Leukemia-associated Rho Guanine Nucleotide Exchange Factor, a ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 29, Issue of July 20, pp. 27145–27151, 2001 Printed in U.S.A.

Leukemia-associated Rho Guanine Nucleotide Exchange Factor, a Dbl Family Protein Found Mutated in Leukemia, Causes Transformation by Activation of RhoA* Received for publication, April 20, 2001, and in revised form, May 18, 2001 Published, JBC Papers in Press, May 23, 2001, DOI 10.1074/jbc.M103565200

Gary W. Reuther,a,b Que T. Lambert,a Michelle A. Booden,a,c Krister Wennerberg,a,d Brian Becknell,e,f Guido Marcucci,e John Sondek,a, g Michael A. Caligiuri,e,h and Channing J. Dera From the aDepartment of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295 and the eDivision of Hematology and Oncology, Department of Internal Medicine, Division of Human Cancer Genetics and Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, College of Medicine and Public Health, Columbus, Ohio 43210

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH2-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias. * This work was supported in part by National Institutes of Health Grants CA42978, CA55008, and CA63071 (all to C. J. D.); RO1GM57391 (to J. S.); and KO8-CA90469 (to G. M.). 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. b Supported by a Cancer Research Institute/Merrill Lynch fellowship. To whom correspondence should be addressed: University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, CB 7295, Chapel Hill, NC 27599-7295. Tel.: 919-962-1057; Fax: 919-9660162; E-mail: [email protected]. c Supported by a University of North Carolina Lineberger Comprehensive Cancer Center fellowship. d Supported by Grant 99/697 from the Swedish Foundation for International Cooperation in Research and Higher Education. f Supported by the Medical Scientist Program and Bennett fellowships from Ohio State University. g Supported by the Pew Charitable Trusts. h Supported by the Coleman Leukemia Research Fund. This paper is available on line at http://www.jbc.org

Dbl family proteins are guanine nucleotide exchange factors (GEFs)1 for the Rho family of small GTPases (1). To date, at least 18 human Rho GTPases have been identified, with RhoA, Rac1, and Cdc42 being the most widely studied and characterized (2, 3). Rho proteins are molecular switches that are active and transduce downstream signals when they are bound to GTP and are inactive when bound to GDP. Dbl proteins activate Rho proteins by catalyzing the exchange of GDP for GTP bound to Rho and are selective toward specific Rho family members (1). Signaling pathways regulated by Rho proteins control cell cycle progression, transcription, and actin cytoskeletal arrangement. Hence, it is not surprising that the aberrant activation of Rho GTPases has been shown to promote the uncontrolled growth as well as the invasive and metastatic properties of tumor cells (2, 3). All Dbl family proteins contain a tandem Dbl homology (DH) domain/pleckstrin homology (PH) domain structure (1). The DH domain is the catalytic region of the protein, whereas the PH domain regulates the DH domain as well as the subcellular localization of the Dbl protein (1, 4). Whereas some DH domains act as GEFs for specific Rho GTPases, others show broad activity and can cause activation of multiple Rho GTPases. For example, Vav can act as a GEF for RhoA, RhoG, Rac1, and Cdc42 (5– 8), whereas Tiam1 is a specific activator of Rac1 (9), p115 RhoGEF/Lsc is an activator of RhoA (10), and Fgd1 is a specific activator of Cdc42 (11). To date, what DH domain residues dictate GTPase specificity has not been determined. Many Dbl family proteins were identified originally in gene transfer screening studies as novel oncoproteins that cause transformation of NIH3T3 cells (e.g. Dbl (1), Vav (12), Ect2 (13), Lfc (14), and Lsc (15)). For a number of Dbl family proteins, activation of transforming activity was a result of either amino-terminal (Dbl, Vav, Ect2, etc.) (1) or carboxyl-terminal truncation (Lbc) (16) of sequences that flank the DH/PH domains. Although some of these screens involved the analyses of DNA from tumor cell lines, the rearrangements that led to activation of transforming activity were due to artifacts of the

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; DH, Dbl homology; PH, pleckstrin homology; LARG, leukemia-associated Rho guanine nucleotide exchange factor; MLL, mixed-lineage leukemia; RGS, regulator of G protein signaling; AML, acute myeloid leukemia; JNK, Jun NH2-terminal kinase; SRF, serum response factor; GST, glutathione S-transferase; GPCR, G protein-coupled receptor; PCR, polymerase chain reaction; HA,. hemagglutinin; WT, wild type; mant, N-methylanthraniloyl; RBD, Ras/Rho binding domain.

