Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia

Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 15, Issue of April 13, pp. 11804 –11811, 2001 Printed in U.S.A. Activation of the Rap1 Guanine Nucleotide Exchange Gene, CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia* Received for publication, October 2, 2000, and in revised form, January 12, 2001 Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008970200 Adam J. Dupuy‡, Kelly Morgan‡, Friederike C. von Lintig§, Haifa Shen¶, Hasan Acar‡, Diane E. Hasz‡, Nancy A. Jenkins¶, Neal G. Copeland¶, Gerry R. Boss§, and David A. Largaespada‡i From the ‡University of Minnesota Cancer Center, Institute of Human Genetics, and Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, the §Department of Medicine, University of California San Diego, La Jolla, California 92093, and ¶Mouse Cancer Genetics Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Here we report the recurrent proviral activation of provide powerful genetic tags for disease gene identification (2, the Rap1-specific guanine nucleotide exchange factor 3). During the past several years, a number of disease genes CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., have been identified in BXH-2 AMLs by proviral tagging. They Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., include a tumor suppressor gene, Neurofibromatosis type 1 Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., (Nf1); a gene with homology to the lymphoid-restricted type II Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. membrane protein Jaw1, Mrv integration site 1 (Mrvi1); a gene Acad. Sci. U. S. A. 95, 13278 –13283; Correction (1999) encoding a hematopoietic cell growth and differentiation factor, Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 myeloblastosis oncogene (Myb); three homeobox genes, ho- acute myeloid leukemia. We also show that CalDAG-GEF meobox A7 (Hoxa7), homeobox A9 (Hoxa9), and myeloid eco- I encodes two protein isoforms, a full-length isoform tropic viral integration site 1 (Meis1); a zinc finger protein (CalDAG-GEF Ia) and a C-terminally truncated isoform gene, ecotropic viral integration site 9 (Evi9); and a novel (CalDAG-GEF Ib). Expression of the full-length CalDAG- cytokine receptor (Evi27) (4 –12). Importantly, two of these GEF Ia isoform in Rat2 fibroblasts enhances growth in genes, NF1 and HOXA9, are known human myeloid leukemia low serum, whereas expression in Swiss 3T3 cells causes disease genes, validating the usefulness of this approach for morphological transformation and increased saturation human disease gene identification (9, 13). density. In FDCP1 myeloid cells, CalDAG-GEF Ia expres- Mutations in the human NF1 gene are responsible for the sion increases growth and saturation density in the cancer predisposition syndrome neurofibromatosis type 1 (13). presence of the diacylglycerol analogs phorbol 12-myris- NF1 encodes neurofibromin, a GTPase-activating protein tate 13-acetate (PMA), which activates CalDAG-GEF Ia (GAP) that is active on the four true Ras proteins (Ha-Ras, exchange activity. Likewise, in 32Dcl3 myeloblast cells, K-RasA, K-RasB, and N-Ras) as well as R-Ras (14 –16). In CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore BXH2 AMLs, about 15% of the AMLs have proviral insertions and allows higher saturation densities and prolonged in the Nf1 gene (4, 5). These proviral insertions inhibit neuro- growth on fibronectin-coated plates. These effects were fibromin expression, and no wild type neurofibromin is ex- correlated with increased Rap1, but not Ras, protein pressed in these AMLs (5). Loss of neurofibromin in myeloid activation following PMA and calcium ionophore treat- cells is associated with increased and prolonged Ras activation ment. Our results suggest that Rap1-GTP delivers sig- after cytokine stimulation in chronic myeloid leukemia and in nals that favor progression through the cell cycle and primary cells (17). These results contribute to a large body of morphological transformation. The identification of evidence implicating aberrant regulation of Ras signaling in CalDAG-GEF I as a proto-oncogene in BXH-2 acute my- myeloid leukemia. eloid leukemia is the first evidence implicating Rap1 The other 85% of BXH-2 AMLs do not have proviral inser- signaling in myeloid leukemia. tions in the Nf1 gene (4, 5). This has led to the suggestion that these AMLs have insertions in or near other genes involved in Ras regulation and that these insertions activate Ras in the Retroviral insertional mutagenesis in BXH-2 recombinant absence of inactivating insertions at Nf1. One class of likely inbred mice induces a high incidence of acute myeloid leukemia targets are the guanine nucleotide exchange factors (GEF) that (AML), and the proviral integration sites in the leukemias catalyze the exchange of GTP for GDP on Ras and consequently turn on Ras. One such GEF is RasGRP (18, 19). RasGRP is one of three guanine exchange factors that have calcium-binding * The costs of publication of this article were defrayed in part by the “EF hands” in addition to diacylglycerol (DAG)-binding do- payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to mains (18 –20). It has been speculated that this class of GEFs indicate this fact. couple cell surface receptors that signal through Ca and DAG i To whom correspondence should be addressed. Tel.: 612-626-4979; to the Ras pathway (18, 20). Fax: 612-626-3941; E-mail: [email protected]. In the studies described here we report the recurrent provi- The abbreviations used are: AML, acute myeloid leukemia; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase ral activation of the calcium- and diacylglycerol-binding gua- chain reaction; GAP, GTPase-activating protein; GEF, guanine nucle- nine nucleotide exchange factor 1 (CalDAG-GEF I) gene in otide exchange factors; P/S, penicillin/streptomycin; DMEM, Dulbecco’s BXH-2 AML. Like RasGRP, CalDAG-GEF I has EF hands and modified Eagle’s medium; FBS, fetal bovine serum; IL-3, interleukin-3; DAG domains. Previous studies, however, have shown that CS, calf serum; CalDAG-GEF I, calcium- and diacylglycerol-binding guanine nucleotide exchange factor 1; REM, Ras exchanger motif. CalDAG-GEF I is not a Ras GEF, rather it appears to be a Rap1 11804 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. CalDAG-GEF I and Myeloid Leukemia 11805 the CalDAG-GEF Ia and CalDAG-GEF Ib expression constructs were GEF (1). We also show that forced overexpression of CalDAG- sequenced for errors. Both expression constructs were used in an in GEF I results in transformation of cultured fibroblasts and vitro transcription/translation experiment (Promega) to confirm that implicate Rap1 signaling in producing these effects. This is the they produced an ;70-kDa V5-tagged protein for CalDAG-GEF Ia and first evidence implicating Rap1 signaling in myeloid leukemia 19-kDa Myc-tagged protein for CalDAG-GEF Ib. The CalDAG-GEF Ia in human or mouse. insert was subcloned into the MSCV2.1 retroviral expression vector and electroporated into the PT67 amphotropic packaging cell line EXPERIMENTAL PROCEDURES (CLONTECH). The electroporated PT67 cells were selected in 400 Molecular Cloning of CalDAG-GEF I Proviral Insertions—Proviral mg/ml G418 in 10% fetal bovine serum (FBS), Dulbecco’s modified insertions in the CalDAG-GEF I first intron were discovered as part of Eagle’s medium (DMEM), and penicillin/streptomycin (P/S). Resist- a large scale cloning effort that utilized a long template, inverse PCR ant colonies were pooled and replated at 1–2 3 10 cells/ml. Viral method, described in detail elsewhere (6). In brief, 5 mg of genomic DNA supernatants were harvested 24 h after cells reached confluence and from individual BXH-2 AMLs was digested with SacII overnight; the stored at 270 °C. enzyme was inactivated by heating at 65 °C for 10 min, and the DNA Cell Culture Assays—The following cell types were transduced with fragments were ligated in 500-ml reactions, using 5 units of T4 DNA MSCV-CalDAG-GEF Ia or MSCV2.1 empty vector virus and selected in ligase (Stratagene) at 4 °C overnight, to produce circular provirus/ G418: Rat2, Swiss 3T3, FDCP1, and 32Dcl3Gr. All lines were obtained cellular DNA templates for PCR amplification. The ligated material from the ATCC, apart from 32Dcl3Gr, which was obtained from D. was precipitated in ethanol and resuspended in 20 ml of Tris-EDTA (pH Askew (University of Cincinnati). Rat2 and Swiss 3T3 were grown in 8.0). Two ml of this precipitated material was used as template in a 10% FBS/DMEM supplemented with P/S and selected in 400 mg/ml primary PCR in a 50-ml reaction volume containing 20 nmol of each G418. FDCP1 and 32Dcl3Gr were grown in 20% WEHI-3 cell line dNTP, 10 pmol each forward and reverse primer, 13 buffer 2, and 2.5 conditioned medium in 10% FBS/DMEM supplemented with 10% TM units of enzyme mix in the Expand Long Template PCR System NCTC-109, P/S, Hepes buffer (pH 7.4), 10 units/ml insulin, glutamine, (Roche Molecular Biochemicals). Amplification was performed with a sodium pyruvate, nonessential amino acids, and 10 M b-mercaptoeth- Omnigene Hybaid thermocycler programmed as follows: 92 °C for 2 anol. All media and supplements were from Life Technologies, Inc., min; 10 cycles of 92 °C for 10 s, 63 °C for 30 s, 68 °C for 10 min; 20 cycles except insulin and b-mercaptoethanol (Sigma). FDCP1 and 32Dcl3Gr of 92 °C for 10 s, 63 °C for 30 s, 68 °C for 10 min with 20-s auto- cells were selected in 1 mg/ml G418. Retroviral transduction was done 6 6 extension. The amount of primary PCR product was semi-quantified by by overnight infection of 10 3 10 (FDCP1 and 32Dcl3Gr) or 3 3 10 1% agarose gel electrophoresis and 0.01 to 1 ml of the primary PCR cells (Rat2 and Swiss 3T3) in 10 ml of a 50:50 mix of high titer retroviral product was used as template in a secondary PCR under the same pool and regular growth medium supplemented with 16 mg/ml Poly- conditions except that the secondary primers were used. The secondary brene (Sigma). Twenty four hours after infection, the cells were PCR product was separated on a 1% agarose gel, purified using the switched to selective media. Pooled G418-resistant populations were Geneclean II kit (Bio 101), and directly cloned using CloneAmp® analyzed for phenotypes after mock-infected parallel cultures in G418 pAMP1 System (Life Technologies, Inc.) according to supplied protocol. had cleared. Rat2 cells were tested for colony formation by plating at These particular clones were obtained using primer pairs designed to 4 3 10 cells in 2 ml of 5 or 0.5% FBS/DMEM/P/S medium in 6-well amplify proviruses located 39 of genomic SacII sites. The primers used plates. After 10 days (5% FBS) or 24 days (0.5% FBS) in culture, the in the primary PCRs are as follows: 59 ECO F1 (59-GGCTGCCATGCAC- colonies were fixed and stained with methylene blue and counted. Rat2 GATGACCTT-39) and 59 ECO R4 (59-CGGCCAGTACTGCAACTGAC- cells were also plated in soft agar colony forming assays as described CAT-39). For the secondary PCR the primer pair was 59 ECO F2-dUMP (22). Ten-centimeter plates of G418-resistant Swiss 3T3 transductants (59-GAGGCCACCTCCACTTCTGAGAT-39) and U3REVD-dUMP (59- were examined for foci of morphologically transformed cells after com- CTCTGTCGCCATCTCCGTCAGA-39. The cloned PCR products were ing to confluence. These cells were trypsinized, counted, and replated at TM TM 5 sequenced using the PRISM BigDye Cycle Sequencing Kit 5 3 10 cells/10-cm plate in 10 ml of 10% FBS/DMEM or 5% calf serum (PerkinElmer Life Sciences) on an ABI model 373A DNA Sequencer (CS)/DMEM. Secondary foci (from 10% FBS/DMEM cultures) and total (Applied Biosystems). SP6 and T7 sequencing primers were purchased cells per plate (from 10% FBS/DMEM and 5% CS/DMEM cultures) were from Life Technologies, Inc. counted 14 days after the cells were replated. For growth rates and Southern and Northern Blot Analysis—Isolation and analysis of saturation density, FDCP1 transductants were plated at 2 3 10 cells in BXH2 genomic DNA and poly(A) RNA, by Southern and Northern 2 ml of growth medium with 0, 10, or 50 ng/ml phorbol 12-myristate blotting, was performed using random primed [ P]dCTP-labeled probe 13-acetate (PMA, from Sigma) and counted every day for 4 days. For (Roche Molecular Biochemicals) as described before (21). apoptosis assays, transduced 32Dcl3Gr cells were washed three times Western Blot Analysis—Stably or transiently transfected cells were in 13 PBS and plated at a density of 1 3 10 cells per ml in normal lysed in RIPA buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM growth medium (previously described) lacking WEHI-3-conditioned me- EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) at a concentra- dium, the IL-3 source. PMA was also included in a subset of plates at a tion of 10 3 10 cells per ml at 4 °C for 10 min. The lysates were clarified concentration of 1 mM. Cells were scored at the indicated time points for by centrifugation for 15 min at 14,000 rpm to remove insoluble mate- total cell count by trypan blue dye exclusion and viability. rial. The supernatants were frozen in liquid nitrogen and stored at Measurement of Ras and Rap1 Activation States—The activation 270 °C. The concentration of protein in the supernatants was deter- states of Ras and Rap1 were measured as described previously in mined using the Bradford assay. Fifty mg of protein was loaded onto coupled enzymatic assays that measure the total amount of GTP and SDS-polyacrylamide gels, run, and transferred to nitrocellulose as de- GTP plus GDP bound to Ras or Rap1 (23–25). Briefly, cells were har- scribed (5). The blots were hybridized with anti-tubulin or anti-V5 vested rapidly and snap-frozen in liquid nitrogen. For measuring Ras antibody (Invitrogen) according to manufacturer’s instructions and vi- activation, the frozen cells were extracted in a Hepes-based buffer sualized with the ECL chemiluminescence kit (Amersham Pharmacia containing 1% Triton X-100, and after removing nuclei and subcellular Biotech). organelles from the extracts by brief centrifugation, the extracts were Expression Constructs, Electroporation, and Retroviral Vectors—The split in half, one-half receiving the pan-Ras rat monoclonal antibody CalDAG-GEF Ia and CalDAG-GEF Ib open reading frames were cloned Y13–259 (the experimental sample) and the other half receiving rat IgG from a mouse bone marrow cDNA library using the HIFI polymerase (the control sample). After shaking for1hat4 °C,Raswas collected on chain reaction kit from Roche Molecular Biochemicals. Primer se- protein G-agarose beads to which goat anti-rat antibody was bound. quences used to amplify full-length CalDAG-GEF Ia are forward 59- The immunoprecipitated Ras was washed four times in lysis buffer, and ACCGGCAGCCATGACGA-39 and reverse 59-GTGGATGTCAAA- GTP and GDP bound to Ras were eluted by heating. The samples were CACTCCGTCCTC-39. PCR products were gel-purified and cloned into split in half, and GTP was measured in one-half by conversion to ATP the pcDNA3.1/V5/His-TOPO vector from Invitrogen. This vector added using ADP, and the enzyme nucleoside diphosphate kinase and the a V5 epitope tag to the C-terminal end of the CalDAG-GEF Ia protein. resulting ATP was measured by the firefly luciferase system that is Primer sequences used to amplify full-length CalDAG-GEF Ib are for- sensitive to 1 fmol. The control sample was subtracted from the exper- ward 59-CTCGAGCCGTGGGAGGCTCTGAGA-39 and reverse 59-TCTA- imental sample, and the amount of GTP was determined from standard GAAGCACATATGGTGTAGCATGCG-39. These primers added a XhoI curves prepared with each set of samples. The sum of GTP plus GDP site and a XbaI site to the 59 and 39 ends of the PCR product, respec- was measured in the other half of the sample by converting GDP to GTP tively. PCR products were gel-purified, digested with XhoI and XbaI, using phosphoenolpyruvate and pyruvate kinase, and then GTP, which and then cloned into the XhoI and XbaI sites of the now represents the sum of GTP plus GDP, was measured as above. pcDNA3.1/Myc-His(1) from Invitrogen. This vector added a Myc Rap1 activation was measured similarly, but since there are no good epitope tag to the C-terminal end of the CalDAG-GEF Ib protein. Both immunoprecipitating antibodies for Rap1, the Rap-binding domain of 11806 CalDAG-GEF I and Myeloid Leukemia FIG.1. A, proviral insertions in the first intron of the CalDAG-GEF I gene. Exons are shown as black boxes, and the position and orientation of the proviruses at CalDAG-GEF I are indicated by an arrow. B, Northern blot analysis of BXH-2 AMLs with proviral insertions at CalDAG-GEF I. BXH-2 poly(A) RNAs are shown hy- bridized with a CalDAG-GEF Ib cDNA probe. The size (in kilobase pairs (kb)) and position of migration of RNA markers are indicated to the left. Tumor 22 has a proviral insertion in the first intron of the CalDAG-GEF I gene. Below the blot are shown the Gapdh-normalized CalDAG- GEF I transcript levels as determined us- ing PhosphorImager analysis. Ral GDS fused to glutathione S-transferase was used to isolate Rap GTP from one-half of the sample on glutathione-Sepharose beads (25). The GTP was then eluted from Rap and measured as described above. To measure the sum of GTP and GDP bound to Rap in the other half of the sample, extracts were incubated in the absence of magnesium with 10 mM GTP to convert Rap-GDP to Rap-GTP. Excess free GTP was removed by a temperature-dependent phase extraction since, as part of the procedure for measuring Rap1 activation, cells were extracted in 0.92% Triton X-114, 0.08% Triton X-45; an aqueous phase containing the free GTP and a detergent phase containing the Rap1 were gener- ated by warming the samples to 15 °C for 2 min. Rap-GTP, which now represented the sum of Rap-GTP plus Rap-GDP, was then isolated by binding to the Rap binding domain glutathione S-transferase fusion protein, and the GTP was eluted and measured as described above. RESULTS Proviral Integration at CalDAG-GEF I in BXH-2 AML Leads to Increased CalDAG-GEF I Expression—Proviral insertions within the first intron of the CalDAG-GEF I gene were identi- fied as part of a large screen to discover proviral insertions that are located near CpG islands (6). Sequence analysis of two of the clones showed that they were located in the first intron of the mouse homolog of the human CDC25-like gene (Fig. 1A) also called CalDAG-GEF I (1). Northern blot analysis of one of the leukemias (number 22) showed that CalDAG-GEF I expres- sion was elevated compared with other BXH-2 leukemias (numbers 13, 29, 106, 117, and 132) that did not contain viral integrations at CalDAG-GEF I (Fig. 1B). These results show that viral integration at CalDAG-GEF I leads to increased CalDAG-GEF I expression. FIG.2. Expression of CalDAG-GEF I mRNA in adult tissues Two CalDAG-GEF I-hybridizing transcripts were detected in and the embryo. Poly(A) RNA, isolated from day 7, 11, 15, and 17 all BXH-2 leukemias tested. This was surprising because the normal mouse embryos or normal adult heart (Hr), brain (Br), spleen shorter transcript has not been reported in humans (1). Inter- (Sp), lung (Lg), liver (Li), skeletal muscle (Sm), kidney (Kd), and testis estingly, both transcripts were up-regulated by viral integra- (Ts), was hybridized with a CalDAG-GEF Ib cDNA probe. CalDAG-GEF Ia and CalDAG-GEF Ib transcripts are indicated. tion at CalDAG-GEF I (Fig. 1B). This prompted us to examine normal mouse tissues for the shorter CalDAG-GEF I tran- script. As shown in Fig. 2, both CalDAG-GEF I transcripts are pression found between 15 and 17 days of development. Sub- expressed in normal mouse tissues. Expression was highest in sequently, we confirmed that the shorter transcript is also brain, heart, and lung followed by spleen, liver and kidney. expressed in humans (data not shown). The shorter transcript Expression was not detected in skeletal muscle. Both tran- hybridized to probes composed of CalDAG-GEF I exons 1–5 but scripts are also expressed in the embryo with the highest ex- not with probes from farther downstream (data not shown). In CalDAG-GEF I and Myeloid Leukemia 11807 FIG.3. Alternate polyadenylation of the CalDAG-GEF I transcript and predicted primary sequence of CalDAG-GEF Ia and CalDAG-GEF Ib isoforms. The predicted protein domain structures for CalDAG-GEF Ia and CalDAG-GEF Ib are shown above. Shown below are the nucleotide sequences surrounding the splice donor site at the end of the mouse and human fifth exon. Exon 5 sequences are shown in bold. The consensus polyadenylation site (AATAAA) present in both mouse and human intron 5 is shown in bold at the end of each sequence. The amino acid sequence of the C terminus of CalDAG-GEF Ib, encoded by intron 5 sequences, is shown for both mouse and human. Significant homology exists between intron 5 encoded amino acid sequence between human and mouse. fact, the size of the shorter transcript is approximately what CalDAG-GEF Ia, and a truncated protein, CalDAG-GEF Ib. would be expected for an mRNA that was polyadenylated Effects of CalDAG-GEF Ia Overexpression in Fibroblasts and shortly after the fifth exon. Quantitation by PhosphorImager Myeloid Cells—Both CalDAG-GEF I transcripts were cloned analysis of normal tissue Northern blots, hybridized with a into mammalian expression vectors through PCR amplification probe containing exons 1–5, showed that the long and short of lymph node cDNA. Primers were designed for both tran- transcripts are usually present at a roughly 1:1 ratio, although scripts to allow the PCR fragments to be cloned in-frame into in some tissues more long form is expressed (Fig. 2). an expression vector and to add an epitope tag to the 39 end of Alternate Polyadenylation Predicts the Expression of a Full- each cDNA. This resulted in a Cdgla clone tagged with a V5 length (CalDAG-GEF Ia) and a C-terminal Truncated epitope and a CalDAG-GEF Ib clone tagged with a Myc epitope. (CalDAG-GEF Ib) Protein—Sequence analysis of the human The cytomegalovirus promoter was used to drive both con- CalDAG-GEF I genomic locus revealed a putative alternate structs. To determine whether the expression vectors func- polyadenylation site that was located just after the splice donor tioned as expected, we transiently transfected each vector into site within intron 5 (Fig. 3). This polyadenylation site is also HeLa cells and then followed their expression using antibodies present in the mouse. Examination of EST data bases revealed to the epitope tag. Surprisingly, we were unable to detect a number of mouse and human ESTs representing cDNA clones CalDAG-GEF Ib protein expression by Western analysis. This in which polyadenylation apparently occurred at this site contrasts with in vitro transcription/translation studies where rather than at the end of the last CalDAG-GEF I exon. These we were able to detect Cgd1b epitope-tagged protein (data not cDNA clones contain CalDAG-GEF I sequences from exon 5, shown). We have also failed to generate stable CalDAG-GEF which then read into the fifth intron and are polyadenylated Ib-expressing transfectants. These results suggest that the after a consensus ATAATA site. Polyadenylation at this site CalDAG-GEF Ib protein is toxic, unstable, or produced at low would produce a transcript of ;0.6 kilobase pairs. levels. The short CalDAG-GEF Ib transcript is predicted to encode To test for possible transforming effects of the CalDAG-GEF a truncated protein. CalDAG-GEF Ib would include the Ras Ia protein, an MSCV2.1-based expression vector for CalDAG- exchanger motif (REM) domain, amino acid sequences between GEF Ia was constructed (MSCV-CalDAG-GEF Ia) and tested the REM domain and CDC25 domain (i.e. the catalytic core for its ability to express V5 epitope-tagged CalDAG-GEF Ia domain similar to that from the yeast CDC25 gene), and 17 protein in FDCP1, Rat2, and 32Dcl3Gr cells. All MSCV- (mouse) or 20 (human) amino acids encoded by intron 5 (Fig. 3). CalDAG-GEF Ia vector transduced cells expressed readily de- Comparison between the human and mouse CalDAG-GEF I tectable protein (Fig. 4). The Rat2 fibroblast cell line was sub- genomic sequences showed conservation in the region sur- sequently transduced with CalDAG-GEF Ia-expressing virus rounding the alternate polyadenylation site in the fifth intron or empty vector virus pools and selected in G418. Stable Rat2 and indeed the whole fifth intron (72% identity). In contrast, transductants were obtained, and pools of greater than 50 other intron sequences showed much less conservation (e.g. colonies of each were assayed for growth in soft agar and fourth intron, 51% identity). These data indicate that the growth rates in 0.5 or 5% FBS, with or without added PMA, a CalDAG-GEF I locus produces two transcripts through alter- diacylglycerol analog, were measured. Neither population con- nate polyadenylation, which encode a full-length protein, tained cells capable of forming large colonies in soft agar (data 11808 CalDAG-GEF I and Myeloid Leukemia cells during this time (Fig. 7). This is consistent with other reports showing that CalDAG-GEF I overexpression does not affect Ras-GTP levels in transiently transected cells (1). Thus, CalDAG-GEF