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• W2093968022 abstract "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. 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. guanine nucleotide exchange factor Dbl homology pleckstrin homology leukemia-associated Rho guanine nucleotide exchange factor mixed-lineage leukemia regulator of G protein signaling acute myeloid leukemia Jun NH2-terminal kinase serum response factor glutathione S-transferase G protein-coupled receptor polymerase chain reaction . hemagglutinin wild type N-methylanthraniloyl Ras/Rho binding domain Dbl family proteins are guanine nucleotide exchange factors (GEFs)1 for the Rho family of small GTPases (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). To date, at least 18 human Rho GTPases have been identified, with RhoA, Rac1, and Cdc42 being the most widely studied and characterized (2Zohn I.M. Campbell S.L. Khosravi-Far R. Rossman K.L. Der C.J. Oncogene. 1998; 17: 1415-1438Crossref PubMed Scopus (319) Google Scholar, 3Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1655) Google Scholar). 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 (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). 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 (2Zohn I.M. Campbell S.L. Khosravi-Far R. Rossman K.L. Der C.J. Oncogene. 1998; 17: 1415-1438Crossref PubMed Scopus (319) Google Scholar, 3Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1655) Google Scholar). All Dbl family proteins contain a tandem Dbl homology (DH) domain/pleckstrin homology (PH) domain structure (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). 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 (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar, 4Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (612) Google Scholar). 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 (5Han J. Das B. Wei W. Van Aelst L. Mosteller R.D. Khosravi-Far R. Westwick J.K. Der C.J. Broek D. Mol. Cell. Biol. 1997; 17: 1346-1353Crossref PubMed Scopus (276) Google Scholar, 6Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (676) Google Scholar, 7Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (708) Google Scholar, 8Schuebel K.E. Movilla N. Rosa J.L. Bustelo X.R. EMBO J. 1998; 17: 6608-6621Crossref PubMed Scopus (223) Google Scholar), whereas Tiam1 is a specific activator of Rac1 (9van Leeuwen F.N. van der Kammen R.A. Habets G.G. Collard J.G. Oncogene. 1995; 11: 2215-2221PubMed Google Scholar), p115 RhoGEF/Lsc is an activator of RhoA (10Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and Fgd1 is a specific activator of Cdc42 (11Zheng Y. Fischer D.J. Santos M.F. Tigyi G. Pasteris N.G. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). 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 (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar), Vav (12Katzav S. Martin-Zanca D. Barbacid M. EMBO J. 1989; 8: 2283-2290Crossref PubMed Scopus (419) Google Scholar), Ect2 (13Miki T. Smith C.L. Long J.E. Eva A. Fleming T.P. Nature. 1993; 362: 462-465Crossref PubMed Scopus (255) Google Scholar), Lfc (14Whitehead I. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 270: 18388-18395Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and Lsc (15Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643-18650Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar)). For a number of Dbl family proteins, activation of transforming activity was a result of either amino-terminal (Dbl, Vav, Ect2, etc.) (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar) or carboxyl-terminal truncation (Lbc) (16Sterpetti P. Hack A.A. Bashar M.P. Park B. Cheng S.D. Knoll J.H. Urano T. Feig L.A. Toksoz D. Mol. Cell. Biol. 1999; 19: 1334-1345Crossref PubMed Scopus (68) Google Scholar) 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 transfection procedure. One Dbl family protein, BCR, is rearranged in the leukemia-associated BCR-Abl translocation gene product (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). However, the transforming function of this oncoprotein has been attributed to the Abl tyrosine kinase portion rather than the rearranged BCR sequences (17Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (353) Google Scholar). 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) (18Kourlas P.J. Strout M.P. Becknell B. Veronese M.L. Croce C.M. Theil K.S. Krahe R. Ruutu T. Knuutila S. Bloomfield C.D. Caligiuri M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2145-2150Crossref PubMed Scopus (199) Google Scholar). 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 α subunits of heterotrimeric G proteins (19Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (913) Google Scholar, 20Siderovski D.P. Strockbine B. Behe C.I. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (96) Google Scholar). 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 Gα13 association with this domain promotes p115-RhoGEF activation of RhoA (21Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar). LARG also contains a PDZ domain that is likely involved in protein-protein interactions (22Saras J. Heldin C.H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (221) Google Scholar). Since the catalytic activity of Dbl proteins can be activated by amino-terminal truncation, the putative exchange activity of LARG may be altered in the MLL-LARG fusion, where the amino-terminal 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. A human prostate cDNA library (CLONTECH) clone containing the entire 4635-base pair LARG open reading frame was modified to includeBamHI 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 truncatedLARG cDNA representing nucleotides 925–4635 in the published sequence (GenBank™ accession no. NM_015313) was generated and encodes the portion of LARG retained in theMLL-LARG chimeric gene identified (designated ΔN308 LARG) (18Kourlas P.J. Strout M.P. Becknell B. Veronese M.L. Croce C.M. Theil K.S. Krahe R. Ruutu T. Knuutila S. Bloomfield C.D. Caligiuri M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2145-2150Crossref PubMed Scopus (199) Google Scholar). 