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LARG Is a RhoA Activator

transfection procedure. One Dbl family protein, BCR, is rearranged in the leukemia-associated BCR-Abl translocation gene product (1). However, the transforming function of this oncoprotein has been attributed to the Abl tyrosine kinase portion rather than the rearranged BCR sequences (17). Thus, to date, no Dbl family protein has been found to be aberrantly activated in human cancers. LARG is a Dbl family member that was identified as a fusion partner with MLL in a patient with acute myeloid leukemia (AML) (18). In addition to a DH and a PH domain, LARG contains a regulator of G-protein signaling (RGS) box, suggesting that it may bind and function as a GTPase-activating protein toward ␣ subunits of heterotrimeric G proteins (19, 20). The presence of the RGS domain also suggests that LARG may link G protein-coupled receptor (GPCR)-mediated signaling with Rho GTPases. p115-RhoGEF also contains an RGS box, and G␣13 association with this domain promotes p115-RhoGEF activation of RhoA (21). LARG also contains a PDZ domain that is likely involved in protein-protein interactions (22). Since the catalytic activity of Dbl proteins can be activated by aminoterminal truncation, the putative exchange activity of LARG may be altered in the MLL-LARG fusion, where the aminoterminal end of LARG is deleted. Consequently, as Rho proteins are known to regulate cellular growth and transformation it, is possible that deregulation of these proteins by mutations in Dbl family proteins plays a role in carcinogenesis. Since not all Dbl family proteins are functional GEFs, whether LARG is an activator of a specific Rho GTPase(s) and whether LARG exhibits growth-promoting activity has not been determined. In this study, we determined that LARG is a specific activator of RhoA, and not Rac1 or Cdc42, in cells and that overexpression of LARG can promote growth transformation. These observations suggest that aberrant LARG and RhoA function may contribute to the development of AML. EXPERIMENTAL PROCEDURES

Molecular Constructs—A human prostate cDNA library (CLONTECH) clone containing the entire 4635-base pair LARG open reading frame was modified to include BamHI restriction sites at 5⬘ and 3⬘ ends by the polymerase chain reaction (PCR) (Expand PCR system, Roche Molecular Biochemicals). The modified cDNA was cloned into the BamHI site of pBluescript (Stratagene) and sequenced completely. A truncated LARG cDNA representing nucleotides 925– 4635 in the published sequence (GenBank娂 accession no. NM_015313) was generated and encodes the portion of LARG retained in the MLL-LARG chimeric gene identified (designated ⌬N308 LARG) (18). This cDNA was modified by PCR to include flanking BamHI sites. The LARG cDNA was further truncated to include nucleotides 2263– 4635 in the published sequence and encodes an amino-terminal truncated protein that lacks essentially all sequences upstream of the DH/PH domains (designated ⌬N754 LARG). This cDNA was modified by PCR to include flanking BamHI sites. Following complete sequencing, the cDNAs for LARG, ⌬N308 LARG, and ⌬N754 LARG were cloned into the BamHI site of the pCGN-hyg eukaryotic expression vector, which is a derivative of pCGN (23). This provided the attachment of an amino-terminal hemagglutinin (HA) epitope tag to the amino terminus of each LARG protein. These cDNAs were also cloned into the BamHI site of pZBE-HA (a gift from Adrienne D. Cox, University of North Carolina at Chapel Hill, Chapel Hill, NC), a derivative of the pZIP-NeoSV(x)1 eukaryotic expression vector (24) containing the coding sequence for a HA epitope tag upstream of the BamHI site. pGEX expression vectors encoding GST fusion proteins of RhoA(17A), Rac1(15A), and Cdc42(15A) were created by site-directed mutagenesis (Stratagene, Inc.). cDNA sequences encoding either the LARG DH (residues 785–1019) domain or the DH/PH (residues 785–1140) domains or human Vav2 DH/PH/CRD (residues 191–573) were generated by PCR and inserted into the NcoI/XhoI sites of the bacterial expression vector pET-28a (Novagen). Transcriptional Reporter and Transformation Assays—NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, and 293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The SRE-

