Characterization, Expression and Chromosomal Localization of a Human Gene Homologous to the Mouse Lsc Oncogene, with Strongest Expression in Hematopoetic Tissues

SHORT REPORT Characterization, expression and chromosomal localization of a human gene homologous to the mouse Lsc oncogene, with strongest expression in hematopoetic tissues

Hans-Christian Aasheim1, Florence Pedeutour2 and Erlend B Smeland1

1Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310, Oslo, Norway; 2URA CNRS 1462, Faculty de Medecine, Avenue de Valombrose, Nice, France

A human cDNA clone, denoted sub1.5, was isolated from McCormick, 1993; Quilliam et al., 1995; Denhardt, cDNA library generated from human T cells. The sub1.5 1996). The Ras family itself is involved in triggering cDNA sequence was novel and was not identical to any cell proliferation in response to mitogens and growth known cDNA sequences in the GenBank. Recently, factors via the Raf/MEK/mitogen-activated protein however, a mouse cDNA (Lsc) with high homology to kinase signalling pathway leading to a phosphoryla- sub1.5 was identi®ed, indicating that the sub1.5 sequence tion cascade which activates transcription factors to may represent the human homologue of the mouse Lsc induce gene expression (Denhardt, 1996). The Rho/Rac gene. The sub1.5 cDNA includes an open reading frame family is involved in cytoskeletal organisation and of 875 amino acids, predicting a protein with molecular focal contacs (Ridley and Hall, 1992; Ridley et al., weight of 97 kDa. Like Lsc, sub1.5 shows homology to 1992) and has been suggested to participate in growth the previous described oncogene Lbc, in particular to two factor signalling (Nobes and Hall, 1995; Coso et al., functional domains in the Lbc protein; the Dbl proto- 1995; Minden et al., 1995; Hill et al., 1995) and to be oncogene homology domain and the pleckstrin homology involved in regulation of receptor mediated endocytosis domain. Lsc is proposed to be an oncogene and is a through coated pits (Lamaze et al., 1996). member of a growing family of proteins that may The Ras superfamily of GTP-binding proteins function as activators of the Rho family GTPases. alternate between alternate active GTP-bound and Members of the Rho family regulates the polymerization inactive GDP-bound states (for review see Quilliam et of actin to produce stress ®bers. Activation of Rho al., 1995). The GTP-binding/GTPase cycles are tightly GTPases by sub1.5 is also indicated by our studies, as controlled, with guanidine nucleotide exchange factors stress ®ber formation is observed in serum-starved stable (GEFs) catalyzing their conversion to the GTP-bound NIH3T3 sub1.5 transfectants. Sub1.5 cDNA hybridizes active state and GTPase-activating proteins (GAPs) to two major transcripts of 3.5 and 5 kb size and the ensuring their return to an inactive, basal state through strongest expression is seen in hematopoietic tissues like the stimulation of GTP hydrolysis (Boguski and thymus, lymph nodes, peripheral blood leukocytes and McCormick, 1993). GEFs have been described for spleen. We also show that both puri®ed B and T cells virtually all the Ras GTPase families (Boguski and express sub1.5. In addition, our data indicate that sub1.5 McCormick, 1993). Recently, the product of the Dbl- mRNA is abundantly expressed in CD34+ human oncogene was shown to serve as a GEF for speci®c progenitor cells. Fluorescent in situ hybridisation, using members of the Rho branch of the Ras superfamily sub1.5 cDNA as a probe on human metaphases, shows (RhoA, Rac1, CDC42HS; Hart et al., 1994). It was that the sub1.5 gene is localized to chromosome shown that both the GEF activity and the transform- 19q13.13. ing activity of the Dbl protein was localized to a 240 amino acid motif between residues 498 and 738 in the Keywords: Lsc homologue; Rho GEF; Dbl-family; protein (Hart et al., 1994). An emerging group of hematopoetic expression proteins conferring homology to this 240 amino acid motif in the Dbl oncogene has been identi®ed including Vav (Katzav et al., 1989), Ect-2 (Miki et al., 1993), Lbc Members of the Ras superfamily of proteins function (Toksoz and Williams, 1994), Lfc (Whitehead et al., as molecular switches in a diversity of cellular 1995), Tim (Chan et al., 1994), Tiam (Habets et al., signalling pathways, in¯uencing processes such as 1994), Cdc24 (from Saccharomysis cerevisia; Drubin, cytoskeletal organization, development, vesicular trans- 1991), the breakpoint cluster region protein (Bcr; port, cell polarity and cell motility (Adams et al., 1990; Hariharan and Adams, 1987), and the mammalian Boguski and McCormic, 1993; Ridley et al., 1992; ras guanidine releasing factor (Shou et al., 1992). Ridley and Hall, 1992). The Ras superfamily consists All Dbl family members possess a second shared of more than 50 members that share the ability to bind domain, designated the plekcstrin homology (PH) and hydrolyze GTP. The superfamily can be divided domain. The PH domain was initially identi®ed as a into several subfamilies, designated Ras, Rho/Rac, region of sequence homology, of approximately 120 Rab, Ran, and Arf/Sar (For review see Boguski and amino acids, which is duplicated in pleckstrin (reviewed in Lemmon et al., 1996; Gibson et al., 1994). Pleckstrin is the major substrate of protein kinase C in platelets. Correspondence: H-C Aasheim Received 8 November 1996; revised 17 December 1996; accepted 17 The PH domain is found in a large variety of proteins December 1996 involved in cellular signalling and cytoskeletal func- Identification of the homologue to the Lsc oncogene H-C Aasheim et al 1748 tions. It is believed that the function of the PH domain is to target the host protein to the cell membrane, by binding to lipids or proteins (Lemmon et al., 1996), to facilitate and regulate enzymatic activities.

