foci of morphologically transformed cells (1). The ECT2 transfectants exhibit anchorage-independent cell growth and efficient tumor formation in nude mice. The transforming ECT2 cdna encodes the C-terminal half of the full-length protein containing Dbl-homology (DH) 1 and pleckstrin homology (PH) domains, which are now found in a number of molecules involved in regulation of the Rho family GTPases. The N-terminal half of ECT2 contains domains related to cell cycle control and repair proteins, including Clb6 and Rad4/Cut5 (2, 3). CLB6 encodes a B-type cyclin of the budding yeast, which promotes the transition from G 1 into S phase (4). Fission yeast cut5, which is identical to the repair gene rad4, is required for both the onset of S phase and the restraint of M phase before the completion of S phase (5). The Cut5-related domain of ECT2 consists of two repeats (6, 7), designated BRCT (BRCA1 C-terminal) repeats, which are widespread in a number of cell-cycle checkpoint control and DNA repair proteins (7). These cell-cycle regulator-related domains of ECT2 play essential roles on the regulation of cytokinesis (2, 3). ECT2 catalyzes guanine nucleotide exchange in vitro on three representative Rho GTPases; RhoA, Rac1, and Cdc42 (2). The Rho family of small GTPases function as molecular switches of diverse biological functions, including cytoplasmic actin reorganization, cell motility, and cell scattering (8). Activation of the Rho proteins is promoted by guanine nucleotide exchange factors, which catalyze the replacement of bound GDP by GTP. The GTP-bound form of Rho proteins can specifically interact with their effectors or targets and transmit signals to downstream molecules. Rho proteins are inactivated through the hydrolysis of bound GTP to GDP by the intrinsic GTPase activity assisted by GTPase-activating proteins (GAPs). RhoA, Rac1, and Cdc42 induce the formation of actin stress fibers, lamellipodia, and filopodia, respectively (9). Among the known guanine nucleotide exchange factors for Rho GTPases, ECT2 shows several unique characteristics. ECT2 expression is induced in S phase and reaches the highest 1 The abbreviations used are: DH, Dbl homology; AP-1, activator protein-1; BRCT, BRCA1 C-terminal; CLB6, cyclin B6; Cut5, cells untimely torn 5; DAPI, 4, 6-diamidino-2 -phenylindole; ECT2, epithelial cell transforming gene 2 (human); ect2, mouse ECT2; Erk, extracellular signal regulated kinase; GAP, GTPase activating protein; GFP, green fluorescent protein; GST, glutathione S-transferase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NLS, nuclear localizing signal; PH, pleckstrin homology; RFP, red fluorescent protein; SRE, serum response element; SRF, serum response factor; ffu, focus-forming units; DN, dominant negative; WT, wild type.

2 7170 Mechanism of ECT2 Activation level in G 2 and M phases in regenerating mouse liver (10). ECT2 protein is specifically phosphorylated in G 2 and M phases (2). ECT2 exhibits nuclear localization in interphase, disperses throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of a dominant negative ECT2 or microinjection of anti-ect2 antibody strongly inhibits cytokinesis, indicating that ECT2 is a critical regulator of cytokinesis (2). Furthermore, the Drosophila pbl gene, whose mutation results in the inhibition of cytokinesis in mitotic cycle 14 during embryogenesis, was found to encode the fly homologue of human ECT2 (11). Although the transforming activity of several DBL family oncogenes is stimulated by N-terminal alterations (12 15), the activation mechanisms are still obscure. Because Rho GTPases play a critical role in cell transformation (16 18), ECT2 may display its transforming activity through the activation of Rho proteins. However, ECT2 is predominantly expressed in the nucleus where no expression of Rho GTPases is reported. In the present study, we examined the activation mechanism of the transforming activity of ECT2. We identified the small central domain containing two tandem nuclear localization signals as a negative regulator of the transforming activity. We show that elimination of these signals and a negative regulatory domain from ECT2 resulted in the activation of Rho GTPases in the cytoplasm, leading to malignant transformation of the cells. EXPERIMENTAL PROCEDURES DNA Constructs Full-length and N-terminally truncated ECT2 cdnas were amplified by PCR using ECT2 clone 1M (2, 3) as template and subcloned between the BamHI and EcoRI sites of the mammalian expression vector pcev29 or its derivative pcev29f3, which contains three tandem copies of FLAG sequence (19). ECT2 N-terminal derivatives, ECT2-N1 (amino acids 1 421), ECT2-N2 (amino acids 1 378), ECT2-N3 (amino acids 1 360), and ECT2-N4 (amino acids 1 333) have been described previously (3). ECT2- S mutant lacking the S domain (amino acids ) was created from two PCR products using the same template and primers I IV with the following sequences: I, 5 - CTC GGA TCC ATG GCT GAA AAT AGT GTA TTA-3 ; II, 5 -CAG ACT CGC GGA GTA TTT GCC TTT TCA TA-3 ; III, 5 -TCA CTC CGC GGT GGC AAG TTG CAA AAG AG-3 ; and IV, 5 -ACT GAA TTC GGT AAC GCT TCA TAT CAA ATG-3. The PCR products synthesized using primer pairs I and II were digested with BamHI and BstUI. The PCR products generated by primers III and IV were digested with BstUI and EcoRI. These products were ligated together with the pcev29 or pcev29f3 vector, which had been digested with BamHI and EcoRI, to create ECT2- S. Two ECT2 mutants, S1 and S2, containing RRR to AAA and R to A mutations in the NLS sequence of the