Luc (25), Gal-Jun-(1–223), and 5xGal-Luc (26) luciferase reporter plasmids have been described previously. pZIP-raf1(Y340D) (27) encodes a point mutation-activated mutant of human Raf-1. pAX142-⌬N186vav (28), pAX142-dbl-HA1 (29), and pcDNA3-tiam1 (c1199) (provided by Gideon Bollag, Onyx Pharmaceuticals) encode amino-terminal truncated and constitutively activated mutants of the mouse Vav1, mouse Dbl, and human Tiam1 Dbl family proteins, respectively. Both the transcriptional reporter assays (30) and cell transformation assays (31, 32) were done in NIH3T3 cells by a standard calcium phosphate transfection procedure as described previously. Guanine Nucleotide Exchange Assays—The pET-28a bacterial expression constructs encoding the LARG DH domain and the LARG DH/PH domains were transformed into the Escherichia coli strain BL21 (DE3), and protein expression was induced with 0.5 mM isopropyl-1thio-␤-D-galactopyranoside at 22 °C. The recombinant proteins were His6-tagged at their carboxyl terminus and were purified from bacterial lysate on a nickel-nitrilotriacetic acid-agarose column (Qiagen) (33). Bacterially expressed RhoA(WT), Rac1(WT), and Cdc42(WT) proteins were produced essentially as described (34). GST-RhoE(WT) and GSTRhoG(WT) proteins were kindly provided by K. Burridge (University of North Carolina at Chapel Hill, Chapel Hill, NC). His6-TC10(WT) was expressed from pET-19b (35) (provided by Gretchen Murphy, University of North Carolina at Chapel Hill, Chapel Hill, NC) in bacteria and purified essentially as described (33). Fluorescence spectroscopic analysis of N-methylanthraniloyl (mant)GDP incorporation into GDP-preloaded Rac1, Cdc42, RhoA, RhoG, RhoE, and TC10 was carried out using a PerkinElmer Life Sciences LS 50 B Spectrometer at 20 °C essentially as described (36). Exchange reaction mixtures containing 20 mM Tris, pH 7.5, 50 mM NaCl, 10% glycerol, 400 nM mant-GDP (Biomol), and 2 ␮M GTPase were prepared and allowed to equilibrate with continuous stirring. After equilibration (300 s), each LARG or Vav2 polypeptide was added to 100 nM, and the relative mant fluorescence (␭ex ⫽ 360 nm, ␭em ⫽ 440 nm) was monitored. All experiments were performed in duplicate. Affinity Precipitation of Dbl Family Proteins—A 70% confluent 100 mm dish of NIH3T3 cells was transfected with pCGN-⌬N754 LARG by using LipofectAMINE Plus (Life Technologies, Inc.). Twenty-four hours after transfection, the cells were lysed with 150 mM NaCl, 50 mM Tris, pH 7.6, 2 mM MgCl2, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin. The lysates were cleared by centrifugation at 16,000 ⫻ g for two min and split into four aliquots (250 ␮g of protein/sample). Thirty ␮g of either bacterially expressed GST, GST-Cdc42(15A), GST-Rac1(15A), or GST-RhoA(17A) immobilized on glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) were added to each sample. The samples were rotated at 4 °C for 45 min, and the beads were washed three times with lysis buffer. Affinity-precipitated proteins were eluted in protein sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-HA antibodies. Measurement of Rho Protein Activation in Vivo—Activation of Rho proteins in vivo was determined by using a modification of the assay originally described by Taylor and Shalloway (37) for the measurement of Ras activation (38 – 42). pGEX expression vectors encoding GST fusion proteins that contain the isolated GTP-dependent binding domains of the Rac and Cdc42 effector PAK1 (amino acids 70 –132 of PAK1; PAK-RBD) (provided by Wang Lu and Bruce Mayer, Harvard, Cambridge, MA) or the RhoA effector rhotekin (amino acids 7– 89 of rhotekin; rhotekin-RBD) (43) (provided by Keith Burridge, University of North Carolina at Chapel Hill, Chapel Hill, NC) were used for the bacterial expression of GST fusion proteins, which were isolated by a procedure described previously (44). GST-rhotekin-RBD fusion (45) was used to specifically affinity precipitate activated RhoA-GTP from cell lysates (43). GST-PAK-RBD fusion protein was used to affinity-precipitate activated Rac1-GTP and Cdc42-GTP from cell lysates. Briefly, 7 h after transfection of 293T cells, these cells were placed in medium containing 0.1% fetal bovine serum. Twenty-four hours after transfection, cells were lysed in 25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 10 mM MgCl2, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin. Lysates were clarified by centrifugation at 16,000 ⫻ g for 10 min. GST-PAK and GST-rhotekin fusion proteins immobilized on glutathione-agarose beads (Sigma) were incubated with 100 –200 ␮g of cell lysates in a final volume of 0.5 ml for 30 min at 4 °C. The beads were washed twice with lysis buffer, and bound proteins were eluted in protein sample buffer and analyzed by SDSpolyacrylamide gel electrophoresis and Western blotting. The following antibodies were used for Western blot analyses to verify expression of transfected genes: anti-HA (16B12, Covance Research Products), antiMyc (9E10, Roche Molecular Biochemicals) anti-RhoA (sc-418), and