cDNA sequence and predicted protein structure of the sub1.5 cDNA sequence We have identi®ed and sequenced a novel cDNA encoding a putative protein with homology to two recently described members of the Dbl-family of oncogenes, Lbc and Lfc (Toksoz and Williams, 1994; Whitehead et al., 1995). A novel 3.4 kb cDNA was isolated from a human TPA-stimulated T cell cDNA library using a subtractive strategy recently described (Aasheim et al., 1994, 1996). The sequence revealed an open reading frame (ORF) of 875 amino acids, starting with an ATG codon at nucleotide 436 and terminating with a stop codon at nucleotide 3044 (data not shown, accession number Y09160). The ®rst ATG codon is in moderately good context for translation initiation with the sequence, CACCTCATGG as compared with the Kozak consensus sequence (XCXGCCATGG;Kozak, 1994). Recently, a mouse cDNA (Lsc, Whitehead et al., 1996) was published with 87% overall homology at the amino acid level to the sub1.5 sequence. The overall amino acid homology, the similar expression pattern and the transcript sizes strongly suggest that sub1.5 is the human homolog of the corresponding mouse gene. The mouse protein is denoted Lsc (Lbc's second cousin) and is suggested to be an oncogene (Whitehead et al., 1996). The Lsc cDNA was isolated based on the ability to induce strong oncogenic transformation when expressed in NIH3T3 ®broblast cells. The Lsc sequence is proposed to start at an ATG 53 amino acids upstream of the start of the sub1.5 sequence (Figure 1). Sub1.5 and Lsc diverge from nucleotide Figure 1 Comparison of the amino acid sequence of sub1.5 and number 334 in the sub1.5 sequence and 5' upstream. Lsc. The sequences were optimally aligned on the basis of identical residues (vertical lines). The DH-domain and the PH- Here, the two sequences do not show any homology to domain is localized from amino acid 359 to 710 in the sub1.5 each other. The sub1.5 sequence does not harbour an sequence ATG for translation start of the corresponding site where Lsc is proposed to start (data not shown). The sub1.5 cDNA encodes a protein with a predicted molecular weight of 97 kDa with several interesting transforming activity and the GEF activity of features. Thus, based on homology to previous known oncogenic Dbl. So far GEF activity has been shown genes, the predicted sub1.5 protein and Lsc show for Dbl (Hart et al., 1994), Ost (Horii et al., 1994), homology to the Dbl family of GEF's, and most Cdc24 (Zheng et al., 1994), Tiam-1 (Van Leeuwen et closely related to the Dbl family member Lbc, an al., 1995) and Lbc (Zheng et al., 1995), all exhibiting exchange factor with speci®city for Rho family exchange activity in vitro on Rho family GTPases. All GTPases (Toksoz and Williams, 1994; Zheng et al., DH family members, including sub1.5 and Lsc, also 1995). Like all Dbl family members, sub1.5 contains a possess a PH domain. PH domains are thought to PH domain in tandem with the DH-domain. Both anchore proteins to cell membranes, either through Sub1.5 and Lsc are distinguished from Lbc by an interaction with lipids or with proteins (Gibson et al., extended N-terminus (more than 400 amino acids) that 1994; Lemmon et al., 1996). Both sub1.5 and Lsc does not appear to be necessary for, or inhibitory to possess a PH domain. The transforming activity of cellular transformation (Whitehead et al., 1996). This both the Lfc and Lbc oncogene has been shown to be area does not align signi®cantly to any other proteins dependent of the PH domain (Whitehead et al., 1995, when performing GenBank searches. 1996) indicating that a PH-dependent recruitment of The DH-domain is found in a number of proteins Dbl family members to the plasma membrane may be a suspected to be involved in cell growth regulation. necessary step for transformation in NIH3T3 cells. These include Cdc24 (Zheng et al., 1994), Tiam-1 (Habets et al., 1994), Vav (Katzav et al., 1989), Ect2 NIH3T3 stable transfectants and stress ®ber formation (Miki et al., 1993), Ost (Horii et al., 1994), Lbc (Zheng et al., 1995) and Lfc (Whitehead et al., 1995). This Whitehead et al. (1996) have shown that the transform- domain has been shown to be both essential for the ing activity of the Lsc protein is dependent of exchange Identification of the homologue to the Lsc oncogene H-C Aasheim et al 1749 activity for Rho family GTPases. The experiments signalling pathways and are consistent with the Rho were performed by cotransfection of Lsc with p190 family exchange activity that has been attributed to GAP, which has GAP activity for several Rho family Dbl family GEF's. In mammalian cells, RhoA GTPases (RhoA, RhoB, Rac1, Rac2, and Cdc42) in in regulates actin stress ®ber formation and focal vitro assays (Settleman et al., 1992), and by use of adhesion assembly following growth factor stimula- dominant-negative mutants of Rac1, RhoA and tion (Ridley and Hall, 1992). The expression of the Cdc42. The transforming activity of Lsc, as observed Lbc oncogene was found to be associated with stress by focus formation of NIH3T3 cells, was blocked by ®ber formation in serum starved NIH3T3 cells (Zheng all the above mentioned constructs. These data et al., 1995), a typical response of Rho stimulation. provide support for Lsc-triggered activation of Rho Rho activation by the sub1.5 gene product is indicated