S domain, respectively, were generated by the similar procedure, but following oligonucleotides were used for PCR to introduce mutations: V, 5 -CAG ACT GCG GCC GCT TTG CGA TTG CTG TTA GGG GT-3 ; VI, 5 -TCA CTC GCG GCC GCT TTA AAA GAA ACA CTT GCT CAG-3 ; VII, 5 -TTT GGC GCG CCC GGG GTG GAA ATG GTG ACA C-3 ; and VIII, 5 -TTT GGC GCG CCC ATC AGC TGA GCA TTC CCT T-3. NotI and AscI were used to create S1 and S2, respectively, instead of BstUI. S3 was created by the similar procedure, but S1 was used as a PCR template instead of ECT2-F. ECT2-F, S1, S2, S3, S, and N5 were also cloned into pegfp-c1 (BD Biosciences/Clontech) to express ECT2 as green fluorescent protein (GFP) fusion proteins. All constructs generated by the use of PCR were sequenced to ensure that no PCR mutation was generated except the desired mutations. An ECT2- N5 derivative containing PVQR to AAAA mutations (amino acids ) in the DH domain was generated by amplifying two PCR fragments. Primers for the first fragment were as follows: a forward primer with a BamHI restriction site, 5 -CCC GGA TCC GCC ACC ATG GTT CCT TCA AAG CAG TCA GCA-3, and a reverse primer with SfiI site, 5 -CAG ACT GGC CGC TGC GGC CCG GAT AAG AAG TTC AAC AAG-3. Primers for the second fragment were: a forward primer with a SfiI site, 5 -TCA CTC GGC CGC AGC GGC CTT ACC CAG TGT TGC ATT ACT-3, and a reverse primer with an EcoRI site, 5 -ACT GAA TTC GGT AAC GCT TCA TAT CAA ATG-3. PCR products were then digested with the indicated restriction enzymes and simultaneously ligated with the pcev29f3 vector digested with BamHI and EcoRI. A new SfiI site was generated as a result of the introduction of PVQR to AAAA mutations. To introduce the SV40 NLS (nuc) into ECT2- N5, ECT2- N5 fragment was subcloned into BamHI site of pecfp-nuc vector (BD Biosciences/Clontech), and then a DNA fragment containing the triple repeats of SV40 large T antigen NLS sequence (GAT CCA AAA AAG AAG AGA AAG GTA GAT CCA AAA AAG AAG AGA AAG GTA GAT CCA AAA AAG AAG AGA AAG GTA) and ECT2- N5 sequence was excised and then cloned in pegfp-c1 and pcev29f3 vectors. Expression of fusion proteins of expected sizes were confirmed by Western blotting. ECT2 NLS sequence (amino acids ) was attached to the 3 end of EGFP sequence (BD Biosciences/Clontech) by PCR and subcloned between BamHI and EcoRI sites of the mammalian EGFP expression vector pcaggfp (20) to create an EGFP-EGFP-ECT2 NLS fusion protein. The expression of 60-kDa ECT2 NLS-tagged tandem GFP protein was confirmed by Western blotting in U-2 OS cells. Focus Forming Assays NIH 3T3 cells were transfected with various amounts ( g) of the eukaryotic expression vector pcev29 or pcev29f3 (19) containing ECT2 cdnas or vector alone by the calcium phosphate transfection method. Focus formation was observed in unselected plates 14 days after transfection and quantified after Giemsa staining. FLAG-tagged ECT2 variants (in pcev29f3) showed slightly lower transforming activity than non-tagged versions (in pcev29). Transforming activity was expressed as the number of foci per picomoles of DNA (ffu/pmol). Comparative efficiency of transfection was confirmed by G-418-resistant colony formation in duplicated plates. Expression levels of the ECT2 variants were examined using anti- FLAG M2 antibody (Sigma, St. Louis, MO) and anti-gfp (BD Biosciences/Clontech) 48 h after transfection with 10:1 mixture of FLAGtagged ECT2 expression vector and pegfp-c1. Transient Expression Reporter Gene Assays The construction of SRF-, and AP-1-luciferase reporter plasmids in pgl2luc containing a minimal c-fos promoter ( 56 to 109) has been described previously (21). The SRF binding sequence is derived from the serum response element of the c-fos gene and corresponds to SRE.mutL (22). Firefly luciferase reporter and TK-Renilla luciferase control plasmids were cotransfected with each expression vector into COS cells. Total amount of DNA was adjusted by the addition of vector DNA h posttransfection, cells were lysed and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, WI) according to the accompanied protocol. Firefly luciferase activity in the lysates was normalized to Renilla luciferase activity and expressed as a relative luciferase activity. No significant increase in luciferase activity was observed following transfection of each expression vector DNA with a reporter plasmid containing only a luciferase cassette and c-fos minimal promoter. In Vitro Invasion Assays Invasion assays were performed using Biocoat Matrigel invasion chambers (BD Biosciences/Clontech) essentially as described in the manufacturer s protocol. Matrigel invasion chambers (24-well) were rehydrated with Dulbecco s modified Eagle s medium containing 0.1% bovine serum albumin for 2 h at room temperature. NIH 3T3 cells transfected with ECT2 or control vectors were seeded at /0.4 ml of medium containing 0.1% bovine serum albumin into the inner well of invasion chambers. The outer chambers were filled with 0.4 ml of medium containing 10% calf serum. Cells were allowed to invade Matrigel matrices for 10 12hat37 C inaco 2 incubator. To count cells that migrated onto the membrane at lower surface, the cells on the upper side of the membrane were scraped off with cotton swipe, then the inserts with membrane were stained with Diff-Quick (Dade Diagnostics). Cells on the lower side of membrane were photographed and counted. Activation Assays for Rho GTPases COS cells were transfected by GFP or GFP-ECT2- N5 expression vectors together with pef4/myc- His (Invitrogen, CA) carrying inserts for AU5-tagged Rho