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FIG. 1. Expression of LARG in NIH3T3 cells leads to activation of SRF but does not activate JNK. A. Schematic representations of LARG, ⌬N308 LARG, and ⌬N754 LARG are shown. HA, hemagglutinin tag; PDZ, postsynaptic density disc-large zo-1 domain; RGS, regulator of G protein signaling domain; NLS, nuclear localization signal; DH, Dbl homology domain; PH, pleckstrin homology domain. B, expression plasmids (0.5 ␮g) encoding LARG proteins were co-transfected into NIH3T3 cells with a luciferase reporter plasmid (0.5 ␮g) that is responsive to activation of serum response factor. All reporter assays utilized the pCGN-hyg expression vector and were done in six-well plates in duplicate. The next day, cells were placed in 0.5% calf serum for an additional 18 h. Cell lysates were then analyzed for luciferase activity. Data shown represent the average of two to four experiments (⫾ standard error of the mean). C, expression plasmids (0.5 ␮g) encoding LARG proteins were co-transfected into NIH3T3 cells with a 5xGal-luciferase (1.25 ␮g)/Gal-Jun (0.125 ␮g) two-plasmid system that measures activation of JNK. Cells were treated and luciferase activity was analyzed as in B. Data represent the average of two experiments (⫾ the standard error of the mean) performed in duplicate.

anti-Cdc42 (sc-87) (Santa Cruz Biotechnology), and anti-Rac1 (Upstate Biotechnology). RESULTS AND DISCUSSION

LARG Is an Activator of Serum Response Factor but Not the JNK Mitogen-activated Protein Kinase Cascade—LARG is a Dbl family protein that was identified as a fusion partner with MLL in a patient with AML (18). LARG contains many previously identified functional domains including a PDZ domain (22, 46), an RGS domain (19, 20), a tandem DH/PH domain structure found in all Dbl family members (1), as well as a putative nuclear localization signal (Fig. 1A). LARG resembles two other Dbl family members: p115RhoGEF/Lsc (10), which has an RGS domain; and PDZ-RhoGEF (47, 48) which has both a PDZ domain and an RGS domain. In order to characterize LARG function, we introduced the cDNA encoding LARG into the pCGN-hyg eukaryotic expression vector. This also facilitated the attachment of an HA epitope tag at the amino terminus of the protein. In addition, we also generated expression vectors encoding two amino-terminally truncated versions of LARG. ⌬N308 LARG represents the portion of LARG still present in the AML-associated MLL-LARG fusion protein (18). ⌬N754 LARG is similar to an activating deletion mutation of PDZ-RhoGEF (Fig. 1A) (48). Since LARG contains the DH/PH domain structures found in all members of the Dbl family of Rho protein activators (1), we first investigated the ability of these LARG proteins to activate signaling pathways known to be stimulated by Rho GTPase activation (49 –51). In transient transfection assays, LARG activated the serum response factor (SRF) (52) as measured by a luciferase reporter gene that is only responsive to SRF activity (Fig. 1B). RhoA, Rac1, and Cdc42 have all been shown to activate SRF (49). Thus, unlike our observations with BCR, another Dbl family member mutated in leukemias, where we