Fluorescence Phase contrast Figure 2 Eects of sub1.5 expression on actin stress ®bers formation. NIH3T3 cells were cotransfected with the sub1.5 cDNA, in the plasmid pCDM8, and the plasmid pPUR (Clontech, CA) conferring resistance to puromycin, using the lipophilic agent DOTAP (Boehringer Mannheim, Germany) essentially as described by the manufacturer (5 mg sub1.5 plasmid and 0.5 mg of pPUR plasmid per 10 cm plate). Control cells were transfected with the pPUR plasmid alone. The cells were grown in selection medium containing 4 mg/ml puromycin. Sub1.5 expression in stable transfectants was con®rmed by Northern analysis (data not shown). Actin ®laments were detected by staining with rhodamine-labelled phalloidin, an actin binding protein as previously described (Ridley et al., 1992). Brie¯y, cells were seeded in 15 mm wells containing 13 mm circular coverslips. The cells were serum starved in DMEM containing 0.5% serum over night, after which they were ®xed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and incubated with rhodamine-labelled phalloidin (Molecular probes), to localize actin. Cells were viewed on a Zeiss microscope and photographed. Phalloidin stained sub1.5 NIH3T3 transfectants (a) and phalloidin stained control transfectants (c). Corresponding phase contrast is shown in (b) (sub1.5 transfectants) and d (control transfectants) Identification of the homologue to the Lsc oncogene H-C Aasheim et al 1750 by our studies showing formation of stress ®bers Expression pattern of the sub1.5 gene ending in focal adhesions in stable sub1.5 NIH3T3 transfectants after over-night serum starvation as Sub1.5 was expressed at relatively high levels in blood compared to control cells (Figure 2). The control and lymphoid tissues (thymus, spleen, tonsils and cells showed few stress ®bers and a punctuated lymph nodes), and at weaker levels in most other distribution of ®lamentous actin at the plasma tissues (Figure 4 a ± c). Two main transcripts were membrane, as previously described for Swiss 3T3 detected with the sizes of 3.5 and 5 kb, respectively, cells (Ridley and Hall, 1992). We also observed a and the relative expression levels of these two better survival of the sub1.5 transfectants after serum transcripts varied between tissues. The expression starvation than control transfectants. These results pattern of sub1.5 roughly parallells the expression of support the data from studies with the mouse Lsc Lsc, which also showed a particular abundant which indicate activation of the Rho signalling expression in murine hematopoetic tissues. In mice, pathways (Whitehead et al., 1996). Lsc showed a high expression in total bone marrow, while the Sub1.5 gene was expressed only at moderate levels in total bone marrow in humans. This dierence Chromosomal localization of the sub1.5 gene may be due to dierent regulation of the genes between Using the FISH technique and the sub1.5 cDNA as a mouse and man, or to dierences in the isolation probe, we demonstrate that the chromosomal localiza- procedure of bone-marrow cells. Highly puri®ed B cells tion of sub1.5 was to chromosome 19q13.13 (Figure 3). and T cells from blood and tonsils expressed sub1.5 at The chromosomal localization was con®rmed by high levels, in agreement with the tissue blot data simultaneous hybridization of sub1.5 with a control (Figure 4d). While more mature B lymphoid cells lines probe located on 19q13.1 and using two-color (Raji, Daudi and U266) all abundantly expressed detection. Signals of the two probes were shown to sub1.5, the pre-B cell lines Nalm-6 and Reh expressed be colocalized on the long arm of chromosome 19, the lower levels of sub1.5 mRNA. A dierence in the level sub1.5 signal appeared to be slightly distal to a control of sub1.5 expression during B lymphopoesis was probe (chromosome 19q13.1 probe, data not shown). further supported by semi-quantitative PCR analysis, Other genes localized to chromosome 19q13.1 are demonstrating low levels of sub1.5 transcripts in early myeloid b (A4) precursor like protein, hepsin, CD22, B lymphoid cells (pro- and pre-B cells) and higher ATP4, Cytochrome C oxidase subunit VIb and VIIa levels of expression in bone marrow derived B cells polypeptide 1, glucose phosphatate isomerase, ryano- expressing surface IgM (Figure 5). From the Northern dine receptor 1 peptidase D and CAAT/Enhancer blot data on thymus, which show strong expression of binding protein a (source LLNL Human Genome both transcripts, one would suspect that T cells express Center). Genomic Southern blot data indicate that the the gene from the early dierentiation stages, indicat- two major transcripts are trancribed from the same ing that the regulation of sub1.5 expression is gene (data not shown). dierently regulated during T cell development versus