Ia expression at high levels can influence the level of endogenous Rap1 that becomes activated in response to stimulation. FIG.4. Expression of epitope-tagged CalDAG-GEF Ia in fibro- As has been reported in HL60 cells (25), Rap1 protein in blast and myeloblast cell lines. Total cell lysates from MSCV2.1 32Dcl3Gr cells shows a relatively high basal state of activation empty vector (M) or MSCV-CalDAG-GEF Ia vector (C)-transduced compared with Ras (Fig. 7). This has been suggested to be due FDCP1, Rat2, and 32Dcl3Gr cells were hybridized with an anti-V5- specific monoclonal antibody. All MSCV-CalDAG-GEF Ia vector-trans- to the presence of a threonine at position 61 in Rap1, which duced cells expressed readily detectable protein. decreases its intrinsic GTPase activity (25). DISCUSSION not shown). The number of colonies formed in liquid culture in 0.5% FBS/DMEM was significantly higher for MSCV-CalDAG- Proviral Activation of a Rap1 GEF Gene—A screen of somatic GEF Ia transductants than for the empty vector transductants murine leukemia virus integration sites in BXH-2 AML has both with and without added PMA (Fig. 5A). At higher serum identified a GEF gene, CalDAG-GEF I, that is a target for concentrations, both populations gave rise to a similar number murine leukemia virus integration in BXH2 AMLs. Proviral of colonies. integration at CalDAG-GEF I leads to increased CalDAG-GEF In contrast to Rat2 cells, Swiss 3T3 cells transduced with I expression suggesting that CalDAG-GEF I is a proto-onco- MSCV-CalDAG-GEF Ia formed multiple, transformed foci gene. CalDAG-GEF I is the second GEF to be implicated as a upon reaching confluence after G418 selection (Fig. 5B). These myeloid leukemia disease gene in humans or mouse. The foci appeared as areas of more spindle-shaped cells, growing at NUP98 gene is fused in frame, by translocation, to the broadly high density and in multiple layers. The size of these foci active GEF gene smgGDS in some human myeloid leukemias, increased with time in culture. Upon replating, many second- the result of which may be an increase in GTPase activation ary foci were present in cultures transduced with the MSCV- levels (29). CalDAG-GEF Ia vector. These transformed foci were more CalDAG-GEF I is closely related to another GEF, RasGRP apparent in 10% FBS than in 5% calf serum (CS) medium. The (1, 18, 19, 20). RasGRP has been shown to have GEF activity CalDAG-GEF Ia-transduced Swiss 3T3 cells also reached a for Ha-Ras but little activity for other Ras superfamily mem- higher saturation density than did empty vector transductants bers such as Rap, Rho, or Rac (1, 18, 19). In contrast, CalDAG- (Fig. 5C). GEF I shows little GEF activity for Ha-Ras but very high The MSCV-CalDAG-GEF Ia and empty vector retroviruses activity for Rap1 (1). More recently, RasGRP, CalDAG-GEF I, were also used to transduce cells from the myeloid cell lines and a newly identified GEF, CalDAG-GEF III, have also been 32Dcl3Gr and FDCP1 to test the effects of CalDAG-GEF Ia shown to have exchange activity for R-Ras and TC21 GTPases overexpression on myeloid cells. The growth and viability of (16, 30). Furthermore, another recent paper (31) suggests that 32Dcl3Gr and FDCP1 cells are dependent on the presence of CalDAG-GEF I can cause exchange on N-Ras and K-Ras but interleukin-3 (IL-3) in the culture medium. If IL-3 is removed not Ha-Ras proteins in cells that are chronically stimulated from the culture medium, these cells will die by apoptosis. with PMA or high serum. Thus, activation of Rap1, Ha-Ras, Previous studies have suggested that Rap1 plays a role in N-Ras, R-Ras, and TC21 GTPases could potentially mediate regulating integrin-mediated cell adhesion to fibronectin (26 – the oncogenic effects of CalDAG-GEF I. 28) and therefore fibronectin was included in some of these Small G Protein Signaling and Cancer—There is abundant experiments. genetic evidence from studies of primary cancers, cancer mod- The addition of PMA (50 ng/ml), in the absence of IL-3, els, and transformation systems for a central role for the so caused a slight delay in apoptosis in MSCV-CalDAG-GEF Ia- called “true” Ras genes (HRAS, NRAS, and KRAS) in cancer transduced but not control empty vector-transduced 32Dcl3Gr development (32, 33). Much less is known, however, about the cells, which was most apparent in fibronectin-coated plates role of other small G proteins of the Ras-like subfamily in (data not shown). PMA and calcium ionophore-treated MSCV- oncogenesis. Rap1 was initially identified in a screen for genes CalDAG-GEF Ia-transduced 32Dcl3Gr cells displayed a sub- that can suppress the transformed phenotype of K-Ras-trans- stantial increase in adherence to fibronectin compared with formed fibroblasts (34). This would suggest that Rap1 signals controls (Fig. 6A). FDCP1 cells expressing CalDAG-GEF Ia at suppress rather than promote oncogenesis. Later publications high levels did not become IL-3-independent but showed a (35–37) showed that the Rap1 effector domain, being virtually subtle increase in growth rate and saturation density compared identical to that of Ras, can bind to many Ras effectors without with empty vector transductants (Fig. 6B). However, the dif- activating them. These data have led to the hypothesis that ference in growth rate and saturation density was evident only Rap1 serves as a Ras antagonist by sequestering Ras effectors. in the presence of 10 or 50 ng/ml PMA. PMA activates the More recent work (38), however, has shown that Ras activity is guanine nucleotide exchange activity of CalDAG-GEF I (1). not inhibited by endogenous Rap1 activation, suggesting that Rap1, but Not Ras, Activation in Cells Expressing CalDAG- Rap1 has distinct biological functions, apart from the inhibition GEF Ia at High Levels—To determine whether enforced ex- of Ras. pression of CalDAG-GEF Ia in cells resulted in increased en- Support for this hypothesis has come from studies of the dogenous Rap activation in myeloid cells, we measured TSC2 tumor suppressor gene. TSC2 encodes tuberin, which endogenous Rap-GDP and Rap-GTP in empty vector or MSCV- has Rap1 GAP activity (39). In addition, two common human CalDAG-GEF Ia transduced populations of 32Dcl3Gr cells. gliomas, astrocytoma and ependymoma, have been shown to These cells were starved of serum and IL-3 for 10 h and stim- have increased Rap1 expression or reduced/absent tuberin ex- ulated with calcium ionophore and PMA to activate CalDAG- pression in 50 – 60% of the tumors examined (40). In Swiss 3T3 GEF Ia for 15 min. CalDAG-GEF Ia-transduced cells showed cells, Rap1-GTP can induce DNA synthesis and a transformed an increase in induced Rap-GTP levels at 15 min compared morphology (41). Expression of wild type Rap1 at high levels in with control cells (Fig. 7). The levels of Ras-GTP did not vary Swiss 3T3 cells causes morphological transformation and tu- significantly between CalDAG-GEF Ia and parental 32Dcl3Gr morigenicity in nude mice (42). This phenotype is similar to our CalDAG-GEF I and Myeloid Leukemia 11809 FIG.5. Activity of CalDAG-GEF Ia in Rat2 and Swiss 3T3 cells. A, colony formation at low serum in Rat2 cells. The number of colonies present after seeding 4 3 10 cells per plate of MSCV2.1-trans- duced Rat 2 cells (white bars) or MSCV- CalDAG-GEF Ia-transduced Rat2 cells (black bars) is indicated. Error bars indi- cate standard deviations. B, transformed foci in CalDAG-GEF Ia-transduced Swiss 3T3 cells. Photomicrograph of parental Swiss 3T3 cells, MSCV2.1-transduced Swiss 3T3 cells (MSCV2.1), and of four different transformed foci of MSCV- CalDAG-GEF Ia-transduced Swiss 3T3 cells (MSCV-CalDAG-GEF Ia). C, pri- mary and secondary foci and saturation density in Swiss 3T3. The number of Swiss 3T3 cells per 10-cm dish at conflu- ency is shown for MSCV2.1-transduced cells (white bars) and for MSCV-CalDAG- GEF Ia-transduced Swiss 3T3 cells (black bars). The experiment was performed in 10% FBS or 5% CS. In addition, the aver- age number of transformed foci of paren- tal Swiss 3T3, MSCV2.1-transduced, or MSCV-CalDAG-GEF Ia-transduced Swiss 3T3 cells is shown. Primary foci are the number that appeared after G418 se- lection and growth to confluency. Second- ary foci are the number that appeared after replating and growth to confluency. Error bars indicate standard deviations. observations with CalDAG-GEF Ia, which causes morphologi- ogous to the effects seen after tuberin depletion in other fibro- cal transformation of Swiss 3T3 cells but not NIH 3T3 or Rat2 blast cell lines (43). cells. It is also possible that R-Ras and/or TC21 mediate the effects Our data suggest that signaling via CalDAG-GEF Ia in my- of CalDAG-GEF Ia overexpression. Indeed, TC21 (43– 45) and eloid cells can promote cellular proliferation and increase ad- R-Ras (46, 47) can cause malignant transformation of rodent herence. This is consistent with other published data, which fibroblasts. TC21 has been shown to be overexpressed in breast also suggest that Rap1 signaling regulates adherence. For ex- carcinoma and activated by amino acid substitution or inser- ample, overexpression of the Rap1 GAP, SPA-1 in 32Dcl3 cells tional mutation in some breast and ovarian cancer cell lines leads to a block in Rap1 activation and adherence during gran- (48 –50). Furthermore, activated R-Ras can suppress apoptosis ulocyte-colony stimulating factor induced differentiation (28). and stimulate adhesion in 32Dcl3 cells (51). Finally, recent Rap1 also seems to mediate adhesion induced by CD31 activa- data suggest that CalDAG-GEF I is produced as both the form tion in lymphoid cells (27) and is a major LFA1 activator, we have identified and a longer form, referred to as RasGRP2, permitting adhesion to fibronectin (26). Our results are consist- that is myristoylated or palmitoylated and can act on N- or ent with a role for Rap1 activation in adherence to fibronectin K-Ras (31). Proviral insertion at this locus may therefore not in myeloid cells. It could be imagined that adhesion to fibronec- only up-regulate CalDAG-GEF Ia but also the longer form tin gives an AML clone a selective advantage, either by sup- called RasGRP2. Both forms were shown to be capable of GEF pression of apoptosis via the so-called “outside-in” integrin activity in cells for N-Ras or K-Ras. However, the shorter signal or by permitting the clone to colonize extramedullary nonfatty acid modified form of CalDAG-GEF I was only capable sites or extravasate more readily. of N- and K-Ras GEF activity after chronic PMA treatment or Other data indirectly implicate Rap1 signaling in cell cycle growth in high serum, when it becomes localized to the plasma control and proliferation. Expression of the Rap1 GAP, tuberin, membrane. However, it is not clear that the longer form of this regulates the abundance and subcellular distribution of p27 protein, called RasGRP2, is actually conserved in the mouse. and cyclin D1 proteins in fibroblasts (43). Our data suggest We have looked for mouse EST clones, analogous to the human that overexpression of CalDAG-GEF Ia can cause inappropri- RasGRP2 isoform, but could find none. Furthermore, the alter- ate cell division in Rat2 cells at low serum. This may be anal- nate exon that encodes most of the additional N-terminal 11810 CalDAG-GEF I and Myeloid Leukemia FIG.7. Ras and Rap activation in CalDAG-GEF Ia-transduced cells. The percentage of total Rap1 protein bound to GTP or total Ras protein bound to GTP is indicated for 32Dcl3Gr cells transduced with MSCV2.1-empty vector or MSCV-CalDAG-GEF Ia viruses. These val- ues are from cells at time 0 (white bars) and 15 min after stimulation (black bars) of IL-3 and serum-starved cells with calcium ionophore and PMA. The average of an experiment done in triplicate is shown. proviral insertion and may be activated (6), and the Ras GAP Nf1 gene is inactivated in BXH2 AML (4, 5). Therefore, it will FIG.6. Activity of CalDAG-GEF Ia in FDCP1 and 32Dcl3Gr be important to determine whether and where Rap1 and N-, K-, cells. A, appearance of adherent cells in PMA plus calcium ionophore or Ha-Ras signalings overlap. Indeed, a lot of data have accu- (white boxes; PMA 1 Ca) and IL-3 plus PMA plus calcium ionophore mulated showing that small G proteins can cooperate in cell (black boxes; IL-3 1 PMA 1 Ca)-treated 32Dcl3Gr cells. The average growth control (56) or must act in concert for cellular transfor- number of cells per field (at 3 magnification) remaining on the tissue culture dish 2 h after replating MSCV2.1-empty vector transductants or mation to occur (37, 51, 57– 60). MSCV-CalDAG-GEF Ia transductants and washing in PBS is