failed to observe activation of SRF,2 we did find that LARG can mediate signaling activities shared with Rho GTPases. Amino-terminal truncation of Dbl family members often results in constitutively activated mutants of these proteins (1). However, we were surprised to find that amino-terminal truncation did not enhance LARG-mediated SRF activity (Fig. 1B). Unlike what has been observed with p115-RhoGEF (21) and PDZ-RhoGEF (48), deletion of the RGS domain did not result in variants of LARG with enhanced signaling activity. These data also indicate that the ability of LARG to activate known Rhoresponsive signaling pathways does not depend on functional PDZ or RGS domains. Activation of Rac1 and Cdc42, but not RhoA (50, 51), RhoD,3 or RhoE,4 causes activation of the JNK family of mitogenactivated protein kinases. These kinases phosphorylate and activate the c-Jun transcription factor (53, 54). In order to determine if LARG activates JNK, a Gal-Jun reporter system was utilized in which the Jun transactivation region is fused to a Gal4 DNA binding domain (26). Activation of JNK leads to the phosphorylation of the transactivation domain leading to activation of the reporter. LARG was unable to activate Gal-Jun, suggesting Rac1 and Cdc42 are not downstream targets of LARG (Fig. 1C). LARG also did not activate extracellular signal-regulated kinase, suggesting it is not an activator of Ras.5 These data suggest that LARG may target other Rho family members that are not upstream activators of JNK. LARG Is a RhoA-specific GEF —We next used three approaches to evaluate the ability of LARG to interact with and activate specific Rho GTPases. First, we expressed recombi2

G. W. Reuther and C. J. Der, unpublished observations. K. Rogers-Graham and C. J. Der, unpublished observations. R. Jain and C. J. Der, unpublished observations. 5 G. W. Reuther, Q. T. Lambert, and C. J. Der, unpublished observations. 3 4

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FIG. 2. The LARG DH domain and DH/PH domains stimulate the incorporation of mant-GDP into RhoA, but not Cdc42 or Rac1, in vitro. The ability of bacterially expressed LARG DH, LARG DH/PH, and Vav2 DH/PH/CRD (100 nM) to stimulate the incorporation of mantGDP into bacterially expressed RhoA (A), Cdc42 (B), and Rac1 (C) (2 ␮M) was measured by fluorescence spectroscopy (␭ex ⫽ 360 nm, ␭em ⫽ 440 nm). Arrows indicate the time point (300 s) at which the Dbl polypeptides were added to the exchange reaction mixture. GTPase alone indicates no Dbl polypeptides were added and shows the uncatalyzed incorporation of mant-GTP. Results are representative of two independent assays.

nant protein corresponding to the isolated DH domain and the DH/PH domains of LARG and we performed in vitro exchange assays using bacterially expressed RhoA, Rac1, and Cdc42. These assays utilized fluorescence spectroscopy to measure the incorporation of mant-GDP into bacterially expressed GTPases. We found that the DH domain of LARG functioned as a strong activator of RhoA in these assays (Fig. 2A). Increased activity was seen with the DH/PH domain, suggesting the PH domain of LARG may influence the intrinsic catalytic activity of the DH domain or affect binding to RhoA (Fig. 2A). The LARG DH domain did not catalyze the incorporation of mantGDP into Cdc42 (Fig. 2B), Rac1 (Fig. 2C), RhoE, RhoG, or TC10 in these assays.6 Although it appeared that addition of the LARG DH domain increased mant-GDP incorporation into Cdc42 (Fig. 2B), the slope of the curve of fluorescence versus time does not increase, suggesting the LARG DH domain exhibits no catalytic activity in this reaction. Second, we determined whether specific Rho family members are capable of binding to LARG. For these analyses, we generated mutant versions of RhoA, Rac1, and Cdc42 that harbor a mutation analogous to the 15A mutation that renders Ras deficient in GTP/GDP binding (55). GEFs show preferential binding to the nucleotide-free state of their target GTPases (56). GST fusion proteins of these mutant GTPases were then tested for their ability to interact with LARG. ⌬N754 LARG was transiently expressed in NIH3T3 cells and tested for its ability to bind to and be affinity precipitated by these nucleotide-free Rho family proteins immobilized on Sepharose beads. We found that LARG formed a stable complex with RhoA(17A) but not Rac1(15A) or Cdc42(15A) (Fig. 3). Vav2, which can activate RhoA, Rac1, and Cdc42 (36, 43), interacted with all three of these nucleotide-free GTPases in this assay.7 Finally, we determined the ability of LARG to activate Rho family proteins in cells using recently developed pull down assays. These assays involve the use of GST fusion proteins that contain the GTP-dependent binding domains (RBDs) from effector proteins that bind the various Rho GTPases (39 – 42). Rhotekin interacts with GTP-bound RhoA, but not Rac1 or Cdc42 (45). Conversely, the PAK serine/threonine kinase interacts with activated Rac1 and Cdc42, but not RhoA (57–59). 6 7