Figure 3 Chromosomal localization of the sub1.5 gene. Fluorescence in situ hybridization (FISH) was performed as previously described (Pedetour et al., 1994). Biotinylated sub1.5 probe was hybridized to 20 metaphases from three unrelated healthy donors (two males and one female). Revelation of the signal was done using avidin-¯uorescein. The chromosomes were counterstained with propidium iodide (a and b) and 4,5-diamino-2-phenyleindole (DAPI, for chromosome identi®cation; c and d). Slides were observed using a Zeiss Axiophot microscope and metaphases were analysed using an image processor (Perspective Scienti®c International, Chester, UK). Arrowheads indicate chromosome 19 with a ¯uorescent signal at 19q13.13 band. The idiogram illustrates the chromosomal localization at 19q13.13 Identification of the homologue to the Lsc oncogene H-C Aasheim et al 1751

H2O CD34+,CD38–

a —7.5 kb —4.4 kb CD34+,CD38+ spleen CD34+,CD19– thymus CD34+,CD19+ prostate CD19+,IgM– CD19+,IgM+ testis PBL B cells ovary 26x 30x 26x small intestine colon sub1.5 actin PBL Figure 5 Semi-quantitative PCR analysis of levels of sub1.5 in dierent hematopoetic cell populations. CD34+ cells were positively selected from adult bone marrow aspirates as described (Rusten et al., 1994) and costained with phycoerythrin (PE)- conjugated anti-CD34 and ¯uorescein (FITC) conjugated anti- b CD38, or PE anti-CD34 and FITC anti-CD19. CD19+B cells heart (depleted for CD34+ cells) were positively selected from adult bone marrow aspirates as described (Funderud et al., 1990; brain Rasmussen et al., 1992) and costained with PE anti-CD19 and placenta FITC anti-IgM. B cells were also isolated from blood as described (Funderud et al., 1990). Cells were sorted on a FACSortTM+ lung instrument (Becton Dickinson) after appropriate compensation, as described elsewhere (Rusten et al., 1994). After sorting, the liver cells were centrifuged and lysed with cold lysis buer (phosphate skeletal muscle buered saline (PBS), 1% NP-40) at concentrations of 26104 to 5 1610 cells/mL. mRNA was isolated and cDNA was generated as kidney described (Aasheim et al., 1996). cDNA generated from mRNA isolated from 5 6 103 cells were used as template in the ®rst PCR pancreas (32 cycles) using sub1.5 speci®c primers 1 and 2 hybridizing to region 2045 ± 2065 and 2382 ± 2400 respectively. One ml of the ®rst PCR reaction was used as template in the second PCR using a second inner set of sub1.5 speci®c primers (primer 3 and 4) c hybrizing to region 2096 ± 2113 and 2178 ± 2199 respectively. fetal liver Primer 1:5'-AAA TTC TAC ACC ACG TCA ACC, Primer 2: bone marrow 5'-GGT CAT GGC GGA GGT GAG C, Primer 3: 5'-GGC TCA AGG ACT ATC AGC G, Primer 4: 5'-CAA TTT CTT CTT PBL GGT GAT GTC C. Actin speci®c primers were used for normalization of the amount of cDNA from each cell population appendix (right panel). Here only a ®rst PCR reaction was performed 3 thymus starting with 2 6 10 cells. Sub1.5 speci®c PCR results are shown in left and middle panel, actin speci®c PCR results are shown in lymph node right panel. Cycle numbers in the second PCR (sub1.5) and ®rst PCR (actin) are shown below the panels. Five ml aliquots were spleen removed from the PCR reaction mixture at the indicated cycle number, seperated on an 2% agarose gel and processed for hybridization with random labelled [32P]dCTP sub1.5 insert or actin cDNA