GTPases. Lysates were prepared 24 h after transfection, and the GTP-bound forms of Rho GTPases were determined by pull-down assays using GST-Rhotekin (for RhoA, RhoB, and RhoC) or GST-PAK PBD according to the manufacturer s protocol (Cytoskeleton, Denver, CO). Subcellular Localization and Cell Morphology NIH 3T3 cells were transfected with the GFP- or FLAG-tagged ECT2 expression vectors using the LipofectAMINE Plus reagent (Invitrogen, Carlsbad, CA). GFP-expressing cells were identified by green fluorescence. Actin and DNA were stained with rhodamine-conjugated phalloidin (Sigma) and 4,6 -diamidino-2-phenylindole (DAPI, Sigma), respectively, as reported previously (2, 19). FLAG-tagged ECT2 derivatives (0.5 g each) were transfected into NIH 3T3 cells using the LipofectAMINE Plus reagent. Cells were fixed with a freshly prepared mixture of methanol:acetone (1:1) for 2 min at room temperature 40 h after transfection. Expressed proteins were visualized using anti-flag M2 monoclonal antibody-cy3 conjugate (Sigma) in the presence of 1 g/ml DAPI. U-2 OS cells were

3 Mechanism of ECT2 Activation 7171 FIG. 1. Induction of foci of transforming cells by ECT2 derivatives. A, morphology of foci induced by ECT2 cdna. 1 g each of vector alone (pcev29) or the expression vector for human fulllength ECT2 (ECT2-F), human ECT2 lacking the N-terminal half (ECT2- N5), mouse ect2 lacking the N-terminal half (ect2-t), H-ras-V12 or c-sis was used to transfect NIH3T3 cells. Cells were stained by Giemsa and then photographed. B, cell morphology of stable ECT2-F, N, N5, and vector alone transfectants. C, expression of FLAG-tagged ECT2-F, N, and N5 in stable transfectants. FLAG-ECT2 fusion proteins were detected by immunoblotting using anti- FLAG M2 monoclonal antibody. also transiently transfected with the expression vectors for GFP-tagged ECT2 mutants using FuGENE 6 reagent (Roche Applied Science) to confirm their subcellular localization. In this case, Hoechst dye was added to culture medium at a final concentration of 10 M, and cells were directly observed under the fluorescence microscope. Images were acquired using a Zeiss Axiovert microscope equipped with a Photometrics digital camera and processed with IPLab software (Signal Analytics). Time-lapse Video Microscopy The RFP-ECT2- N5 expression vector was constructed by inserting ECT2- N5 into prsred2-c1 (BO Biosciences/Clontech) at the BglII and EcoRI sites. pegfp-actin was obtained from Clontech. NIH 3T3 cells were transfected with the equal mixture of both the plasmids using FuGENE 6 transfection reagent (Roche Applied Science). Cells were cultured on 35-mm plates in an environmental chamber on a stage of Zeiss Axiovert S-100 microscope equipped with motorized X-Y-Z stages. Images were taken at 3-min intervals by using a photometric digital camera controlled by OpenLab software (Improvision, Lexington, MA). RESULTS Deletion of the N-terminal Half of Human ECT2 Induces Cell Transformation and Invasiveness The mouse ect2 cdna, ect2-t, which carries an N-terminal truncation, exhibits a high transforming activity, whereas the full-length clone does not significantly induce transformation (23). To test if N-terminal truncation can also activate the transforming activity of human ECT2, we generated a FLAG epitope-tagged full-length human ECT2 and its derivative ECT2- N5, which has a similar N- terminal deletion to mouse ect2-t. Like ect2-t, ECT2- N5 exhibited a high transforming activity in NIH 3T3 cells, whereas the full-length ECT2, ECT2-F, did not show any detectable activity (Fig. 1A). Both mouse and human ECT2 similarly induced tiny foci of transforming cells with stellate morphology, which was distinct from ras- orsis-induced foci (Fig. 1, A and B). We previously reported that ect2-t stimulates anchorageindependent growth of NIH 3T3 cells and tumorigenicity in nude mice (23). To examine additional oncogenic activities of ECT2, we established NIH 3T3 clones expressing FLAG-tagged derivatives of ECT2-F, ECT2- N5, and ECT2-N2. ECT2-N2 carries the region from the N terminus to the S domain (see Fig. 6A). Western blot analysis with anti-flag antibody showed that these stable transfectants expressed FLAG-tagged ECT2-F, ECT2-N2, and ECT2- N5 at comparable levels (Fig. 1C). Upon plating, ECT2- N5-expressing cells formed secondary foci with stellate morphology, whereas the morphology of ECT2-F and ECT2-N2 clones was indistinguishable from the vector alone transfectants (Fig. 1B). When cultured in the presence of 10% serum, all of the stable ECT2 clones exhibited similar growth properties (data not shown). However, in medium containing 1% serum, vector alone, ECT2-N2 and ECT2-F transfectants did not grow well and the number of viable cells gradually decreased (Fig. 2A). In contrast, ECT2- N5 transfectants continued to grow for at least 48 h under these conditions, suggesting that these transfectants acquired low serum dependence. To test if ECT2 can induce cell invasiveness, NIH 3T3 cells expressing ECT2-F, ECT2- N5, or vector alone were placed on the surface of an artificial basement membrane, Matrigel, and the number of the cells that had migrated through the membrane was counted. Interestingly, ECT2- N5 transfectants exhibited a strong invasion activity, whereas ECT2-F or the vector alone transfectants did not show significant activity (Fig. 2B). Additionally, mouse ect2-t, which corresponds to human ECT2- N5, also exhibited a high activity of cell invasiveness. These results indicate that oncogenic ECT2 is an efficient activator of cell invasiveness.