M. A. Booden and C. J. Der, unpublished observations. K. Wennerberg and C. J. Der, unpublished observations.

FIG. 3. LARG interacts with RhoA but not Rac1 or Cdc42. LARG binds to nucleotide-free RhoA, but not nucleotide-free Rac1 or Cdc42, in vitro. pCGN-⌬N754 LARG was transfected into 293T cells. Twenty-four hours later, transfected cells were lysed and lysates were incubated with GST, GST-Rac1(15A), GST-Cdc42(15A), and GSTRhoA(17A) immobilized on glutathione-Sepharose beads. Following washing, bound ⌬N754 LARG was detected by Western blotting with anti-HA antibodies. The migration of molecular size standards is indicated in kDa.

LARG proteins were expressed in 293T cells, and RhoA activation was assayed by affinity precipitation with GST-rhotekinRBD. Following affinity precipitation, Western blot analysis using a RhoA-specific antibody determined that expression of LARG caused a significant increase in the amount of activated GTP-bound endogenous RhoA (Fig. 4, A and B). In contrast, when a GST-PAK-RBD fusion protein was used to affinityprecipitate activated Rac1 and Cdc42, we found that expression of LARG in 293T cells did not lead to an increase in activated Rac1 or Cdc42 (Fig. 4B). In the same experiment, expression of the Rac1 activators, Vav1 (6) and Tiam1 (9) led to an increase in activated, GTP-bound, endogenous Rac1. Similarly, expression of Dbl, an activator of RhoA, Rac1, and Cdc42 (60, 61), caused activation of these GTPases. Our results with Vav1, Tiam1, and Dbl in this assay are in agreement with the established GTPase specificities of these Dbl family proteins. Our signaling analyses indicated that the deletion of amino-


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FIG. 4. LARG activates RhoA but not Rac1 or Cdc42 in cells. A, expression of LARG in 293T cells leads to an increase in the level of RhoA-GTP. Expression plasmids (1 ␮g) containing cDNAs that encode LARG, ⌬N308 LARG, and ⌬N754 LARG were transfected into 293T cells. After overnight culturing in low serum (0.1%), cells were lysed and the lysates were used in GST pull-down assays using GST-rhotekin-RBD immobilized on glutathione-agarose beads. Bound proteins and total cell lysates were analyzed by Western blotting with anti-RhoA antibodies (bottom two panels) and LARG was detected by Western blotting of total cell lysates with anti-HA antibodies (top). B, amino terminal-truncation does not enhance LARG-stimulated RhoA-GTP formation and LARG does not activate Cdc42 or Rac1. Expression plasmids containing cDNAs that encode LARG (5 ␮g), ⌬N308 LARG (1 ␮g), ⌬N754 LARG (0.1 ␮g), ⌬N186 Vav1 (1 ␮g), activated Tiam1 (C1199) (1 ␮g), and activated Dbl (1 ␮g) were transfected into 293T cells. After overnight culturing in low serum (0.1%), cells were lysed and the lysates were used in GST pull-down assays using GST-rhotekin-RBD or GST-PAK-RBD immobilized on glutathione-agarose beads. Bound proteins and total cell lysates were analyzed by Western blotting with anti-RhoA, anti-Rac1, and anti-Cdc42 antibodies as indicated (lower panels). The expression of Dbl family proteins was analyzed by Western blotting of total cell lysates with anti-HA antibodies and anti-Myc epitope tag antibodies (for Tiam1) (top panel). The migration of molecular size standards is indicated in kDa.