d —3.5 kb B cells B cells TPA B cell development. However, we can not exclude the B cells tonsils possibility that the 5 kb transcript is expressed during CD4 cells early B cell dierentiation, but is not recognized by the CD4 cells TPA primer combinations we have used, due to lack of CD8 cells knowledge about where the sequence dierence between the two dierent transcripts is located. CD8 cells TPA Interestingly, we also demonstrated high levels of sub1.5 expression in early hematopoetic progenitor Figure 4 Expression of sub1.5 mRNAs in dierent human tissues. (a, b and c) Represent dierent commercial tissue blots cells (Figure 5). Thus, sub1.5 was abundantly expressed (Multiple human tissue blot 1 and 2, human immunsystem in both CD34+CD387 cells, which are highly enriched multiple tissue blot; Clontech, CA). Each lane shows hybridiza- in the most primitive hematopoetic progenitor cells, as tion to 2 mg mRNA of the indicated tissue. (d) Represents a well as CD34+CD38+ cells, containing primarily early Northern blot of B cells, CD4 cells and CD8 cells isolated from blood or tonsils by positive selection using magnetic Dynabeads dierentiation stages of the lymphoid and myeloid coated with anti-CD19, anti-CD4 and anti-CD8, respectively lineages. However, it should be noted that these semi- (Funderud et al., 1990, Rasmussen et al., 1992). mRNA was isolated from the cells using Dynabeads oligo-(dT)25 essentially as described (Aasheim et al., 1994). Each lane in d shows hybridization to 1 mg mRNA of the indicated cell type. Molecular weights are indicated in kb on top of a. PBL = 13-acetate. The Northern blots were hybridized with random peripheral blood leucocytes, TPA = 12-O-tetra decanoylphorbol- labelled [32P]dCTP sub1.5 insert Identification of the homologue to the Lsc oncogene H-C Aasheim et al 1752 quantitative PCR data only give indications with highly homologous to the previously published mouse regard to the expression of the sub1.5 gene and have cDNA, Lsc. Based on the similarities in the amino acid to be further con®rmed by protein expression studies sequence, and in the expression pattern of sub1.5 and using antibodies. We are currently in the process of Lsc, we propose that sub1.5 most probably represents generating such antibodies. the human homologue of Lsc. In conclusion a human cDNA clone, sub1.5, was isolated from a T cell cDNA library, and showed to be strongest expressed in lymphoid tissues. The predicted protein confers homology to the Dbl-family of GEF's, Acknowledgements This work was supported by the Norwegian Cancer harbouring both a DH-domain and a PH-domain in Society. We are grateful to Toril Larsen and Ruth Solem addition to an extended N-terminus without known for technical assistance, and Raisa Gurvitsj for secretarial function. Stable NIH3T3 transfectants showed stress help. We also wish to thank Dr Harald Stenmark for ®ber formation upon serum starvation, indicating valuable discussions and suggestions. Sub1.5 accession activation of Rho-like GTPases by sub1.5. Sub1.5 is number is Y09160.