shown. Functional Role of a Truncated GEF—CalDAG-GEF Ib, the Standard deviations are indicated. B, fold increase at saturation den- truncated form of CalDAG-GEF Ia, is unique in that no other sity of FDCP1 cells growing in IL-3. The maximal fold increases in cell GEFs identified thus far have a truncated form. The simplest number for MSCV2.1-transduced FDCP1 cells (white boxes) or MSCV- CalDAG-GEF Ia-transduced FDCP1 cells (black boxes) are shown. Cells model to explain the function of CalDAG-GEF Ib is that it acts were initially plated in normal growth media with IL-3 at 0, 10, or 50 as a dominant-negative form of CalDAG-GEF Ia, serving to mg/ml PMA, and the cell number was determined every day for 4 days. modulate its activity. The only identified domain within Error bars indicate standard deviations. CalDAG-GEF Ib is the REM domain. A REM-like domain is found N-terminal to the core catalytic CDC25-like domain of amino acids in RasGRP2 is not well conserved at the mouse many GEF proteins. Although the function of the REM domain CalDAG-GEF I locus (data not shown). In addition, we have not is not entirely clear, an intact REM domain is required for the ever detected increased Ras activation in CalDAG-GEF I over- transforming effects of RasGRP overexpression in fibroblasts expressing 32Dcl3 or FDCP1 cells. Nevertheless, it is conceiv- (19). The CDC25-like core catalytic domain of CDC25Mm will able, and seems appealing, to consider that the combined acti- catalyze the exchange of GDP for GTP on purified Ras protein, vation of multiple small GTPases by CalDAG-GEF I is but the inclusion of the REM domain increases the efficiency of responsible for its oncogenicity. this reaction (61). It is at present unclear which of the signaling pathways The crystallization and structural determination of Ha-Ras downstream of Rap1, TC21, Ha-, N- or R-Ras activation can be protein complexed with a fragment of the SOS protein lends linked to apoptosis suppression, adherence, or hyperprolifera- some insight into the role of the REM domain (62). The REM tion seen in CalDAG-GEF Ia-overexpressing cells. Rap1 signal- domain of SOS contains three a-helices. The first two of the ing has been shown to activate the mitogen-activated protein helices interact with, and may stabilize, a portion of the kinase pathway independently of Ras signaling via B-Raf (52, CDC25-like domain (62). The third a-helix and the region of the 53). In addition, Rap1-GTP, like Ras can bind to the Ral gua- protein between the REM domain and the CDC25-like domain nine exchange factors RalGDS and Rgl1 (35, 36). The function interact with neither the core catalytic CDC25-like domain nor of Ral-GTP is not known, but Ral dominant-negative mutants Ha-Ras. This portion of SOS may thus interact with other block R-Ras-induced adhesion to fibronectin in 32D cells (51). proteins. The amino acid sequence of the region between the Ral is also thought to be downstream of Ras signaling and REM and CDC25-like domains is not conserved between SOS forms a required component of the Ras transformation re- and CalDAG-GEF I. The conservation of a short form of sponse (54, 55). TC21 and R-Ras have been shown to activate CalDAG-GEF I, CalDAG-GEF Ib, in both mouse and human the stress-activated protein kinases, p38 and JNK, as well as that includes the REM domain and most of the amino acid phosphatidylinositol 3-kinase (44 – 47). Further work will be sequence between the REM domain and the CDC25-like do- required to determine which of these pathways is important for main, implies that this region of the protein may have an CalDAG-GEF I-induced leukemia. Furthermore, the CalDAG- unidentified and unique regulatory function. For example, GEF II (RasGRP1) Ras GEF gene is found at a common site of CalDAG-GEF Ib may regulate CalDAG-GEF Ia activity by CalDAG-GEF I and Myeloid Leukemia 11811 Mushinski, J. F., and Risser, R. (1992) Oncogene 7, 811– 819 interacting with and titrating a protein(s) that also binds to 23. Scheele, J. S., Rhee, J. M., and Boss, G. R. (1995) Proc. Natl. Acad. Sci. U. S. A. and regulates CalDAG-GEF Ia and perhaps other GEFs. 92, 1097–1100 24. Sharma, P. M., Egawa, K., Huang, Y., Martin, J. L., Huvar, I., Boss, G. R., and CalDAG-GEF I in Other Malignancies—CalDAG-GEF I Olefsky, J. M. (1998) J. Biol. Chem. 273, 18528 –18537 maps to human chromosome 11q13 (20). The 11q13 region has 25. Von Lintig, F. C., Pilz, R. B., and Boss, G. R. (2000) Oncogene, 19, 4029 – 4034 been implicated in a variety of malignancies including breast 26. Katagiri, K., Hattori, M., Minato, N., Irie, S., Takatsu, K., and Kinashi, T. (2000) Mol. Cell. Biol. 20, 1956 –1969 adenocarcinoma, bladder carcinoma, gastric cancer, B cell fol- 27. Reedquist, K. A., Ross, E., Koop, E. A., Wolthuis, R. M., Zwartkruis, F. J., van licular lymphoma, and oral squamous cell carcinoma (63, 64). Kooyk, Y., Salmon, M., Buckley, C. D., and Bos, J. L. (2000) J. Cell Biol. 148, Both translocations and gene amplifications have been ob- 1151–1158 28. Tsukamoto, N., Hattori, M., Yang, H., Bos, J. L., and Minato, N. (1999) J. Biol. served in these cases. It is widely believed that activation of the Chem. 274, 18463–18469 cyclin D1 gene (CYCD1) or fibroblast growth factor superfamily 29. Hussey, D. J., Nicola, M., Moore, S., Peters, G. B., and Dobrovic, A. (1999) Blood 94, 2072–2079 genes, such as FGF and HIS, are the targets for genomic 30. Yamashita, S., Mochizuki, N., Ohba, Y., Tobiume, M., Okada, Y., Sawa, H., alterations in this region (64). However, our identification of Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, 25488 –25493 31. Clyde-Smith, J., Silins, G., Gartside, M., Grimmond, S., Etheridge, M., Apol- CalDAG-GEF I as a proto-oncogene in BXH-2 AML suggests loni, A., Hayward, N., and Hancock, J. F. (2000) J. Biol. Chem. 275, that its activation may also be selected in some of these cases. 32260 –32267 Regarding these tumors, it seems possible that selection is 32. Barbacid, M. (1990) Eur. J. Clin. Invest. 20, 225–235 33. McCormick, F. (1999) Trends Cell Biol. 9, M53—M56 occurring due to effects on multiple genes. Rap activity has 34. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) been implicated in changes in the adhesion and migratory Cell 56, 77– 84 capacity of cells in culture (26 –28). Thus, it seems plausible 35. Ikeda, M., Koyama, S., Okazaki, M., Dohi, K., and Kikuchi, A. (1995) FEBS Lett. 375, 37– 40 that CalDAG-GEF I gene activation could play a role in the 36. Wolthuis, R. M., Bauer, B., van’t Veer, L. J., de Vries-Smits, A. M., Cool, R. H., metastatic behavior of these carcinomas. Spaargaren, M., Wittinghofer, A., Burgering, B. M., and Bos, J. L. (1996) Oncogene 13, 353–362 REFERENCES 37. Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Oncogene 17, 1415–38 1. Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumen- 38. Zwartkruis, F. J., Wolthuis, R. M., Nabben, N. M., Franke, B., and Bos, J. L. stiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., (1998) EMBO J. 17, 5905–5912 Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. 39. Wienecke, R., Konig, A., and DeClue, J. E. (1995) J. Biol. Chem. 270, Acad. Sci. U. S. A. 95, 13278 –13283; Correction (1999) Proc. Natl. Acad. 16409 –16414 Sci. U. S. A. 96, 318 40. Gutmann, D. H., Saporito-Irwin, S., DeClue, J. E., Wienecke, R., and Guha, A. 2. Jonkers, J., and Berns, A. (1996) Biochim. Biophys. Acta 1287, 29 –57 (1997) Oncogene 15, 1611–1616 3. Wolff, L. (1997) Biochim. Biophys. Acta 1332, F67–F104 41. Yoshida, Y., Kawata, M., Miura, Y., Musha, T., Sasaki, T., Kikuchi, A., and 4. Buchberg, A. M., Bedigian, H. G., Jenkins, N. A., and Copeland, N. G. (1990) Takai, Y. (1992) Mol. Cell. Biol. 12, 3407–3414 Mol. Cell. Biol. 10, 4658 – 4666 42. Altschuler, D. L., and Ribeiro-Neto, F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5. Largaespada, D. A., Shaughnessy, J. D., Jr., Jenkins, N. A., and Copeland, 7475–7479 N. G. (1995) J. Virol. 69, 5095–5102 43. Soucek, T., Pusch, O., Wienecke, R., DeClue, J. E., and Hengstschlager, M. 6. Li, J., Shen, H., Himmel, K. L., Dupuy, A. J., Largaespada, D. A., Nakamura, (1997) J. Biol. Chem. 272, 29301–29308 T., Shaughnessy, J. D., Jr., Jenkins, N. A., and Copeland, N. G. (1999) Nat. 44. Graham, S. M., Oldham, S. M., Martin, C. B., Drugan, J. K., Zohn, I. E., Genet. 23, 348 –33 Campbell, S., and Der, C. J. (1999) Oncogene 18, 2107–2116 7. Moskow, J. J., Bullrich, F., Huebner, K., Daar, I. O., and Buchberg, A. M. 45. Movilla, N., Crespo, P., and Bustelo, X. R. (1999) Oncogene 18, 5860 –5869 (1995) Mol. Cell. Biol. 15, 5434 –5443 46. Cox, A. D., Brtva, T. R., Lowe, D. G., and Der, C. J. (1994) Oncogene 9, 8. Nakamura, T., Largaespada, D. A., Shaughnessy, J. D., Jr., Jenkins, N. A., and 3281–3288 Copeland, N. G. (1996) Nat. Genet. 12, 149 –153 9. Nakamura, T., Largaespada, D. A., Lee, M. P., Johnson, L. A., Ohyashiki, K., 47. Huff, S. Y., Quilliam, L. A., Cox, A. D., and Der, C. J. (1997) Oncogene 14, 133–143 Toyama, K., Chen, S. J., Willman, C. L., Chen, I. M., Feinberg, A. P., Jenkins, N. A., Copeland, N. G., and Shaughnessy, J. D., Jr. (1996) Nat. 48. Barker, K. T., and Crompton, M. R. (1998) Br. J. Cancer 78, 296 –300 49. Clark, G. J., Kinch, M. S., Gilmer, T. M., Burridge, K., and Der, C. J. (1996) Genet. 12, 154 –158 10. Shaughnessy, J. D., Jr., Largaespada, D. A., Tian, E., Fletcher, C. F., Cho, Oncogene 12, 169 –176 50. Huang, Y., Saez, R., Chao, L., Santos, E., Aaronson, S. A., and Chan, A. M. B. C., Vyas, P., Jenkins, N. A., and Copeland, N. G. (1999) Oncogene 18, 2069 –2084 (1995) Oncogene 11, 1255–1260 51. Osada, M., Tolkacheva, T., Li, W., Chan, T. O., Tsichlis, P. N., Saez, R., 11. Nakamura, T., Yamazaki, Y., Saiki, Y., Moriyama, M., Largaespada, D. A., Jenkins, N. A., and Copeland, N. G. (2000) Mol. Cell. Biol. 20, 3178 –3186 Kimmelman, A. C., and Chan, A. M. (1999) Mol. Cell. Biol. 19, 6333– 6344 52. Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. 12. Tian, E., Sawyer, J. R., Largaespada, D. A., Jenkins, N. A., Copeland, N. G., and Shaughnessy, J. D., Jr. (2000) Oncogene 19, 2098 –2109 (1997) Cell 89, 73– 82 53. York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and 13. Gutmann, D. H., and Collins, F. S. (1993) Neuron 10, 335–343 14. DeClue, J. E., Papageorge, A. G., Fletcher, J. A., Diehl, S. R., Ratner, N., Vass, Stork, P. J. (1998) Nature 392, 622– 626 54. Urano, T., Emkey, R., and Feig, L. A. (1996) EMBO J. 15, 810 – 816 W. C., and Lowy, D. R. (1992) Cell 69, 265–273 15. Martin, G. A., Viskochil, D., Bollag, G., McCabe, P. C., Crosier, W. J., 55. White, M. A., Vale, T., Camonis, J. H., Schaefer, E., and Wigler, M. H. (1996) J. Biol. Chem. 271, 16439 –16442 Haubruck, H., Conroy, L., Clark, R., O’Connell, P., Cawthon, R. M., Innis, M. A., and McCormick, F. (1990) Cell 63, 843– 849 56. Tang, Y., Yu, J., and Field, J. (1999) Mol. Cell. Biol. 19, 1881–1891 57. Prendergast, G. C., Khosravi-Far, R., Solski, P. A., Kurzawa, H., Lebowitz, 16. Ohba, Y., Mochizuki, N., Yamashita, S., Chan, A. M., Schrader, J. W., Hattori, S., Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, P. F., and Der, C. J. (1995) Oncogene 10, 2289 –2296 58. Qiu, R. G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) Nature 20020 –20026 17. Largaespada, D. A., Brannan, C. I., Jenkins, N. A., and Copeland, N. G. (1996) 374, 457– 459 59. Qiu, R. G., Chen, J., McCormick, F., and Symons, M. (1995) Proc. Natl. Acad. Nat. Genet. 12, 137–143 18. Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J., and Sci. U. S. A. 92, 11781–11785 60. Roux, P., Gauthier-Rouviere, C., Doucet-Brutin, S., and Fort, P. (1997) Curr. Stone, J. C. (1998) Science 280, 1082–1086 19. Tognon, C. E., Kirk, H. E., Passmore, L. A., Whitehead, I. P., Der, C. J., and Biol. 7, 629 – 637 61. Coccetti, P. Mauri, I., Alberghina, L., Martegani, E., and Parmeggiani, A. Kay, R. J. (1998) Mol. Cell. Biol. 18, 6995–7008 20. Kedra, D., Seroussi, E., Fransson, I., Trifunovic, J., Clark, M., Lagercrantz, J., (1995) Biochem. Biophys. Res. Commun. 206, 253–259 62. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Blennow, E., Mehlin, H., and Dumanski, J. (1997) Hum. Genet. 100, 611– 619 Nature 394, 337–343 63. Lammie, G. A., and Peters, G. (1991) Cancer Cells 3, 413– 420 21. Jenkins, N. A., Copeland, N. G., Taylor, B. A., Bedigian, H. G., and Lee, B. K. (1982) J. Virol. 42, 379 –388 64. Mitelman, F., Mertens, F., and Johansson, B. (1997) Nat. Genet. 15 Spec. No., 417– 474 22. Largaespada, D. A., Kaehler, D. A., Mishak, H., Weissinger, E., Potter, M., http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia

Download PDF
8 pages

Loading next page...