FIG. 5. LARG cooperates with Raf1(Y340D) to transform NIH3T3 cells. pZBE-HA expression vector (1 ␮g) that contains cDNA sequences encoding fulllength LARG (FL), ⌬N308 LARG, or ⌬N754 LARG were co-transfected with the empty pZIP-NeoSV(x)1 expression vector or this vector encoding activated Raf-1(Y340D) (pZIP-raf-1(Y340D)) (0.5 ␮g) into NIH3T3 cells. pZIP-rhoA(63L) (0.5 ␮g) was used as a control for transformation cooperation with Raf(Y340D). Cells were fed three times per week to allow primary foci of transformed cells to form. Primary focus-forming activity was determined 18 days after transfection through a Nikon phase contrast inverted microscope. The data shown represent the average number of foci (⫾ the standard error of the mean) of two experiments performed in duplicate.

terminal sequences did not enhance LARG function (Fig. 1B). To further evaluate the role of the amino-terminal sequences in regulating LARG function, we compared the ability of the wild type and truncated proteins to stimulate formation of RhoAGTP. For our initial analyses, we transfected an equal mass of plasmid DNA encoding the different LARG proteins (Fig. 4A). We consistently observed that full-length LARG was expressed

at much lower levels than ⌬N754 LARG. This was confirmed by metabolic labeling of cells expressing LARG proteins.2 Therefore, we altered the amount of LARG DNA used to account for this different expression, so that equivalent levels of protein expression for each LARG protein can be compared (Fig. 4B). In this experiment, we saw equivalent levels of LARG protein expression, which in turn caused similar levels of RhoA-GTP

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activation (Fig. 4B). The amount of LARG-mediated RhoA activation observed in these experiments is directly related to the amount of LARG expression. Furthermore, under similar conditions to Fig. 4B where equivalent RhoA activation is seen, all three LARG proteins produced similar activation of SRF in 293T cells.2 LARG Can Promote Growth Transformation of NIH 3T3 Cells—A transforming function has been described for many, but not all, Dbl family proteins (1). To determine if LARG possesses growth-promoting activity, we utilized a cooperation focus formation assay using NIH3T3 cells. Although activated Rho GTPases lack potent focus-forming activity in NIH3T3 transformation assays, others and we have found that activated Rho family proteins can have a synergistic effect on NIH3T3 cell transformation when co-expressed with an activated form of the Raf-1 kinase (eg. RafCAAX or Raf(Y340D)) (27, 62, 63). We found that co-expression of LARG proteins with Raf(Y340D) in NIH3T3 cells resulted in synergistic focus-forming activity (Fig. 5). Expression of Raf(Y340D) or LARG alone did not induce NIH3T3 cell transformation (Fig. 5) and NIH3T3 cells stably expressing LARG proteins were not transformed.5 Additionally, the morphology of the transformed foci elicited by coexpression of Raf(Y340D) with LARG proteins resembled that induced by coexpression of Raf(Y340D) and activated RhoA.5 The lack of detectable transforming activity when LARG was expressed alone is also consistent with LARG only activating RhoA, and not Rac1 or Cdc42, in vivo. To our knowledge, this is the first demonstration of a non-transforming RhoA-specific Dbl family member that can cooperate with Raf to transform NIH3T3 cells. It is possible other RhoAspecific Dbl proteins, such as Ect2 and Lfc, actually activate other GTPases and therefore are transforming on their own. Dbl family proteins, such as Dbl and Dbs, among others, which activate multiple Rho family members typically display potent focus-forming activity alone in NIH3T3 transformation assays (29). These data, when taken together with our demonstration that LARG is a RhoA-specific GEF, suggest that LARG, when coexpressed with Raf(Y340D), causes transformation by sustained activation of RhoA. Additionally, since we have failed to detect a transforming activity for BCR or any activity of the BCR DH domain,2 these results establish LARG as the first Dbl family protein whose translocation in human cancers may promote oncogenesis by activation of Rho GTPases. p115RhoGEF and PDZ-RhoGEF, like LARG, also contain RGS domains (47, 48, 64). Hence, these Dbl family proteins may link GPCRs with RhoA. The RGS domain of p115-RhoGEF has been shown to function as a GTPase activating protein that specifically inactivates G␣12 and G␣13 (64). Additionally, GTPbound G␣13, but not G␣12, binding to the RGS domain stimulates the GEF activity of p115-RhoGEF in vitro (21). Thus, p115-RhoGEF may serve as a link that promotes RhoA activation by GPCRs that stimulate G␣13 activation. Activated versions of G␣12 and G␣13 have both been shown to activate RhoA (65, 66). G␣q may also play a role in the activation of RhoA by GPCRs (67– 69). However, a Dbl family protein that serves as a mediator of RhoA activation by G␣12 or G␣q has not been determined. Our current studies are evaluating the possibility that LARG may promote such connections. During the course of our studies, Gutkind and colleagues showed that LARG activated RhoA, that the RGS domain of LARG interacted with both G␣12 and G␣13, and that the RGS domain could block GPCR signaling (70). However, although we have detected G␣12 and G␣13 interactions with the RGS domain of LARG, we could not augment the ability of LARG to activate RhoA by co-expressing activated forms of G␣12 or G␣13