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 ΔN754 LARG). This cDNA was modified by PCR to include flankingBamHI sites. Following complete sequencing, the cDNAs for LARG, ΔN308 LARG, and ΔN754 LARG were cloned into theBamHI site of the pCGN-hyg eukaryotic expression vector, which is a derivative of pCGN (23Tanaka M. Herr W. Cell. 1990; 60: 375-386Abstract Full Text PDF PubMed Scopus (517) Google Scholar). 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 theBamHI 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 (24Cepko C.L. Roberts B.E. Mulligan R.C. Cell. 1984; 37: 1053-1062Abstract Full Text PDF PubMed Scopus (637) Google Scholar) containing the coding sequence for a HA epitope tag upstream of theBamHI 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 theNcoI/XhoI sites of the bacterial expression vector pET-28a (Novagen). 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 (25Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (384) Google Scholar), Gal-Jun-(1–223), and 5xGal-Luc (26Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (847) Google Scholar) luciferase reporter plasmids have been described previously. pZIP-raf1(Y340D) (27Khosravi-Far R. Solski P.A. Clark G.J. Kinch M.S. Der C.J. Mol. Cell. Biol. 1995; 15: 6443-6453Crossref PubMed Scopus (638) Google Scholar) encodes a point mutation-activated mutant of human Raf-1. pAX142-ΔN186vav (28Abe K. Whitehead I.P. O'Bryan J.P. Der C.J. J. Biol. Chem. 1999; 274: 30410-30418Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), pAX142-dbl-HA1 (29Westwick J.K. Lee R.J. Lambert Q.T. Symons M. Pestell R.G. Der C.J. Whitehead I.P. J. Biol. Chem. 1998; 273: 16739-16747Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), 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 (30Hauser C.A. Westwick J.K. Quilliam L.A. Methods Enzymol. 1995; 255: 412-426Crossref PubMed Scopus (45) Google Scholar) and cell transformation assays (31Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (180) Google Scholar, 32Solski P.A. Abe K. Der C.J. Methods Enzymol. 2000; 325: 425-441Crossref PubMed Google Scholar) were done in NIH3T3 cells by a standard calcium phosphate transfection procedure as described previously. 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 mmisopropyl-1-thio-β-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) (33Self A.J. Hall A. Methods Enzymol. 1995; 256: 3-10Crossref PubMed Scopus (167) Google Scholar). Bacterially expressed RhoA(WT), Rac1(WT), and Cdc42(WT) proteins were produced essentially as described (34Worthylake D.K. Rossman K.L. Sondek J. Nature. 2000; 408: 682-688Crossref PubMed Scopus (303) Google Scholar). GST-RhoE(WT) and GST-RhoG(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 (35Murphy G.A. Solski P.A. Jillian S.A. Perez de la Ossa P. D'Eustachio P. Der C.J. Rush M.G. Oncogene. 1999; 18: 3831-3845Crossref PubMed Scopus (68) Google Scholar) (provided by Gretchen Murphy, University of North Carolina at Chapel Hill, Chapel Hill, NC) in bacteria and purified essentially as described (33Self A.J. Hall A. Methods Enzymol. 1995; 256: 3-10Crossref PubMed Scopus (167) Google Scholar). 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 (36Abe K. Rossman K.L. Liu B. Ritola K.D. Chiang D. Campbell S.L. Burridge K. Der C.J. J. Biol. Chem. 2000; 275: 10141-10149Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Exchange reaction mixtures containing 20 mm Tris, pH 7.5, 50 mm NaCl, 10% glycerol, 400 nm mant-GDP (Biomol), and 2 µm 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 (λex = 360 nm, λem = 440 nm) was monitored. All experiments were performed in duplicate. A 70% confluent 100 mm dish of NIH3T3 cells was transfected with pCGN-ΔN754LARG 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 mmMgCl2, 1% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The lysates were cleared by centrifugation at 16,000 ×g for two min and split into four aliquots (250 µg of protein/sample). Thirty µg 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. Activation of Rho proteins in vivo was determined by using a modification of the assay originally described by Taylor and Shalloway (37Taylor S.J. Shalloway D. Curr. Biol. 1996; 6: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) for the measurement of Ras activation (38Reid T. Bathoorn A. Ahmadian M.R. Collard J.G. J. Biol. Chem. 1999; 274: 33587-33593Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 39Ren X.D. Schwartz M.A. Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar, 40Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1350) Google Scholar, 41Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 42Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). 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) (43Liu B.P. Burridge K. Mol. Cell. Biol. 2000; 20: 7160-7169Crossref PubMed Scopus (170) Google Scholar) (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 (44Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5028) Google Scholar). GST-rhotekin-RBD fusion (45Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) was used to specifically affinity precipitate activated RhoA-GTP from cell lysates (43Liu B.P. Burridge K. Mol. Cell. Biol. 2000; 20: 7160-7169Crossref PubMed Scopus (170) Google Scholar). 