References

Aasheim H-C, Deggerdal A, Smeland EB and Hornes E. Lemmon MA, Ferguson KM and Schlessinger J. (1996). (1994). Biotechniques, 16, 716 ± 721. Cell, 85, 621 ± 624. Aasheim H-C, Logtenberg T and Larsen F. (1996). Methods Miki T, Smith CL, Long JE, Eva A and Fleming TP. (1993). in Molecular Biology ± cDNA Library Protocols.CowellI Nature, 362, 462 ± 465. and Austin C. (eds.). Humana Press. Vol 69. Minden A, Lin A, Claret F-X, Abo A and Karin M. (1995). Adams AEM, Johnson DI, Longnecker RM, Sloat BF and Cell, 81, 1147 ± 1157. Pringle JR. (1990). J. Cell Biol., 111, 131 ± 142. Nobes CD and Hall A (1995). Cell, 81, 53 ± 62. Boguski MS and McCormick F. (1993). Nature, 366, 643 ± Pedetour F, Szpirer C and Nahon JL. (1994). Genomics, 19, 654. 31 ± 33. Chan AML, McGovern ES, Catalano G, Fleming TP and Rasmussen A-M, Smeland EB, Erikstein B, Caignault L and Miki T. (1994). Oncogene, 9, 1057 ± 1063. Funderud S. (1992). J. Imm. Methods., 146, 195 ± 202. Coso OA, Chiariello M, Yu J-C, Teramoto H, Crespo AÊ ,Xu Ridley AJ and Hall A. (1992). Cell, 7, 389 ± 399. N, Miki N and Gutkind JS. (1995). Cell, 81, 1137 ± 1146. Ridley AJ, Paterson HF, Johnston CL, Diekman D and Hall Denhardt DT. (1996). Biochem. J., 318, 729 ± 747. A. (1992). Cell, 70, 401 ± 410. Drubin DG. (1991). Cell, 65, 1093 ± 1096. Rusten LS, Jacobsen SEW, Kaalhus O, Veiby OP, Funderud Funderud S, Eriksten B, AÊ sheim H-C, Nustad K, Blomho SF and Smeland EB. (1994). Blood, 84, 1473 ± 1481. HK, Holte H and Smeland EB. (1990). Eur. J. Immunol., Settleman J, Albright CF, Foster LC and Weinberg RA. 20, 201 ± 206. (1992). Nature, 359, 153 ± 154. Gibson JB, HyvoÈ nen M, Musacchio A and Saraste M. Shou C, Farnsworth CL, Neel BG and Feig LA. (1992). (1994). Trends Biochem. Sci., 19, 349 ± 353. Nature, 358, 351 ± 354. Habets GGM, Scholtes EHM, Zuydgeest D, van der Toksoz D and Williams DA. (1994). Oncogene, 9, 621 ± 628. Kammen RA, Stam JC, Berns Allmenn and Collard JG. Quilliam LA, Khosravi-Far R, Hu SY and Der CJ. (1995). (1994). Cell, 77, 537 ± 549. BioEssays, 17, 395 ± 404. Hariharan IK and Adams JM. (1987). EMBO J., 6, 115 ± Van Leeuwen FN, van der Kammen RA, Habets GGM and 119. Collard JG. (1995). Oncogene, 11, 2215 ± 2221. Hart MJ, Eva A, Zangrilli D, Aaronsen SA, Evans T, Whitehead I, Kirk H, Tognon C, Trigo-Conzales G and Kay Cerione RA and Zheng Y. (1994). J. Biol. Chem., 269, 62 ± R. (1995). J. Biol. Chem., 270, 18388 ± 18395. 65. Whitehead I, Khosravi-Far R, Kirk H, Trigo-Conzales G, Hill CS et al. (1995). Cell, 81, 1159 ± 1170. Der CJ and Kay R. (1996). J. Biol. Chem., 271, 18643 ± Horii Y, Beeler JF, Sakaguchi K, Tachibana M and Miki T. 18650. (1994). EMBO J., 13, 4776 ± 4786. Zheng Y, Olson MF, Hall Allmenn, Cerione RA and Toksoz Katzav S, Martin-Zanca D and Barbacid M. (1989). EMBO D. (1995). J. Biol. Chem., 270, 9031 ± 9034. J., 8, 2283 ± 2290. Zheng Y, Cerione R and Bender A. (1994). J. Biol. Chem., Kozak M. (1994). J. Biol. Chem., 266, 19867 ± 19873. 269, 2369 ± 2372. Lamaze C, Chuang T-H, Terlecky LJ, Bokoch GM and Schmid S. (1996). Nature, 382, 177 ± 179.