with LARG in cells.8 Thus, there are no experimental data from our studies or those by Fukuhara et al. (70) that indicate G␣12 or G␣13 mediates activation of LARG in vivo. It is possible that other G␣ proteins interact with the RGS domain of LARG and alter the activity of the LARG DH domain. It is intriguing to postulate that expression of an MLL-LARG fusion protein in a patient with AML resulted in aberrant activation of this RhoA activator and that the subsequent RhoA activation played a mechanistic signaling role in the development of the leukemic state (18). This notion is supported by the observation of the inactivation of a GAP for RhoA in leukemia. Borkhardt et al. (71) described the formation of an MLL fusion protein with GRAF, which functions as a GAP for RhoA. This fusion results in the deletion of the GAP domain of GRAF. These analyses also identified point mutations in the GAP domain of GRAF, as well as mutations that caused truncated forms of GRAF in the remaining undeleted GRAF allele of several patients. Inactivation of a RhoA GAP, which functions to convert active GTP-bound Rho proteins to the inactive GDPbound form, would potentially have a similar RhoA-activating effect as activation of a RhoA GEF such as LARG. It is possible that LARG protein is not normally expressed highly in hematopoietic cells and that fusion to MLL resulted in increased levels of this RhoA activator. MLL fusion to LARG may eliminate putative amino-terminal sequences of LARG that regulate expression level and thus lead to aberrant LARG protein levels. An amino-terminally truncated form of Dbl has a longer half-life than the full-length protein, suggesting higher levels of expression may, in part, play a role in transformation by oncogenic Dbl (72). We have consistently found that amino-terminally truncated forms of LARG are expressed at higher steady-state protein levels when compared with full-length LARG. However, pulsechase analyses did not indicate that amino-terminal truncation caused a significant increase in the half-life of the protein.2 Additionally, we did identify putative PEST sequences (73) in the amino terminus of LARG, but our preliminary analyses failed to indicate that the full-length protein was targeted for ubiquitin-mediated proteolysis to account for its reduced level of protein expression. Thus, we presently have no clear explanation for why amino-terminal truncated LARG is expressed at higher levels. Additionally, MLLLARG would be under the control of the MLL promoter and not the promoter for LARG (18), thereby altering expression control of LARG. It is also possible that fusion of MLL to LARG alters the subcellular localization of LARG, affecting its ability to activate RhoA. Development of further reagents such as LARG antibodies and an MLL-LARG cDNA, which has proven difficult to clone because of its size, would be required to further investigate these possibilities. Acknowledgments—We thank Adrienne D. Cox, Bruce Mayer, Gideon Bollag, Keith Burridge, and Gretchen Murphy for the gifts of plasmids and proteins. REFERENCES 1. Whitehead, I. P., Campbell, S., Rossman, K. L., and Der, C. J. (1997) Biochim. Biophys. Acta 1332, F1–F23 2. Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Oncogene 17, 1415–1438 3. Bishop, A. L., and Hall, A. (2000) Biochem. J. 348, 241–255 4. Lemmon, M. A., and Ferguson, K. M. (2000) Biochem. J. 350, 1–18 5. Han, J., Das, B., Wei, W., Van Aelst, L., Mosteller, R. D., Khosravi-Far, R., Westwick, J. K., Der, C. J., and Broek, D. (1997) Mol. Cell. Biol. 17, 1346 –1353 6. Crespo, P., Schuebel, K. E., Ostrom, A. A., Gutkind, J. S., and Bustelo, X. R. (1997) Nature 385, 169 –172 7. Han, J., Luby-Phelps, K., Das, B., Shu, X., Xia, Y., Mosteller, R. D., Krishna,

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