4 7172 Mechanism of ECT2 Activation FIG. 2.Characterization of NIH 3T3 cells transfected with ECT2 variants. A, growth of ECT2 transfectants in low serum conditions. Cells were cultured in Dulbecco s modified Eagle s medium containing 1% serum, and viable cells were scored at the indicated time points. B, induction of cell invasiveness by ECT2. Stable NIH 3T3 transfectants expressing the indicated cdna were used for Matrigel assays to estimate cell invasiveness in vitro. The cells that invaded the Matrigels were stained and photographed (left half). The number of cells passed through the Matrigel is summarized (right half). ect2-t is a mouse cdna with an N-terminal deletion similar to human ECT2- N5. Oncogenic ECT2 Activates Rho Signaling Pathways Because Rho GTPases are known to regulate the JNK and p38 MAPK pathways (24, 25), we examined whether these pathways are activated in ECT2 transfectants. We first analyzed endogenous JNK activity in these ECT2 transfectants using an antibody specific to the activated form of c-jun, which is phosphorylated at serine 63. In vector alone transfectants, a very low level of JNK activity was observed (Fig. 3A, top). The activity of JNK in these cells was increased by the stimulation with sorbitol, an activator of the JNK pathway. The activity of JNK was also elevated in cells expressing ECT2- N5. In contrast, ECT2-F or ECT2-N2 expression did not significantly affect the JNK activity. The JNK activity was induced by sorbitol to a similar level in all the transfectants (data not shown), indicating that all of the transfectants maintained the ability to induce JNK activity. In contrast, we did not observe significant activation of p38 or Erk MAPKs by ECT2 and its derivatives (Fig. 3A, middle and bottom). These results indicate that ECT2- N5 preferentially activates the JNK signaling pathway. Rho proteins can stimulate the transcriptional activity regulated by serum response factor (SRF) (22). To examine whether ECT2 can stimulate SRF-regulated transcription, a serum response element (SRE)-luciferase plasmid was used as a reporter. Upon coexpression of the reporter plasmid with either the full-length or truncated ECT2 expression vector, luciferase activity was estimated. As shown in Fig. 3B, upper panel, expression of ECT2- N5 potently induced the transcriptional activity of the SRE reporter plasmid. In contrast, expression of ECT2-F exhibited the activity slightly higher than the vector alone control. The SRE-regulated transcriptional activity induced by ECT2- N5 was efficiently inhibited by either of dominant negative RhoA, Rac1, or Cdc42. Moreover, either of constitutively active RhoA, Rac1, or Cdc42 efficiently enhanced SRE-mediated transcription in this system. These results suggest that ECT2- N5 can stimulate SRE-mediated transcription through the activation of Rho GTPases. We previously showed that Ost, a guanine nucleotide exchange factor for RhoA and Cdc42, activates the transcriptional activity regulated by activator protein-1 (AP-1) (19). To examine whether ECT2 can also stimulate AP-1-regulated transcriptional activity, an AP-1-binding site-luciferase plasmid was utilized as a reporter. As shown in Fig. 3B, lower panel, expression of ECT2- N5 moderately elevated AP-1-regulated transcriptional activity, whereas ECT2-F or ECT2-N2 failed to stimulate the activity. Coexpression of dominant negative RhoA, Rac1, or Cdc42 reduced the ECT2- N5-mediated stimulation of AP-1-regulated transcription, albeit at lower levels as compared with their effects on SRE-regulated transcription. We also found that constitutively active RhoA, Rac1, or Cdc42 efficiently stimulated AP-1-mediated transcription. Among these GTPases, Rac1 displayed the highest level of stimulation of AP-1-regulated transcriptional activity. All of these results indicate that ECT2 can regulate the transcriptional events mediated by SRE and AP-1 through the activation of Rho GTPases. Oncogenic ECT2 Induces Cell Rounding in NIH 3T3 Fibroblasts Rho family proteins are involved in the organization of actin-based cytoskeletal structures. In fibroblasts, RhoA activates actin stress fiber formation, whereas Rac1 and Cdc42 induce lamellipodia and filopodia formation, respectively (8). To test which actin-based structures ECT2 can induce, we

5 Mechanism of ECT2 Activation 7173 FIG. 3. Stimulation of MAPK activity and AP-1 or SRF-regulated transcriptional activation by ECT2. A, effects of ECT2 derivative expression on MAPK activity in NIH 3T3 cells. MAPK activity in the stable transfectants expressing indicated plasmids (see Fig. 1C) was measured using phosphospecific antibodies, and endogenous proteins were detected by specific antibodies (Cell Signaling Technology, Beverly, MA) according to the accompanied protocols. JNK activation was detected as phosphorylated c-jun at Ser-63, and activated Erk and p38 proteins were detected as their phosphorylated forms. B, effects of ECT2 derivative expression on AP-1- or SRF-regulated transcription. COS cells were cotransfected with the indicated ECT2 expression vector and SRE-LUC (upper) or AP-1-LUC (lower) reporter plasmid. Activation of AP-1- or SRF-regulated transcriptional activity was estimated by the luciferase activity. prk-tk-renilla luciferase vector was used to cotransfect the cells together with the reporter plasmid for an internal control to normalize the transfection/expression efficiency. transiently expressed GFP-tagged ECT2-F, ECT2- N5, or GFP vector alone in NIH 3T3 cells. A population ( 20%) of cells expressing GFP-ECT2- N5 showed a flat phenotype with moderately enhanced actin stress fiber formation (Fig. 4A, GFP- ECT2- N5, left panels), suggesting that Rho was preferentially activated by ECT2- N5 in these cells. However, the majority of NIH 3T3 cells expressing GFP-ECT2- N5 exhibited a compacted structure with saturated F-actin staining (Fig. 4A, GFP- ECT2- N5, right panels). In contrast, the morphology of the surrounding cells, which were not expressing the GFP fusion protein, was similar to that of vector alone transfectants. These findings are consistent with the previous results that oncogenic ECT2 induces foci containing both fusiforms and rounded cells (23). To examine how these compacted cells were generated by ECT2- N5 expression, NIH 3T3 cells were transfected with red fluorescent protein (RFP)-fused ECT2- N5 and GFP-fused actin expression vectors. Cells expressing RFP-ECT2- N5 were identified with red fluorescence, and their morphologies were examined by time-lapse video microscopy. In most of the cells expressing RFP-ECT2- N5, actin stress fibers were