 
/lp/unpaywall/activation-of-the-rap1-guanine-nucleotide-exchange-gene-caldag-gef-i-60vGLacW5C

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.m008970200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 15, Issue of April 13, pp. 11804 –11811, 2001 Printed in U.S.A. Activation of the Rap1 Guanine Nucleotide Exchange Gene, CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia* Received for publication, October 2, 2000, and in revised form, January 12, 2001 Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M008970200 Adam J. Dupuy‡, Kelly Morgan‡, Friederike C. von Lintig§, Haifa Shen¶, Hasan Acar‡, Diane E. Hasz‡, Nancy A. Jenkins¶, Neal G. Copeland¶, Gerry R. Boss§, and David A. Largaespada‡i From the ‡University of Minnesota Cancer Center, Institute of Human Genetics, and Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, the §Department of Medicine, University of California San Diego, La Jolla, California 92093, and ¶Mouse Cancer Genetics Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Here we report the recurrent proviral activation of provide powerful genetic tags for disease gene identification (2, the Rap1-specific guanine nucleotide exchange factor 3). During the past several years, a number of disease genes CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., have been identified in BXH-2 AMLs by proviral tagging. They Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., include a tumor suppressor gene, Neurofibromatosis type 1 Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., (Nf1); a gene with homology to the lymphoid-restricted type II Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. membrane protein Jaw1, Mrv integration site 1 (Mrvi1); a gene Acad. Sci. U. S. A. 95, 13278 –13283; Correction (1999) encoding a hematopoietic cell growth and differentiation factor, Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 myeloblastosis oncogene (Myb); three homeobox genes, ho- acute myeloid leukemia. We also show that CalDAG-GEF meobox A7 (Hoxa7), homeobox A9 (Hoxa9), and myeloid eco- I encodes two protein isoforms, a full-length isoform tropic viral integration site 1 (Meis1); a zinc finger protein (CalDAG-GEF Ia) and a C-terminally truncated isoform gene, ecotropic viral integration site 9 (Evi9); and a novel (CalDAG-GEF Ib). Expression of the full-length CalDAG- cytokine receptor (Evi27) (4 –12). Importantly, two of these GEF Ia isoform in Rat2 fibroblasts enhances growth in genes, NF1 and HOXA9, are known human myeloid leukemia low serum, whereas expression in Swiss 3T3 cells causes disease genes, validating the usefulness of this approach for morphological transformation and increased saturation human disease gene identification (9, 13). density. In FDCP1 myeloid cells, CalDAG-GEF Ia expres- Mutations in the human NF1 gene are responsible for the sion increases growth and saturation density in the cancer predisposition syndrome neurofibromatosis type 1 (13). presence of the diacylglycerol analogs phorbol 12-myris- NF1 encodes neurofibromin, a GTPase-activating protein tate 13-acetate (PMA), which activates CalDAG-GEF Ia (GAP) that is active on the four true Ras proteins (Ha-Ras, exchange activity. Likewise, in 32Dcl3 myeloblast cells, K-RasA, K-RasB, and N-Ras) as well as R-Ras (14 –16). In CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore BXH2 AMLs, about 15% of the AMLs have proviral insertions and allows higher saturation densities and prolonged in the Nf1 gene (4, 5). These proviral insertions inhibit neuro- growth on fibronectin-coated plates. These effects were fibromin expression, and no wild type neurofibromin is ex- correlated with increased Rap1, but not Ras, protein pressed in these AMLs (5). Loss of neurofibromin in myeloid activation following PMA and calcium ionophore treat- cells is associated with increased and prolonged Ras activation ment. Our results suggest that Rap1-GTP delivers sig- after cytokine stimulation in chronic myeloid leukemia and in nals that favor progression through the cell cycle and primary cells (17). These results contribute to a large body of morphological transformation. The identification of evidence implicating aberrant regulation of Ras signaling in CalDAG-GEF I as a proto-oncogene in BXH-2 acute my- myeloid leukemia. eloid leukemia is the first evidence implicating Rap1 The other 85% of BXH-2 AMLs do not have proviral inser- signaling in myeloid leukemia. tions in the Nf1 gene (4, 5). This has led to the suggestion that these AMLs have insertions in or near other genes involved in Ras regulation and that these insertions activate Ras in the Retroviral insertional mutagenesis in BXH-2 recombinant absence of inactivating insertions at Nf1. One class of likely inbred mice induces a high incidence of acute myeloid leukemia targets are the guanine nucleotide exchange factors (GEF) that (AML), and the proviral integration sites in the leukemias catalyze the exchange of GTP for GDP on Ras and consequently turn on Ras. One such GEF is RasGRP (18, 19). RasGRP is one of three guanine exchange factors that have calcium-binding * The costs of publication of this article were defrayed in part by the “EF hands” in addition to diacylglycerol (DAG)-binding do- payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to mains (18 –20). It has been speculated that this class of GEFs indicate this fact. couple cell surface receptors that signal through Ca and DAG i To whom correspondence should be addressed. Tel.: 612-626-4979; to the Ras pathway (18, 20). Fax: 612-626-3941; E-mail: [email protected]. In the studies described here we report the recurrent provi- The abbreviations used are: AML, acute myeloid leukemia; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase ral activation of the calcium- and diacylglycerol-binding gua- chain reaction; GAP, GTPase-activating protein; GEF, guanine nucle- nine nucleotide exchange factor 1 (CalDAG-GEF I) gene in otide exchange factors; P/S, penicillin/streptomycin; DMEM, Dulbecco’s BXH-2 AML. Like RasGRP, CalDAG-GEF I has EF hands and modified Eagle’s medium; FBS, fetal bovine serum; IL-3, interleukin-3; DAG domains. Previous studies, however, have shown that CS, calf serum; CalDAG-GEF I, calcium- and diacylglycerol-binding guanine nucleotide exchange factor 1; REM, Ras exchanger motif. CalDAG-GEF I is not a Ras GEF, rather it appears to be a Rap1 11804 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. CalDAG-GEF I and Myeloid Leukemia 11805 the CalDAG-GEF Ia and CalDAG-GEF Ib expression constructs were GEF (1). We also show that forced overexpression of CalDAG- sequenced for errors. Both expression constructs were used in an in GEF I results in transformation of cultured fibroblasts and vitro transcription/translation experiment (Promega) to confirm that implicate Rap1 signaling in producing these effects. This is the they produced an ;70-kDa V5-tagged protein for CalDAG-GEF Ia and first evidence implicating Rap1 signaling in myeloid leukemia 19-kDa Myc-tagged protein for CalDAG-GEF Ib. The CalDAG-GEF Ia in human or mouse. insert was subcloned into the MSCV2.1 retroviral expression vector and electroporated into the PT67 amphotropic packaging cell line EXPERIMENTAL PROCEDURES (CLONTECH). The electroporated PT67 cells were selected in 400 Molecular Cloning of CalDAG-GEF I Proviral Insertions—Proviral mg/ml G418 in 10% fetal bovine serum (FBS), Dulbecco’s modified insertions in the CalDAG-GEF I first intron were discovered as part of Eagle’s medium (DMEM), and penicillin/streptomycin (P/S). Resist- a large scale cloning effort that utilized a long template, inverse PCR ant colonies were pooled and replated at 1–2 3 10 cells/ml. Viral method, described in detail elsewhere (6). In brief, 5 mg of genomic DNA supernatants were harvested 24 h after cells reached confluence and from individual BXH-2 AMLs was digested with SacII overnight; the stored at 270 °C. enzyme was inactivated by heating at 65 °C for 10 min, and the DNA Cell Culture Assays—The following cell types were transduced with fragments were ligated in 500-ml reactions, using 5 units of T4 DNA MSCV-CalDAG-GEF Ia or MSCV2.1 empty vector virus and selected in ligase (Stratagene) at 4 °C overnight, to produce circular provirus/ G418: Rat2, Swiss 3T3, FDCP1, and 32Dcl3Gr. All lines were obtained cellular DNA templates for PCR amplification. The ligated material from the ATCC, apart from 32Dcl3Gr, which was obtained from D. was precipitated in ethanol and resuspended in 20 ml of Tris-EDTA (pH Askew (University of Cincinnati). Rat2 and Swiss 3T3 were grown in 8.0). Two ml of this precipitated material was used as template in a 10% FBS/DMEM supplemented with P/S and selected in 400 mg/ml primary PCR in a 50-ml reaction volume containing 20 nmol of each G418. FDCP1 and 32Dcl3Gr were grown in 20% WEHI-3 cell line dNTP, 10 pmol each forward and reverse primer, 13 buffer 2, and 2.5 conditioned medium in 10% FBS/DMEM supplemented with 10% TM units of enzyme mix in the Expand Long Template PCR System NCTC-109, P/S, Hepes buffer (pH 7.4), 10 units/ml insulin, glutamine, (Roche Molecular Biochemicals). Amplification was performed with a sodium pyruvate, nonessential amino acids, and 10 M b-mercaptoeth- Omnigene Hybaid thermocycler programmed as follows: 92 °C for 2 anol. All media and supplements were from Life Technologies, Inc., min; 10 cycles of 92 °C for 10 s, 63 °C for 30 s, 68 °C for 10 min; 20 cycles except insulin and b-mercaptoethanol (Sigma). FDCP1 and 32Dcl3Gr of 92 °C for 10 s, 63 °C for 30 s, 68 °C for 10 min with 20-s auto- cells were selected in 1 mg/ml G418. Retroviral transduction was done 6 6 extension. The amount of primary PCR product was semi-quantified by by overnight infection of 10 3 10 (FDCP1 and 32Dcl3Gr) or 3 3 10 1% agarose gel electrophoresis and 0.01 to 1 ml of the primary PCR cells (Rat2 and Swiss 3T3) in 10 ml of a 50:50 mix of high titer retroviral product was used as template in a secondary PCR under the same pool and regular growth medium supplemented with 16 mg/ml Poly- conditions except that the secondary primers were used. The secondary brene (Sigma). Twenty four hours after infection, the cells were PCR product was separated on a 1% agarose gel, purified using the switched to selective media. Pooled G418-resistant populations were Geneclean II kit (Bio 101), and directly cloned using CloneAmp® analyzed for phenotypes after mock-infected parallel cultures in G418 pAMP1 System (Life Technologies, Inc.) according to supplied protocol. had cleared. Rat2 cells were tested for colony formation by plating at These particular clones were obtained using primer pairs designed to 4 3 10 cells in 2 ml of 5 or 0.5% FBS/DMEM/P/S medium in 6-well amplify proviruses located 39 of genomic SacII sites. The primers used plates. After 10 days (5% FBS) or 24 days (0.5% FBS) in culture, the in the primary PCRs are as follows: 59 ECO F1 (59-GGCTGCCATGCAC- colonies were fixed and stained with methylene blue and counted. Rat2 GATGACCTT-39) and 59 ECO R4 (59-CGGCCAGTACTGCAACTGAC- cells were also plated in soft agar colony forming assays as described CAT-39). For the secondary PCR the primer pair was 59 ECO F2-dUMP (22). Ten-centimeter plates of G418-resistant Swiss 3T3 transductants (59-GAGGCCACCTCCACTTCTGAGAT-39) and U3REVD-dUMP (59- were examined for foci of morphologically transformed cells after com- CTCTGTCGCCATCTCCGTCAGA-39. The cloned PCR products were ing to confluence. These cells were trypsinized, counted, and replated at TM TM 5 sequenced using the PRISM BigDye Cycle Sequencing Kit 5 3 10 cells/10-cm plate in 10 ml of 10% FBS/DMEM or 5% calf serum (PerkinElmer Life Sciences) on an ABI model 373A DNA Sequencer (CS)/DMEM. Secondary foci (from 10% FBS/DMEM cultures) and total (Applied Biosystems). SP6 and T7 sequencing primers were purchased cells per plate (from 10% FBS/DMEM and 5% CS/DMEM cultures) were from Life Technologies, Inc. counted 14 days after the cells were replated. For growth rates and Southern and Northern Blot Analysis—Isolation and analysis of saturation density, FDCP1 transductants were plated at 