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 mmMgCl2, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Lysates were clarified by centrifugation at 16,000 × gfor 10 min. GST-PAK and GST-rhotekin fusion proteins immobilized on glutathione-agarose beads (Sigma) were incubated with 100–200 µg 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 SDS-polyacrylamide 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), anti-Myc (9E10,Roche Molecular Biochemicals) anti-RhoA (sc-418), and anti-Cdc42 (sc-87) (Santa Cruz Biotechnology), and anti-Rac1 (Upstate Biotechnology). LARG is a Dbl family protein that was identified as a fusion partner with MLL in a patient with AML (18Kourlas P.J. Strout M.P. Becknell B. Veronese M.L. Croce C.M. Theil K.S. Krahe R. Ruutu T. Knuutila S. Bloomfield C.D. Caligiuri M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2145-2150Crossref PubMed Scopus (199) Google Scholar). LARG contains many previously identified functional domains including a PDZ domain (22Saras J. Heldin C.H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 46Ponting C.P. Phillips C. Davies K.E. Blake D.J. Bioessays. 1997; 19: 469-479Crossref PubMed Scopus (352) Google Scholar), an RGS domain (19Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (913) Google Scholar, 20Siderovski D.P. Strockbine B. Behe C.I. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (96) Google Scholar), a tandem DH/PH domain structure found in all Dbl family members (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar), as well as a putative nuclear localization signal (Fig.1 A). LARG resembles two other Dbl family members: p115RhoGEF/Lsc (10Hart M.J. Sharma S. elMasry N. Qiu R.G. McCabe P. Polakis P. Bollag G. J. Biol. Chem. 1996; 271: 25452-25458Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), which has an RGS domain; and PDZ-RhoGEF (47Rumenapp U. Blomquist A. Schworer G. Schablowski H. Psoma A. Jakobs K.H. FEBS Lett. 1999; 459: 313-318Crossref PubMed Scopus (47) Google Scholar, 48Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar) 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. ΔN308 LARG represents the portion of LARG still present in the AML-associated MLL-LARG fusion protein (18Kourlas P.J. Strout M.P. Becknell B. Veronese M.L. Croce C.M. Theil K.S. Krahe R. Ruutu T. Knuutila S. Bloomfield C.D. Caligiuri M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2145-2150Crossref PubMed Scopus (199) Google Scholar). ΔN754 LARG is similar to an activating deletion mutation of PDZ-RhoGEF (Fig. 1 A) (48Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Since LARG contains the DH/PH domain structures found in all members of the Dbl family of Rho protein activators (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar), we first investigated the ability of these LARG proteins to activate signaling pathways known to be stimulated by Rho GTPase activation (49Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1197) Google Scholar, 50Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1442) Google Scholar, 51Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1555) Google Scholar). In transient transfection assays, LARG activated the serum response factor (SRF) (52Johansen F.E. Prywes R. Biochim. Biophys. Acta. 1995; 1242: 1-10Crossref PubMed Scopus (105) Google Scholar) as measured by a luciferase reporter gene that is only responsive to SRF activity (Fig. 1 B). RhoA, Rac1, and Cdc42 have all been shown to activate SRF (49Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). Thus, unlike our observations with BCR, another Dbl family member mutated in leukemias, where we failed to observe activation of SRF, 2G. W. Reuther and C. J. Der, unpublished observations. 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 (1Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar). However, we were surprised to find that amino-terminal truncation did not enhance LARG-mediated SRF activity (Fig. 1 B). Unlike what has been observed with p115-RhoGEF (21Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar) and PDZ-RhoGEF (48Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), 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 Rho-responsive signaling pathways does not depend on functional PDZ or RGS domains. Activation of Rac1 and Cdc42, but not RhoA (50Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1442) Google Scholar, 51Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1555) Google Scholar), RhoD, 3K. Rogers-Graham and C. J. Der, unpublished observations. or RhoE, 4R. Jain and C. J. Der, unpublished observations. causes activation of the JNK family of mitogen-activated protein kinases. These kinases phosphorylate and activate the c-Jun transcription factor (53Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar, 54Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar). 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 (26Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (847) Google Scholar). 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. 1 C). LARG also did not activate extracellular signal-regulated kinase, suggesting it is not an activator of Ras. 5G. W. Reuther, Q. T. Lambert, and C. J. Der, unpublished observations. These data suggest that LARG may target other Rho family members that are not upstream activators of JNK. We next used three approaches to evaluate the ability of LARG to interact with and activate specific Rho GTPases. First, we expressed recombinant protein corresponding to the isolated DH domain and the DH/PH domains of LARG and we performedin vitro exchange assays using bacterially expressed RhoA, Rac1, and Cdc42. These assays utilized fluorescence spectroscopy to measure" @default.
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• W2093968022 date "2001-07-01" @default.
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• W2093968022 title "Leukemia-associated Rho Guanine Nucleotide Exchange Factor, a Dbl Family Protein Found Mutated in Leukemia, Causes Transformation by Activation of RhoA" @default.
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• W2093968022 doi "https://doi.org/10.1074/jbc.m103565200" @default.
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