disrupted h after transfection, and the cells rounded up like the M phase cells (Fig. 4B, see the cell shown by an arrow), but most of them did not divide in a next few hours. The cell that once rounded nearly completely appeared to flatten to some extent (30 32 h panels) and then rounded again (33 35 h after transfection panels). RFP-ECT2- N5 was detected in the entire cell of these transfectants (data not shown). In Fig. 4B, another cell expressing ECT2- N5 at similar level as determined by the red fluorescence also exhibited a similar morphology with additional cortical activities (lower right corner). In contrast, cells expressing ECT2- N5 weakly did not round up, although they expressed GFP-actin at a similar level (see the rightmost cell in Fig. 4B). These results suggest that oncogenic ECT2 stimulates cellular transformation by dramatically changing their actin cytoskeletal morphology in NIH 3T3 cells. ECT2 Transformation Is Dependent on the Activation of Rho GTPases ECT2 can activate RhoA, Rac1, and Cdc42 through guanine nucleotide exchange in vitro (2). Rho GTPases have been shown to play a critical role in cellular transformation (16 18). To test whether ECT2 transformation involves Rho activation, we cotransfected NIH 3T3 cells with the ECT2- N5 expression vector (0.1 g) and each of the expression vectors encoding the dominant negative (DN) forms of Rho proteins (0.5 g). Because the addition of a large amount of DNA usually inhibits focus formation presumably due to the competition for the available transcription and translation machinery in the cells, we compared the effects of wild type (WT) and DN constructs whose difference is in a single amino acid residue. As shown in Fig. 5A, either of dominant negative RhoA, Rac1, or Cdc42 more strongly inhibited ECT2-mediated focus formation in NIH 3T3 cells than the WT counterparts. Inhibition of ECT2 transformation by the empty vector was at the similar level by the WT Rho GTPase expression vectors, and a lower amount of WT Rho GTPases did not exhibit the inhibitory effect on ECT2 transformation (data not shown). These results suggest that ECT2- N5 induces malignant transformation through the activation of Rho GTPases. The S Domain of ECT2 Plays a Critical Role in the Regulation of Transformation To determine which domains in the N-terminal half of ECT2 regulate the transforming activity, we generated a set of overlapping N-terminal truncation mutants, and expressed these constructs in NIH 3T3 cells as FLAGtagged proteins. Unexpectedly, the N-terminal deletions extending to the N-terminal most region (N), CLB6-homology domain, or each of the two BRCT domains did not activate the transforming activity of ECT2 (Fig. 6A). However, two ECT2 derivatives containing the N-terminal deletions extended to

6 7174 Mechanism of ECT2 Activation FIG. 4. Induction of cytoskeletal actin remodeling by oncogenic ECT2. A, actin organization of ECT2 transfectants. NIH 3T3 were transfected with the GFP-ECT2- N5 expression vector or GFP vector. Cells expressing GFP-ECT2- N5 or GFP (indicated by arrows) were identified by green fluorescence and stained with rhodamineconjugated phalloidin for F-actin. B, time-lapse video recording of the morphological changes of NIH 3T3 cells upon oncogenic ECT2 transfection. NIH 3T3 fibroblasts were transiently transfected with GFP-actin and RFP-ECT2- N5, and cells with green fluorescence were photographed using time-lapse video microscopy. An arrow indicates one of the RFP-ECT2- N5-expressing cells, which showed morphological changes. The number in each panel indicates time after transfection (h). Bar, 10 M. FIG. 5. Inhibition of ECT2- N5-induced transformation by dominant negative Rho GTPases. NIH 3T3 cells were cotransfected with ECT2- N5 and indicated Rho expression vectors. Foci of morphologically transformed cells were scored 14 days after transfection and expressed as percentages of the number of foci induced by ECT2- N5 alone. Similar transfection efficiency of NIH 3T3 cells with the vectors used was confirmed by the formation of G418-resistant colonies. Shown is one of the results reproduced three times with an identical pattern. the small central region, designated the S domain, exhibited a markedly high transforming activity (Fig. 6A; see N9 and N5). To determine if the S domain itself regulates the transforming activity, a derivative of ECT2-F lacking the S domain was generated. This mutant ( S) exhibited a high level of transforming activity in NIH 3T3 cells, suggesting that the S domain plays a critical role in the regulation of the transforming activity of ECT2. When the N-terminal deletions extended to the neighboring DH domain, the mutant ECT2 did not show detectable transforming activity (Fig. 6A, N6 and N8), suggesting that the DH domain is required for the transforming activity of ECT2. To confirm this, point mutations (PVQR 3 AAAA, amino acids ) were introduced into the most conserved region of the DH domain in ECT2- N5. This mutant plasmid, ECT2- N5 DH, did not exhibit any detectable transforming activity (Fig. 6A). Therefore, the DH domain, and in turn the exchange activity for the Rho GTPases, appeared to be essential for the transforming activity of ECT2. We also generated a series of C-terminal deletion mutants of ECT2- N5. A deletion extended to the PH domain ( C1) eliminated the transforming activity from ECT2- N5, suggesting that the PH domain is also essential for the transforming activity of ECT2. A small C- terminal deletion ( C3) did not affect the transforming activity of ECT2- N5. A mutant protein lacking most of the C-terminal domain ( C2) was weakly transforming, but the morphology of the foci was less aggressive than the foci induced by ECT2- N5. The expression level of the exogenous ECT2 proteins in transiently transfected NIH 3T3 cells was examined by immunoblotting using anti-flag monoclonal antibody (Fig. 6B). All of the ECT2 derivatives were detected as proteins of expected sizes. The expression level of all the ECT2 derivatives was comparable with an exception of ECT2- N5 C2, which showed markedly high expression. The reason for this high expression is not known, but this expression level might contribute to the transforming activity of ECT2- N5 C2. Transforming ECT2 Derivatives Partially Localize in the Cytoplasm We have found that ECT2 is localized in the nucleus in interphase cells (2). Several putative nuclear localization signals (NLSs) were found in the predicted ECT2 sequence, including RKRRR (amino acids ) and PRKRP ( ) located in the S domain (Fig. 6A). Like endogenous ECT2, exogenously expressed