2 3 10 cells in BXH2 genomic DNA and poly(A) RNA, by Southern and Northern 2 ml of growth medium with 0, 10, or 50 ng/ml phorbol 12-myristate blotting, was performed using random primed [ P]dCTP-labeled probe 13-acetate (PMA, from Sigma) and counted every day for 4 days. For (Roche Molecular Biochemicals) as described before (21). apoptosis assays, transduced 32Dcl3Gr cells were washed three times Western Blot Analysis—Stably or transiently transfected cells were in 13 PBS and plated at a density of 1 3 10 cells per ml in normal lysed in RIPA buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM growth medium (previously described) lacking WEHI-3-conditioned me- EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) at a concentra- dium, the IL-3 source. PMA was also included in a subset of plates at a tion of 10 3 10 cells per ml at 4 °C for 10 min. The lysates were clarified concentration of 1 mM. Cells were scored at the indicated time points for by centrifugation for 15 min at 14,000 rpm to remove insoluble mate- total cell count by trypan blue dye exclusion and viability. rial. The supernatants were frozen in liquid nitrogen and stored at Measurement of Ras and Rap1 Activation States—The activation 270 °C. The concentration of protein in the supernatants was deter- states of Ras and Rap1 were measured as described previously in mined using the Bradford assay. Fifty mg of protein was loaded onto coupled enzymatic assays that measure the total amount of GTP and SDS-polyacrylamide gels, run, and transferred to nitrocellulose as de- GTP plus GDP bound to Ras or Rap1 (23–25). Briefly, cells were har- scribed (5). The blots were hybridized with anti-tubulin or anti-V5 vested rapidly and snap-frozen in liquid nitrogen. For measuring Ras antibody (Invitrogen) according to manufacturer’s instructions and vi- activation, the frozen cells were extracted in a Hepes-based buffer sualized with the ECL chemiluminescence kit (Amersham Pharmacia containing 1% Triton X-100, and after removing nuclei and subcellular Biotech). organelles from the extracts by brief centrifugation, the extracts were Expression Constructs, Electroporation, and Retroviral Vectors—The split in half, one-half receiving the pan-Ras rat monoclonal antibody CalDAG-GEF Ia and CalDAG-GEF Ib open reading frames were cloned Y13–259 (the experimental sample) and the other half receiving rat IgG from a mouse bone marrow cDNA library using the HIFI polymerase (the control sample). After shaking for1hat4 °C,Raswas collected on chain reaction kit from Roche Molecular Biochemicals. Primer se- protein G-agarose beads to which goat anti-rat antibody was bound. quences used to amplify full-length CalDAG-GEF Ia are forward 59- The immunoprecipitated Ras was washed four times in lysis buffer, and ACCGGCAGCCATGACGA-39 and reverse 59-GTGGATGTCAAA- GTP and GDP bound to Ras were eluted by heating. The samples were CACTCCGTCCTC-39. PCR products were gel-purified and cloned into split in half, and GTP was measured in one-half by conversion to ATP the pcDNA3.1/V5/His-TOPO vector from Invitrogen. This vector added using ADP, and the enzyme nucleoside diphosphate kinase and the a V5 epitope tag to the C-terminal end of the CalDAG-GEF Ia protein. resulting ATP was measured by the firefly luciferase system that is Primer sequences used to amplify full-length CalDAG-GEF Ib are for- sensitive to 1 fmol. The control sample was subtracted from the exper- ward 59-CTCGAGCCGTGGGAGGCTCTGAGA-39 and reverse 59-TCTA- imental sample, and the amount of GTP was determined from standard GAAGCACATATGGTGTAGCATGCG-39. These primers added a XhoI curves prepared with each set of samples. The sum of GTP plus GDP site and a XbaI site to the 59 and 39 ends of the PCR product, respec- was measured in the other half of the sample by converting GDP to GTP tively. PCR products were gel-purified, digested with XhoI and XbaI, using phosphoenolpyruvate and pyruvate kinase, and then GTP, which and then cloned into the XhoI and XbaI sites of the now represents the sum of GTP plus GDP, was measured as above. pcDNA3.1/Myc-His(1) from Invitrogen. This vector added a Myc Rap1 activation was measured similarly, but since there are no good epitope tag to the C-terminal end of the CalDAG-GEF Ib protein. Both immunoprecipitating antibodies for Rap1, the Rap-binding domain of 11806 CalDAG-GEF I and Myeloid Leukemia FIG.1. A, proviral insertions in the first intron of the CalDAG-GEF I gene. Exons are shown as black boxes, and the position and orientation of the proviruses at CalDAG-GEF I are indicated by an arrow. B, Northern blot analysis of BXH-2 AMLs with proviral insertions at CalDAG-GEF I. BXH-2 poly(A) RNAs are shown hy- bridized with a CalDAG-GEF Ib cDNA probe. The size (in kilobase pairs (kb)) and position of migration of RNA markers are indicated to the left. Tumor 22 has a proviral insertion in the first intron of the CalDAG-GEF I gene. Below the blot are shown the Gapdh-normalized CalDAG- GEF I transcript levels as determined us- ing PhosphorImager analysis. Ral GDS fused to glutathione S-transferase was used to isolate Rap GTP from one-half of the sample on glutathione-Sepharose beads (25). The GTP was then eluted from Rap and measured as described above. To measure the sum of GTP and GDP bound to Rap in the other half of the sample, extracts were incubated in the absence of magnesium with 10 mM GTP to convert Rap-GDP to Rap-GTP. Excess free GTP was removed by a temperature-dependent phase extraction since, as part of the procedure for measuring Rap1 activation, cells were extracted in 0.92% Triton X-114, 0.08% Triton X-45; an aqueous phase containing the free GTP and a detergent phase containing the Rap1 were gener- ated by warming the samples to 15 °C for 2 min. Rap-GTP, which now represented the sum of Rap-GTP plus Rap-GDP, was then isolated by binding to the Rap binding domain glutathione S-transferase fusion protein, and the GTP was eluted and measured as described above. RESULTS Proviral Integration at CalDAG-GEF I in BXH-2 AML Leads to Increased CalDAG-GEF I Expression—Proviral insertions within the first intron of the CalDAG-GEF I gene were identi- fied as part of a large screen to discover proviral insertions that are located near CpG islands (6). Sequence analysis of two of the clones showed that they were located in the first intron of the mouse homolog of the human CDC25-like gene (Fig. 1A) also called CalDAG-GEF I (1). Northern blot analysis of one of the leukemias (number 22) showed that CalDAG-GEF I expres- sion was elevated compared with other BXH-2 leukemias (numbers 13, 29, 106, 117, and 132) that did not contain viral integrations at CalDAG-GEF I (Fig. 1B). These results show that viral integration at CalDAG-GEF I leads to increased CalDAG-GEF I expression. FIG.2. Expression of CalDAG-GEF I mRNA in adult tissues Two CalDAG-GEF I-hybridizing transcripts were detected in and the embryo. Poly(A) RNA, isolated from day 7, 11, 15, and 17 all BXH-2 leukemias tested. This was surprising because the normal mouse embryos or normal adult heart (Hr), brain (Br), spleen shorter transcript has not been reported in humans (1). Inter- (Sp), lung (Lg), liver (Li), skeletal muscle (Sm), kidney (Kd), and testis estingly, both transcripts were up-regulated by viral integra- (Ts), was hybridized with a CalDAG-GEF Ib cDNA probe. CalDAG-GEF Ia and CalDAG-GEF Ib transcripts are indicated. tion at CalDAG-GEF I (Fig. 1B). This prompted us to examine normal mouse tissues for the shorter CalDAG-GEF I tran- script. As shown in Fig. 2, both CalDAG-GEF I transcripts are pression found between 15 and 17 days of development. Sub- expressed in normal mouse tissues. Expression was highest in sequently, we confirmed that the shorter transcript is also brain, heart, and lung followed by spleen, liver and kidney. expressed in humans (data not shown). The shorter transcript Expression was not detected in skeletal muscle. Both tran- hybridized to probes composed of CalDAG-GEF I exons 1–5 but scripts are also expressed in the embryo with the highest ex- not with probes from farther downstream (data not shown). In CalDAG-GEF I and Myeloid Leukemia 11807 FIG.3. Alternate polyadenylation of the CalDAG-GEF I transcript and predicted primary sequence of CalDAG-GEF Ia and CalDAG-GEF Ib isoforms. The predicted protein domain structures for CalDAG-GEF Ia and CalDAG-GEF Ib are shown above. Shown below are the nucleotide sequences surrounding the splice donor site at the end of the mouse and human fifth exon. Exon 5 sequences are shown in bold. The consensus polyadenylation site (AATAAA) present in both mouse and human intron 5 is shown in bold at the end of each sequence. The amino acid sequence of the C terminus of CalDAG-GEF Ib, encoded by intron 5 sequences, is shown for both mouse and human. Significant homology exists between intron 5 encoded amino acid sequence between human and mouse. fact, the size of the shorter transcript is approximately what CalDAG-GEF Ia, and a truncated protein, CalDAG-GEF Ib. would be expected for an mRNA that was polyadenylated Effects of CalDAG-GEF Ia Overexpression in Fibroblasts and shortly after the fifth exon. Quantitation by PhosphorImager Myeloid Cells—Both CalDAG-GEF I transcripts were cloned analysis of normal tissue Northern blots, hybridized with a into mammalian expression vectors through PCR amplification probe containing exons 1–5, showed that the long and short of lymph node cDNA. Primers were designed for both tran- transcripts are usually present at a roughly 1:1 ratio, although scripts to allow the PCR fragments to be cloned in-frame into in some tissues more long form is expressed (Fig. 2). an expression vector and to add an epitope tag to the 39 end of Alternate Polyadenylation Predicts the Expression of a Full- each cDNA. This resulted in a Cdgla clone tagged with a V5 length (CalDAG-GEF Ia) and a C-terminal Truncated epitope and a CalDAG-GEF Ib clone tagged with a Myc epitope. (CalDAG-GEF Ib) Protein—Sequence analysis of the human The cytomegalovirus promoter was used to drive both con- CalDAG-GEF I genomic locus revealed a putative alternate structs. To determine whether the expression vectors func- polyadenylation site that was located just after the splice donor tioned as expected, we transiently transfected each vector into site within intron 5 (Fig. 3). This polyadenylation site is also HeLa cells and then followed their expression using antibodies present in the mouse. Examination of EST data bases revealed to the epitope tag. Surprisingly, we were unable to detect a number of mouse and human ESTs representing cDNA clones CalDAG-GEF Ib protein expression by Western analysis. This in which polyadenylation apparently occurred at this site contrasts with in vitro transcription/translation studies where rather than at the end of the last CalDAG-GEF I exon. These we were able to detect Cgd1b epitope-tagged protein (data not cDNA clones contain CalDAG-GEF I sequences from exon 5, shown). We have also failed to generate stable CalDAG-GEF which then read into the fifth intron and are polyadenylated Ib-expressing transfectants. These results suggest that the after a consensus ATAATA site. Polyadenylation at this site CalDAG-GEF Ib protein is toxic, unstable, or produced at low would produce a transcript of ;0.6 kilobase pairs. levels. The short CalDAG-GEF Ib transcript is predicted to encode To test for possible transforming effects of the CalDAG-GEF a truncated protein. CalDAG-GEF Ib would include the Ras Ia protein, an MSCV2.1-based expression vector for CalDAG- exchanger motif (REM) domain, amino acid sequences between GEF Ia was constructed (MSCV-CalDAG-GEF Ia) and tested the REM domain and CDC25 domain (i.e. the catalytic core for its ability to express V5 epitope-tagged CalDAG-GEF Ia domain similar to that from the yeast CDC25 gene), and 17 protein in FDCP1, Rat2, and 32Dcl3Gr cells. All MSCV- (mouse) or 20 (human) amino acids encoded by intron 5 (Fig. 3). CalDAG-GEF Ia vector transduced cells expressed readily de- Comparison between the human and mouse CalDAG-GEF I tectable protein (Fig. 4). The Rat2 fibroblast cell line was sub- genomic sequences showed conservation in the region sur- sequently transduced with CalDAG-GEF Ia-expressing virus rounding the alternate polyadenylation site in the fifth intron or empty vector virus pools and selected in G418. Stable Rat2 and indeed the whole fifth intron (72% identity). In contrast, transductants were obtained, and pools of greater than 50 other intron sequences showed much less conservation (e.g. colonies of each were assayed for growth in soft agar and fourth intron, 51% identity). These data indicate that the growth rates in 0.5 or 5% FBS, with or without added PMA, a CalDAG-GEF I locus produces two transcripts through alter- diacylglycerol analog, were measured. Neither population con- nate polyadenylation, which encode a full-length protein, tained cells capable of forming large colonies in soft agar (data 11808 CalDAG-GEF I and Myeloid Leukemia cells during this time (Fig. 7). This is consistent with other reports showing that CalDAG-GEF I overexpression does not affect Ras-GTP levels in transiently transected cells (1). Thus, CalDAG-GEF Ia expression at high levels can influence the level of endogenous Rap1 that becomes activated in response to stimulation. FIG.4. Expression of epitope-tagged CalDAG-GEF Ia in fibro- As has been reported in HL60 cells (25), Rap1 protein in blast and myeloblast cell lines. Total cell lysates from MSCV2.1 32Dcl3Gr cells shows a relatively high basal state of activation empty vector (M) or MSCV-CalDAG-GEF Ia vector (C)-transduced compared with Ras (Fig. 7). This has been suggested to be due FDCP1, Rat2, and 32Dcl3Gr cells were hybridized with an anti-V5- specific monoclonal antibody. All MSCV-CalDAG-GEF Ia vector-trans- to the presence of a threonine at position 61 in Rap1, which duced cells expressed readily detectable protein. decreases its intrinsic GTPase activity (25). DISCUSSION not shown). The number of colonies formed in liquid culture in 0.5% FBS/DMEM was significantly higher for MSCV-CalDAG- Proviral Activation of a Rap1 GEF Gene—A screen of somatic GEF Ia transductants than for the empty vector transductants murine leukemia virus integration sites in BXH-2 AML has both with and without added PMA (Fig. 5A). At higher serum identified a GEF gene, CalDAG-GEF I, that is a target for concentrations, both populations gave rise to a similar number murine leukemia virus integration in BXH2 AMLs. Proviral of colonies. integration at CalDAG-GEF I leads to increased CalDAG-GEF In contrast to Rat2 cells, Swiss 3T3 cells transduced with I expression suggesting that CalDAG-GEF I is a proto-onco- MSCV-CalDAG-GEF Ia formed multiple, transformed foci gene. CalDAG-GEF I is the second GEF to be implicated as a upon reaching confluence after G418 selection (Fig. 5B). These myeloid leukemia disease gene in humans or mouse. The foci appeared as areas of more spindle-shaped cells, growing at NUP98 gene is fused in frame, by translocation, to the broadly high density and in multiple layers. The size of these foci active GEF gene smgGDS in some human myeloid leukemias, increased with time in culture. Upon replating, many second- the result of which may be an increase in GTPase activation ary foci were present in cultures transduced with the MSCV- levels (29). CalDAG-GEF Ia vector. These transformed foci were more CalDAG-GEF I is closely related to another GEF, RasGRP apparent in 10% FBS than in 5% calf serum (CS) medium. The (1, 18, 19, 20). RasGRP has been shown to have GEF activity CalDAG-GEF Ia-transduced Swiss 3T3 cells also reached a for Ha-Ras but little activity for other Ras superfamily mem- higher saturation density than did empty vector transductants bers such as Rap, Rho, or Rac (1, 18, 19). In contrast, CalDAG- (Fig. 5C). GEF I shows little GEF activity for Ha-Ras but very high The MSCV-CalDAG-GEF Ia and empty vector retroviruses activity for Rap1 (1). More recently, RasGRP, CalDAG-GEF I, were also used to transduce cells from the myeloid cell lines and a newly identified GEF, CalDAG-GEF III, have also been 32Dcl3Gr and FDCP1 to test the effects of CalDAG-GEF Ia shown to have exchange activity for R-Ras and TC21 GTPases overexpression on myeloid cells. The growth and viability of (16, 30). Furthermore, another recent paper (31) suggests that 32Dcl3Gr and FDCP1 cells are dependent on the presence of CalDAG-GEF I can cause exchange on N-Ras and K-Ras but interleukin-3 (IL-3) in the culture medium. If IL-3 is removed not Ha-Ras proteins in cells that are chronically stimulated from the culture medium, these cells will die by apoptosis. with PMA or high serum. Thus, activation of Rap1, Ha-Ras, Previous studies have suggested that Rap1 plays a role in N-Ras, R-Ras, and TC21 GTPases could potentially mediate regulating integrin-mediated cell adhesion to fibronectin (26 – the oncogenic effects of CalDAG-GEF I. 28) and therefore fibronectin was included in some of these Small G Protein Signaling and Cancer—There is abundant experiments. genetic evidence from studies of primary cancers, cancer mod- The addition of PMA (50 ng/ml), in the absence of IL-3, els, and transformation systems for a central role for the so caused a slight delay in apoptosis in MSCV-CalDAG-GEF Ia- called “true” Ras genes (HRAS, NRAS, and KRAS) in cancer transduced but not control empty vector-transduced 32Dcl3Gr development (32, 33). Much less is known, however, about the cells, which was most apparent in fibronectin-coated plates role of other small G proteins of the Ras-like subfamily in (data not shown). PMA and calcium ionophore-treated MSCV- oncogenesis. Rap1 was initially identified in a screen for genes CalDAG-GEF Ia-transduced 32Dcl3Gr cells displayed a sub- that can suppress the transformed phenotype of K-Ras-trans- stantial increase in adherence to fibronectin compared with formed fibroblasts (34). This would suggest that Rap1 signals controls (Fig. 6A). FDCP1 cells expressing CalDAG-GEF Ia at suppress rather than promote oncogenesis. Later publications high levels did not become IL-3-independent but showed a (35–37) showed that the Rap1 effector domain, being virtually subtle increase in growth rate and saturation density compared identical to that of Ras, can bind to many Ras effectors without with empty vector transductants (Fig. 6B). However, the dif- activating them. These data have led to the hypothesis that ference in growth rate and saturation density was evident only Rap1 serves as a Ras antagonist by sequestering Ras effectors. in the presence of 10 or 50 ng/ml PMA. PMA activates the More recent work (38), however, has shown that Ras activity is guanine nucleotide exchange activity of CalDAG-GEF I (1). not inhibited by endogenous Rap1 activation, suggesting that Rap1, but Not Ras, Activation in Cells Expressing CalDAG- Rap1 has distinct biological functions, apart from the inhibition GEF Ia at High Levels—To determine whether enforced ex- of Ras. pression of CalDAG-GEF Ia in cells resulted in increased en- Support for this hypothesis has come from studies of the dogenous Rap activation in myeloid cells, we measured TSC2 tumor suppressor gene. TSC2 encodes tuberin, which endogenous Rap-GDP and Rap-GTP in empty vector or MSCV- has Rap1 GAP activity (39). In addition, two common human CalDAG-GEF Ia transduced populations of 32Dcl3Gr cells. gliomas, astrocytoma and ependymoma, have been shown to These cells were starved of serum and IL-3 for 10 h and stim- have increased Rap1 expression or reduced/absent tuberin ex- ulated with calcium ionophore and PMA to activate CalDAG- pression in 50 – 60% of the tumors examined (40). In Swiss 3T3 GEF Ia for 15 min. CalDAG-GEF Ia-transduced cells showed cells, Rap1-GTP can induce DNA synthesis and a transformed an increase in induced Rap-GTP levels at 15 min compared morphology (41). Expression of wild type Rap1 at high levels in with control cells (Fig. 7). The levels of Ras-GTP did not vary Swiss 3T3 cells causes morphological transformation and tu- significantly between CalDAG-GEF Ia and parental 32Dcl3Gr morigenicity in nude mice (42). This phenotype is similar to our CalDAG-GEF I and Myeloid Leukemia 11809 FIG.5. Activity of CalDAG-GEF Ia in Rat2 and Swiss 3T3 cells. A, colony formation at low serum in Rat2 cells. The number of colonies present after seeding 4 3 10 cells per plate of MSCV2.1-trans- duced Rat 2 cells (white bars) or MSCV- CalDAG-GEF Ia-transduced Rat2 cells (black bars) is indicated. Error bars indi- cate standard deviations. B, transformed foci in CalDAG-GEF Ia-transduced Swiss 3T3 cells. Photomicrograph of parental Swiss 3T3 cells, MSCV2.1-transduced Swiss 3T3 cells (MSCV2.1), and of four different transformed foci of MSCV- CalDAG-GEF Ia-transduced Swiss 3T3 cells (MSCV-CalDAG-GEF Ia). C, pri- mary and secondary foci and saturation density in Swiss 3T3. The number of Swiss 3T3 cells per 10-cm dish at conflu- ency is shown for MSCV2.1-transduced cells (white bars) and for MSCV-CalDAG- GEF Ia-transduced Swiss 3T3 cells (black bars). The experiment was performed in 10% FBS or 5% CS. In addition, the aver- age number of transformed foci of paren- tal Swiss 3T3, MSCV2.1-transduced, or MSCV-CalDAG-GEF Ia-transduced Swiss 3T3 cells is shown. Primary foci are the number that appeared after G418 se- lection and growth to confluency. Second- ary foci are the number that appeared after replating and growth to confluency. Error bars indicate standard deviations. observations with CalDAG-GEF Ia, which causes morphologi- ogous to the effects seen after tuberin depletion in other fibro- cal transformation of Swiss 3T3 cells but not NIH 3T3 or Rat2 blast cell lines (43). cells. It is also possible that R-Ras and/or TC21 mediate the effects Our data suggest that signaling via CalDAG-GEF Ia in my- of CalDAG-GEF Ia overexpression. Indeed, TC21 (43– 45) and eloid cells can promote cellular proliferation and increase ad- R-Ras (46, 47) can cause malignant transformation of rodent herence. This is consistent with other published data, which fibroblasts. TC21 has been shown to be overexpressed in breast also suggest that Rap1 signaling regulates adherence. For ex- carcinoma and activated by amino acid substitution or inser- ample, overexpression of the Rap1 GAP, SPA-1 in 32Dcl3 cells tional mutation in some breast and ovarian cancer cell lines leads to a block in Rap1 activation and adherence during gran- (48 –50). Furthermore, activated R-Ras can suppress apoptosis ulocyte-colony stimulating factor induced differentiation (28). and stimulate adhesion in 32Dcl3 cells (51). Finally, recent Rap1 also seems to mediate adhesion induced by CD31 activa- data suggest that CalDAG-GEF I is produced as both the form tion in lymphoid cells (27) and is a major LFA1 activator, we have identified and a longer form, referred to as RasGRP2, permitting adhesion to fibronectin (26). Our results are consist- that is myristoylated or palmitoylated and can act on N- or ent with a role for Rap1 activation in adherence to fibronectin K-Ras (31). Proviral insertion at this locus may therefore not in myeloid cells. It could be imagined that adhesion to fibronec- only up-regulate CalDAG-GEF Ia but also the longer form tin gives an AML clone a selective advantage, either by sup- called RasGRP2. Both forms were shown to be capable of GEF pression of apoptosis via the so-called “outside-in” integrin activity in cells for N-Ras or K-Ras. However, the shorter signal or by permitting the clone to colonize extramedullary nonfatty acid modified form of CalDAG-GEF I was only capable sites or extravasate more readily. of N- and K-Ras GEF activity after chronic PMA treatment or Other data indirectly implicate Rap1 signaling in cell cycle growth in high serum, when it becomes localized to the plasma control and proliferation. Expression of the Rap1 GAP, tuberin, membrane. However, it is not clear that the longer form of this regulates the abundance and subcellular distribution of p27 protein, called RasGRP2, is actually conserved in the mouse. and cyclin D1 proteins in fibroblasts (43). Our data suggest We have looked