FLAG-ECT2-F also localized in the nucleus, whereas FLAG-ECT2- N5, which lacks both the NLSs, was detected in both the nucleus and cytoplasm (Fig. 6C). GFP-tagged ECT2 derivatives also showed similar localization patterns to their FLAG-tagged counterparts (data not shown). ECT2- N5 contains a putative NLS at the C-terminal domain. GFP-ECT2-C, which consisted of the C-terminal domain alone (amino acids ), was localized predominantly in the nucleus (data not shown), suggesting that the NLS in the C-terminal domain is functional. Like ECT2- N5, ECT2- N9, which lacks the N-terminal NLS, had a high transforming activity, whereas ECT2- N4, which contains both the NLSs in the S domain, did not exhibit detectable transforming activity (Fig. 6A). ECT2- N9 also exhibited both the nuclear and cytoplasmic localization (data not shown). Therefore, the transforming activity of ECT2 derivatives coincided with partial nuclear localization. Loss of Nuclear Localization Signals Affects the Transforming Activity of ECT2 To test whether the NLS sequences located in the S domain are responsible for the activation of the transforming activity of ECT2, we mutated the first (N-terminal) NLS (RKRRRLK) to RKAAALK in ECT2-F. Because NLSs are usually rich in the positively charged amino acids Lys and Arg, Arg to Ala changes in NLSs should reduce the nuclear localization of the protein. This mutant, ECT2-S1, exhibited a significant transforming activity, whereas ECT2-F did not have any detectable activity (Fig. 6A). ECT2-S2, an ECT2-F derivative containing a mutation in the second NLS also exhibited a weak transforming activity. We also generated ECT2- S3, which contains mutations in both the NLSs in the S domain. This mutant also showed a significant transforming activity in NIH 3T3 cells. Subcellular localization analysis re-

7 Mechanism of ECT2 Activation 7175 FIG. 6. Mapping of the domains that regulate transforming activity of ECT2. A, deletion mapping of domains that affect transforming activity. Regions carried by the ECT2 derivatives are shown by horizontal bars under a schematic representation of the human ECT2 protein. BRCT-1 and BRCT-2 indicate BRCA1 C-terminal repeats (7). The numbers at the ends of each clone represent the amino acid numbers relative to ECT2-F. S contains an internal deletion of the S domain. N5 DH contains PVQR to AAAA substitutions at amino acids , whose location is also indicated by x. Transforming activity was shown as follows:, ffu/pmol; ffu/pmol;, ffu/pmol; and, ffu/pmol. B, identification of exogenously expressed FLAG-ECT2 fusion proteins by immunoblotting. NIH 3T3 cells were transiently transfected with indicated FLAG-tagged ECT2 derivatives together with the GFP expression vector pegfp-c1. Forty-eight hours after transfection, cells were lysed and the proteins expressed from the vectors were analyzed by immunoblotting with anti-flag (M2), anti-gfp, and anti- tubulin antibodies. The GFP expression level was measured to monitor the transfection efficiency of each ECT2 variant and to detect the possible effect of ECT2 derivatives on protein expression. -Tubulin expression was measured as a loading control. Bands with expected sizes are indicated by dots at the right side. C, subcellular localization of the FLAG-ECT2 fusion proteins. The fusion proteins (red) and nuclei (blue) were detected by anti-flag antibody and DAPI, respectively. Merged images are shown at the bottom. vealed that ECT2-S3 localized in the cytoplasm as well as in the nucleus (Fig. 6C). ECT2-S1 and -S2 also exhibited a similar localization to ECT2-S3 (data not shown). The expression levels of the wild type and mutant proteins were comparable (Fig. 6B). These results suggest that the two NLSs in the S domain can function in vivo and that the impairment of NLSs can activate the transforming activity of ECT2. To further verify that ECT2 NLSs can function as nuclear localization signals, these NLSs were introduced into tandem GFP. Whereas GFP exhibited both the cytoplasmic and nuclear

8 7176 Mechanism of ECT2 Activation FIG. 7.Functional comparison of ECT2 NLS and SV40 NLS. A, subcellular localization of ECT2 NLS-tagged tandem GFP protein in U-2 OS cells. U-2 OS cells were transiently transfected with the indicated expression vectors using FuGENE 6 reagent. Hoechst dye was added to culture medium at a final concentration of 10 M 24 h after transfection, and cells were directly observed under the fluorescence microscope and photographed. B, subcellular localization of SV40 NLS (nuc)-tagged ECT2- N5 in U-2 OS cells. U-2 OS and NIH 3T3 (data not shown) cells were transiently transfected with the indicated expression vectors using FuGENE 6 and LipofectAMINE PLUS reagent, respectively. Hoechst dye was added to culture medium at a final concentration of 10 M 24 h after transfection, and cells were directly observed under the fluorescence microscope and photographed. localization due to its relatively small size, tandem GFP showed reduced nuclear localization (Fig. 7A). In contrast, the derivative of tandem GFP containing ECT2 NLSs displayed predominant nuclear localization, indicating that ECT2 NLSs are functional nuclear localization signals. To test whether cytoplasmic localization of ECT2- N5 can be reduced by the addition of another nuclear localization signal, we introduced SV40 nuclear localization signal (nuc) into ECT2- N5. As shown in Fig. 7B, ECT2- N5 nuc displayed predominant nuclear localization, whereas ECT2- N5 exhibited both the cytoplasmic and nuclear localization. Additionally, GFP-tagged ECT2- N5 displayed a relatively weak but significant transforming activity in NIH 3T3 cells, whereas GFP- ECT2- N5 nuc did not show detectable transforming activity (data not shown). All of these results strongly suggest that cytoplasmic localization of ECT2 is required for its transforming activity. The N-terminal Domain of ECT2 Interacts with the Catalytic Domain Although the N-terminal truncations that did not extend to the S domain did not activate the transforming activity of ECT2, the introduction of NLS mutations to ECT2-F FIG. 8.Interaction of the N- and C-terminal domains of ECT2. A, association of ECT2-N with ECT2- N5 or ECT2-DH. Lysates prepared from COS cells expressing indicated proteins were subjected to immunoprecipitation by anti-gfp or anti-flag antibodies, and blotted by anti-flag antibody. *, location of immunoglobulin heavy chain. V, FLAG vector; F, FLAG-ECT2-F; N4, FLAG-ECT2-N4; N, FLAG- ECT2- N5; DH, FLAG-ECT2-DH (amino acids ). B, inhibition of ECT2- N5-transforming activity in NIH 3T3 cells by ECT2-N. NIH 3T3 cells were transfected with ECT2- N5 together with the indicated expression vectors in triplicate, and foci of transforming cells were scored 14 days after transfection. The number of foci induced by ECT2- N5 plus vector alone was normalized to 100%. Similar experiments were performed using H-rasV12 as control. Shown are representative results reproduced twice with an identical pattern. stimulated its transforming activity. However, the level of activation by NLS mutations was very low compared with the deletion of the N-terminal half ( N5). The deletion of the entire S domain ( S) markedly stimulated the transforming activity, but the transforming activity of ECT2- S was still lower than ECT2- N5, suggesting that the sequence of the S domain other than the NLSs also negatively regulates the transforming activity. We reasoned that, although the deletions of N-terminal domains themselves cannot induce the transforming activity of ECT2, these domains may inhibit the catalytic activity of ECT2 by the interaction with the C-terminal half. To test this possibility, GFP-tagged ECT2-N4 was coexpressed with FLAGtagged ECT2-F, -N4, - N5, or -DH in COS cells. When GFP- ECT2-N4 was immunoprecipitated with anti-gfp antibody, FLAG-tagged ECT2-F, - N5, and -DH were detected in the immunoprecipitates (Fig. 8A). Particularly, FLAG-ECT2-DH exhibited a strong association with GFP-ECT2-N4. In contrast, FLAG-ECT2-N4 was not coimmunoprecipitated with GFP- ECT2-N4. These results suggest that the N-terminal domain of ECT2 can associate with the catalytic domain. We further examined whether ECT2 N-terminal derivatives possess the capability to inhibit the transforming activity of ECT2- N5. Because we previously found that ECT2-N4

9 Mechanism of ECT2 Activation 7177 FIG. 9.Effects of mutations in the nuclear localization signals on the transforming activity of ECT2- N4. NIH 3T3 cells were transfected with the indicated plasmids, and foci of morphologically transformed cells were scored 14 days after transfection. strongly inhibits cytokinesis (2), we used ECT2-N1, which contains the entire S domain, and ECT2-N3, which contains only the N-terminal NLS in the S domain, for this analysis. As shown in Fig. 8B, either of ECT2-N1 or -N3 significantly inhibited the transforming activity of ECT2- N5. In contrast, either ECT2-N1 or -N3 did not significantly affect the transforming activity of the H-ras oncogene. Transforming Activity of N-terminally Truncated ECT2 Is Strongly Stimulated by the Loss of Nuclear Localization Signals As ECT2-N can associate with the catalytic domain, such an intramolecular association may inhibit the catalytic activity of ECT2. This raised the possibility that the loss of NLSs from ECT2- N4, which lacks the N-terminal domains but retains the S domain, activates the transforming activity. To explore this possibility, the NLS mutations were introduced into the S domain of ECT2- N4, and their transforming activity was determined (Fig. 9). Whereas ECT2- N4 did not exhibit any detectable activity, ECT2- N4S1, which contains mutations in the first NLS showed a strong focus formation in NIH 3T3 cells. The introduction of S3 mutations (S1 S2) to ECT2- N4 also exhibited a strong transforming activity. The transforming activity of ECT2- N4S1 and ECT2- N4S3 was comparable to that of ECT2- S. As ECT2- N5, which lacks the entire S domain, showed a higher activity, the NLS mutations might not strong enough to fully activate the transforming activity. These results suggest that loss of NLSs strongly activates the transforming activity when the N-terminal domain is not present. RhoA Is Strongly Activated by Cytoplasmic ECT2 The above results suggest that the cytoplasmic localization of ECT2 derivatives lacking the N-terminal cell cycle regulator-related domains activate cytoplasmic Rho GTPases leading to malignant transformation. To test which Rho GTPases are activated in the cytoplasm by such the ECT2 derivatives in vivo, we cotransfected COS cells with expression vectors for AU5-tagged Rho GTPases and GFP-tagged ECT2- N5 or GFP alone, and GTP-bound Rho GTPases were pulled down with GST-tagged Rho-binding domain of Rhotekin (GST-RBD) or p21-binding domain of PAK (GST-PBD). However, the initial results indicated that the expression level of exogenous Rho GTPases was affected by the ECT2 expression vector and thus the amount of Rho GTPases pulled down with GST-RBD or GST-PBD did not FIG. 10. Identification of Rho GTPases activated by oncogenic ECT2. A, COS cells were transfected with the indicated AU5-tagged Rho expression vectors together with the GFP vector ( ) or GFP-ECT2- N5 expression vector ( ). The GTP-bound forms of Rho GTPases were pulled down by GST-RBD (for RhoA, RhoB, and RhoC) or GST-PBD (for Rac1 and Rac2) and detected by anti-au5 antibody. The amount of GST-RBD and GST-PBD in the reaction mixtures was also determined by protein staining. B, effects of ECT2-N derivatives on ECT2- N5- induced GTP-RhoA accumulation. COS cells were transfected with ECT2- N5 (0.5 g) and indicated ECT2 derivatives (, 1.5 g;, 3.5 g). The GTP-bound forms of Rho GTPases were analyzed as in panel A. Similar results were reproduced three times. reflect the exchange activity of ECT2 (data not shown). Therefore, we subcloned the AU5-Rho inserts into the EF-1 promoter-based expression vector and transfected them together with the ECT2- N5 expression vector into COS cells. As shown in Fig. 10A, the expression level of exogenous Rho GTPases were not affected by ECT2- N5 expression. RhoA was efficiently activated by ECT2- N5. RhoB and RhoC were also activated by ECT2- N5 albeit less efficiently. In these experiments we used AU5-tagged Rho GTPases to avoid possible cross reactivity of specific antibodies. However, the detection of activation of endogenous RhoA, RhoB, and RhoC by ECT2- N5 using specific antibodies revealed similar results (data not shown). Rac2 was also dramatically activated by ECT2- N5, and Rac1 was activated moderately. In contrast, activation of Cdc42 and TC10 was below the detectable level under these conditions (data not shown). To test whether ECT2-N affects the exchange activity of ECT2 in vivo, we cotransfected COS cells with ECT2- N5 and ECT2-N. GTP-bound RhoA was pulled down by GST-RBD and then detected with anti-rhoa antibody (Fig. 10B). ECT2-N4, which lacks NLSs, relatively weakly but significantly reduced the accumulation of GTP-RhoA by ECT2- N5. In contrast, ECT2-N2, which contains NLSs, did not affect the GTP-RhoA accumulation. We also tested a higher amount of ECT2-N DNAs (Fig. 10B; ), but they nonspecifically inhibited GTP- RhoA accumulation.

10 7178 Mechanism of ECT2 Activation DISCUSSION In the present study, we characterized the malignant transformation induced by the human ECT2 protooncogene. Like many other Dbl family proteins, ECT2 also contains the DH and PH domains. However, ECT2 is unique among these proteins in that it contains cell cycle control domains in its N- terminal half and nuclear localization signals in the central and C-terminal domains. The transforming activity of ECT2 can be activated by the deletion of the N-terminal half. In addition to the previously reported oncogenic properties, we also found that ECT2- N5 transfectants exhibited elevated cell invasiveness and reduced serum dependence. We found that oncogenic ECT2 activates several Rho GTPases-regulated signaling pathways. It has been shown that Rac1 and Cdc42 can activate JNK and p38 MAPK cascades (24, 25). ECT2 efficiently activated JNK, but not p38 and Erk pathways, at a detectable level. Although the differences of experimental conditions and sensitivity of the detection may explain why ECT2 did not activate p38 efficiently, ECT2 may associate with cellular components, which are specifically involved in JNK signaling. It is also possible that stimulation of the cycling of GDP- and GTP-bound forms of Rho GTPases by exchange factors may activate the JNK pathways more efficiently than p38 pathway, whereas mutationally activated Rac1 and Cdc42 can efficiently stimulate both the pathways. Because Rac2 is specifically expressed in cells of hematopoietic lineages (26), ECT2 may activate JNK through Rac1. However, we cannot rule out the possibility that ECT2 activates JNK through Cdc42 or other Rho family of GTPases, as these GTPases may be activated by ECT2 under certain conditions. We have demonstrated that activated ECT2 can induce SREor AP-1-regulated transcriptional activity. Although the activation of SRE-regulated transcription by ECT2 was efficiently inhibited by DN-Rho GTPases, AP-1-regulated activity was marginally inhibited by these mutant GTPases. This indicates that activation of AP-1-regulated transcription by ECT2 is partly attributed to the activation of Rho GTPases. Therefore, ECT2 may also activate other pathways to stimulate AP-1- mediated transcription. It has been reported that microinjection of porcine aortic endothelial cells with an oncogenic form of mouse ect2 induced lamellipodia formation (27). We also observed a similar phenotype in COS cells expressing ECT2- N5 (data not shown). However, a population of NIH 3T3 fibroblasts expressing ECT2- N5 exhibited enhanced stress fiber formation. These results may suggest that different Rho GTPases are activated by ECT2- N5 in different cell types. We also found that the majority of the NIH 3T3 cells expressing ECT2- N5 were completely rounded up and actin stress fibers appeared to have been disrupted. These results were consistent with the previous observations that ECT2-induced foci contained both rounded cells and fusiforms (23). Time-lapse microscopy analysis revealed that the morphological change upon ECT2- N5 expression is a dynamic event, which oscillates between rounded and partially flatten cell shapes. It is possible that activation of Rho GTPases was controlled in a temporal manner: upon the expression of oncogenic ECT2, Rho might be activated to induce stress fibers, but at a later stage these stress fibers might have been disrupted by unknown mechanisms, which can generate rounded cells. Because untransformed cells also round up immediately before cell division in their normal cell cycle, it would be of interest to investigate whether or not the cytokinesis regulator ECT2 has an additional role in the control of cell rounding. We utilized focus formation assays to determine the region that regulates the oncogenic activity of ECT2. We found that FIG. 11. A model of ECT2 activation and malignant transformation. The catalytic domain (DH) of ECT2 is inhibited by the binding of the N-terminal domain. ECT2 cannot activate Rho GTPases, because it is sequestered in the nucleus in interphase cells (inactive, nuclear). An ECT2 derivative lacking the N-terminal domain, but retaining the S domain, may be active, but it still cannot activate the Rho GTPases, because it localizes in the nucleus (active, nuclear). An ECT2 derivative having the S deletion lacks two NLSs and, therefore, becomes partially cytoplasmic (active, partially cytoplasmic). This allows the ECT2 derivative to activate Rho GTPases in the cytoplasm and thus causes morphological transformation of the cells (lower part). mutations in the conserved amino acids in the DH domain efficiently abolished the transforming activity of ECT2. These results indicate that the activation of Rho GTPases is critical for cell transformation by ECT2. This conclusion is further supported by the finding that the transforming activity of ECT2 was efficiently inhibited by DN Rho GTPases. The DN RhoA, Rac1, and Cdc42 inhibited ECT2 transformation at a similar level and no significant difference was observed among the GTPases. Because DN GTPases were thought to tightly bind their exchange factors to inhibit downstream signaling (28), these results are consistent with our previous findings that ECT2 activates RhoA, Rac1, and Cdc42 in vitro. We found that deletion of cell cycle regulator-related domains at the N-terminal half of ECT2 alone did not activate the transforming activity. Unexpectedly, deletion of the S domain was a critical factor for the transforming activity. The S domain contains two tandem nuclear localization signals and the ECT2 derivatives lacking the S domain partially localized in the cytoplasm. Because Rho proteins are known to localize in the cytoplasm and membrane fractions, a spill over of ECT2 into the cytoplasm might result in untimely Rho activation and eventually cause malignant transformation. The result that the ECT2 derivatives containing mutations at the NLSs in the S domain partially localized to the cytoplasm and exhibited an elevated level of the transforming activity further suggests the cytoplasmic localization of ECT2 as a major cause of the transforming activity. However, the activation level of the transforming activity of ECT2 by the NLS mutants was very weak when compared with that of ECT2- N5. Therefore, cytoplasmic localization itself might not be sufficient to fully activate the transforming activity of ECT2. The introduction of the NLS mutants into the N-terminally truncated ECT2, N4, dramatically induced its transforming activity (Fig. 9). These results suggest that, although the deletion of the N-terminal cell cycle