for mouse EST clones, analogous to the human that overexpression of CalDAG-GEF Ia can cause inappropri- RasGRP2 isoform, but could find none. Furthermore, the alter- ate cell division in Rat2 cells at low serum. This may be anal- nate exon that encodes most of the additional N-terminal 11810 CalDAG-GEF I and Myeloid Leukemia FIG.7. Ras and Rap activation in CalDAG-GEF Ia-transduced cells. The percentage of total Rap1 protein bound to GTP or total Ras protein bound to GTP is indicated for 32Dcl3Gr cells transduced with MSCV2.1-empty vector or MSCV-CalDAG-GEF Ia viruses. These val- ues are from cells at time 0 (white bars) and 15 min after stimulation (black bars) of IL-3 and serum-starved cells with calcium ionophore and PMA. The average of an experiment done in triplicate is shown. proviral insertion and may be activated (6), and the Ras GAP Nf1 gene is inactivated in BXH2 AML (4, 5). Therefore, it will FIG.6. Activity of CalDAG-GEF Ia in FDCP1 and 32Dcl3Gr be important to determine whether and where Rap1 and N-, K-, cells. A, appearance of adherent cells in PMA plus calcium ionophore or Ha-Ras signalings overlap. Indeed, a lot of data have accu- (white boxes; PMA 1 Ca) and IL-3 plus PMA plus calcium ionophore mulated showing that small G proteins can cooperate in cell (black boxes; IL-3 1 PMA 1 Ca)-treated 32Dcl3Gr cells. The average growth control (56) or must act in concert for cellular transfor- number of cells per field (at 3 magnification) remaining on the tissue culture dish 2 h after replating MSCV2.1-empty vector transductants or mation to occur (37, 51, 57– 60). MSCV-CalDAG-GEF Ia transductants and washing in PBS is shown. Functional Role of a Truncated GEF—CalDAG-GEF Ib, the Standard deviations are indicated. B, fold increase at saturation den- truncated form of CalDAG-GEF Ia, is unique in that no other sity of FDCP1 cells growing in IL-3. The maximal fold increases in cell GEFs identified thus far have a truncated form. The simplest number for MSCV2.1-transduced FDCP1 cells (white boxes) or MSCV- CalDAG-GEF Ia-transduced FDCP1 cells (black boxes) are shown. Cells model to explain the function of CalDAG-GEF Ib is that it acts were initially plated in normal growth media with IL-3 at 0, 10, or 50 as a dominant-negative form of CalDAG-GEF Ia, serving to mg/ml PMA, and the cell number was determined every day for 4 days. modulate its activity. The only identified domain within Error bars indicate standard deviations. CalDAG-GEF Ib is the REM domain. A REM-like domain is found N-terminal to the core catalytic CDC25-like domain of amino acids in RasGRP2 is not well conserved at the mouse many GEF proteins. Although the function of the REM domain CalDAG-GEF I locus (data not shown). In addition, we have not is not entirely clear, an intact REM domain is required for the ever detected increased Ras activation in CalDAG-GEF I over- transforming effects of RasGRP overexpression in fibroblasts expressing 32Dcl3 or FDCP1 cells. Nevertheless, it is conceiv- (19). The CDC25-like core catalytic domain of CDC25Mm will able, and seems appealing, to consider that the combined acti- catalyze the exchange of GDP for GTP on purified Ras protein, vation of multiple small GTPases by CalDAG-GEF I is but the inclusion of the REM domain increases the efficiency of responsible for its oncogenicity. this reaction (61). It is at present unclear which of the signaling pathways The crystallization and structural determination of Ha-Ras downstream of Rap1, TC21, Ha-, N- or R-Ras activation can be protein complexed with a fragment of the SOS protein lends linked to apoptosis suppression, adherence, or hyperprolifera- some insight into the role of the REM domain (62). The REM tion seen in CalDAG-GEF Ia-overexpressing cells. Rap1 signal- domain of SOS contains three a-helices. The first two of the ing has been shown to activate the mitogen-activated protein helices interact with, and may stabilize, a portion of the kinase pathway independently of Ras signaling via B-Raf (52, CDC25-like domain (62). The third a-helix and the region of the 53). In addition, Rap1-GTP, like Ras can bind to the Ral gua- protein between the REM domain and the CDC25-like domain nine exchange factors RalGDS and Rgl1 (35, 36). The function interact with neither the core catalytic CDC25-like domain nor of Ral-GTP is not known, but Ral dominant-negative mutants Ha-Ras. This portion of SOS may thus interact with other block R-Ras-induced adhesion to fibronectin in 32D cells (51). proteins. The amino acid sequence of the region between the Ral is also thought to be downstream of Ras signaling and REM and CDC25-like domains is not conserved between SOS forms a required component of the Ras transformation re- and CalDAG-GEF I. The conservation of a short form of sponse (54, 55). TC21 and R-Ras have been shown to activate CalDAG-GEF I, CalDAG-GEF Ib, in both mouse and human the stress-activated protein kinases, p38 and JNK, as well as that includes the REM domain and most of the amino acid phosphatidylinositol 3-kinase (44 – 47). Further work will be sequence between the REM domain and the CDC25-like do- required to determine which of these pathways is important for main, implies that this region of the protein may have an CalDAG-GEF I-induced leukemia. Furthermore, the CalDAG- unidentified and unique regulatory function. For example, GEF II (RasGRP1) Ras GEF gene is found at a common site of CalDAG-GEF Ib may regulate CalDAG-GEF Ia activity by CalDAG-GEF I and Myeloid Leukemia 11811 Mushinski, J. F., and Risser, R. (1992) Oncogene 7, 811– 819 interacting with and titrating a protein(s) that also binds to 23. Scheele, J. S., Rhee, J. M., and Boss, G. R. (1995) Proc. Natl. Acad. Sci. U. S. A. and regulates CalDAG-GEF Ia and perhaps other GEFs. 92, 1097–1100 24. Sharma, P. M., Egawa, K., Huang, Y., Martin, J. L., Huvar, I., Boss, G. R., and CalDAG-GEF I in Other Malignancies—CalDAG-GEF I Olefsky, J. M. (1998) J. Biol. Chem. 273, 18528 –18537 maps to human chromosome 11q13 (20). The 11q13 region has 25. Von Lintig, F. C., Pilz, R. B., and Boss, G. R. (2000) Oncogene, 19, 4029 – 4034 been implicated in a variety of malignancies including breast 26. Katagiri, K., Hattori, M., Minato, N., Irie, S., Takatsu, K., and Kinashi, T. (2000) Mol. Cell. Biol. 20, 1956 –1969 adenocarcinoma, bladder carcinoma, gastric cancer, B cell fol- 27. Reedquist, K. A., Ross, E., Koop, E. A., Wolthuis, R. M., Zwartkruis, F. J., van licular lymphoma, and oral squamous cell carcinoma (63, 64). Kooyk, Y., Salmon, M., Buckley, C. D., and Bos, J. L. (2000) J. Cell Biol. 148, Both translocations and gene amplifications have been ob- 1151–1158 28. Tsukamoto, N., Hattori, M., Yang, H., Bos, J. L., and Minato, N. (1999) J. Biol. served in these cases. It is widely believed that activation of the Chem. 274, 18463–18469 cyclin D1 gene (CYCD1) or fibroblast growth factor superfamily 29. Hussey, D. J., Nicola, M., Moore, S., Peters, G. B., and Dobrovic, A. (1999) Blood 94, 2072–2079 genes, such as FGF and HIS, are the targets for genomic 30. Yamashita, S., Mochizuki, N., Ohba, Y., Tobiume, M., Okada, Y., Sawa, H., alterations in this region (64). However, our identification of Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, 25488 –25493 31. Clyde-Smith, J., Silins, G., Gartside, M., Grimmond, S., Etheridge, M., Apol- CalDAG-GEF I as a proto-oncogene in BXH-2 AML suggests loni, A., Hayward, N., and Hancock, J. F. (2000) J. Biol. Chem. 275, that its activation may also be selected in some of these cases. 32260 –32267 Regarding these tumors, it seems possible that selection is 32. Barbacid, M. (1990) Eur. J. Clin. Invest. 20, 225–235 33. McCormick, F. (1999) Trends Cell Biol. 9, M53—M56 occurring due to effects on multiple genes. Rap activity has 34. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) been implicated in changes in the adhesion and migratory Cell 56, 77– 84 capacity of cells in culture (26 –28). Thus, it seems plausible 35. Ikeda, M., Koyama, S., Okazaki, M., Dohi, K., and Kikuchi, A. (1995) FEBS Lett. 375, 37– 40 that CalDAG-GEF I gene activation could play a role in the 36. Wolthuis, R. M., Bauer, B., van’t Veer, L. J., de Vries-Smits, A. M., Cool, R. H., metastatic behavior of these carcinomas. Spaargaren, M., Wittinghofer, A., Burgering, B. M., and Bos, J. L. (1996) Oncogene 13, 353–362 REFERENCES 37. Zohn, I. M., Campbell, S. L., Khosravi-Far, R., Rossman, K. L., and Der, C. J. (1998) Oncogene 17, 1415–38 1. Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumen- 38. Zwartkruis, F. J., Wolthuis, R. M., Nabben, N. M., Franke, B., and Bos, J. L. stiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., (1998) EMBO J. 17, 5905–5912 Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. 39. Wienecke, R., Konig, A., and DeClue, J. E. (1995) J. Biol. Chem. 270, Acad. Sci. U. S. A. 95, 13278 –13283; Correction (1999) Proc. Natl. Acad. 16409 –16414 Sci. U. S. A. 96, 318 40. Gutmann, D. H., Saporito-Irwin, S., DeClue, J. E., Wienecke, R., and Guha, A. 2. Jonkers, J., and Berns, A. (1996) Biochim. Biophys. Acta 1287, 29 –57 (1997) Oncogene 15, 1611–1616 3. Wolff, L. (1997) Biochim. Biophys. Acta 1332, F67–F104 41. Yoshida, Y., Kawata, M., Miura, Y., Musha, T., Sasaki, T., Kikuchi, A., and 4. Buchberg, A. M., Bedigian, H. G., Jenkins, N. A., and Copeland, N. G. (1990) Takai, Y. (1992) Mol. Cell. Biol. 12, 3407–3414 Mol. Cell. Biol. 10, 4658 – 4666 42. Altschuler, D. L., and Ribeiro-Neto, F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5. Largaespada, D. A., Shaughnessy, J. D., Jr., Jenkins, N. A., and Copeland, 7475–7479 N. G. (1995) J. Virol. 69, 5095–5102 43. Soucek, T., Pusch, O., Wienecke, R., DeClue, J. E., and Hengstschlager, M. 6. Li, J., Shen, H., Himmel, K. L., Dupuy, A. J., Largaespada, D. A., Nakamura, (1997) J. Biol. Chem. 272, 29301–29308 T., Shaughnessy, J. D., Jr., Jenkins, N. A., and Copeland, N. G. (1999) Nat. 44. Graham, S. M., Oldham, S. M., Martin, C. B., Drugan, J. K., Zohn, I. E., Genet. 23, 348 –33 Campbell, S., and Der, C. J. (1999) Oncogene 18, 2107–2116 7. Moskow, J. J., Bullrich, F., Huebner, K., Daar, I. O., and Buchberg, A. M. 45. Movilla, N., Crespo, P., and Bustelo, X. R. (1999) Oncogene 18, 5860 –5869 (1995) Mol. Cell. Biol. 15, 5434 –5443 46. Cox, A. D., Brtva, T. R., Lowe, D. G., and Der, C. J. (1994) Oncogene 9, 8. Nakamura, T., Largaespada, D. A., Shaughnessy, J. D., Jr., Jenkins, N. A., and 3281–3288 Copeland, N. G. (1996) Nat. Genet. 12, 149 –153 9. Nakamura, T., Largaespada, D. A., Lee, M. P., Johnson, L. A., Ohyashiki, K., 47. Huff, S. Y., Quilliam, L. A., Cox, A. D., and Der, C. J. (1997) Oncogene 14, 133–143 Toyama, K., Chen, S. J., Willman, C. L., Chen, I. M., Feinberg, A. P., Jenkins, N. A., Copeland, N. G., and Shaughnessy, J. D., Jr. (1996) Nat. 48. Barker, K. T., and Crompton, M. R. (1998) Br. J. Cancer 78, 296 –300 49. Clark, G. J., Kinch, M. S., Gilmer, T. M., Burridge, K., and Der, C. J. (1996) Genet. 12, 154 –158 10. Shaughnessy, J. D., Jr., Largaespada, D. A., Tian, E., Fletcher, C. F., Cho, Oncogene 12, 169 –176 50. Huang, Y., Saez, R., Chao, L., Santos, E., Aaronson, S. A., and Chan, A. M. B. C., Vyas, P., Jenkins, N. A., and Copeland, N. G. (1999) Oncogene 18, 2069 –2084 (1995) Oncogene 11, 1255–1260 51. Osada, M., Tolkacheva, T., Li, W., Chan, T. O., Tsichlis, P. N., Saez, R., 11. Nakamura, T., Yamazaki, Y., Saiki, Y., Moriyama, M., Largaespada, D. A., Jenkins, N. A., and Copeland, N. G. (2000) Mol. Cell. Biol. 20, 3178 –3186 Kimmelman, A. C., and Chan, A. M. (1999) Mol. Cell. Biol. 19, 6333– 6344 52. Vossler, M. R., Yao, H., York, R. D., Pan, M. G., Rim, C. S., and Stork, P. J. 12. Tian, E., Sawyer, J. R., Largaespada, D. A., Jenkins, N. A., Copeland, N. G., and Shaughnessy, J. D., Jr. (2000) Oncogene 19, 2098 –2109 (1997) Cell 89, 73– 82 53. York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and 13. Gutmann, D. H., and Collins, F. S. (1993) Neuron 10, 335–343 14. DeClue, J. E., Papageorge, A. G., Fletcher, J. A., Diehl, S. R., Ratner, N., Vass, Stork, P. J. (1998) Nature 392, 622– 626 54. Urano, T., Emkey, R., and Feig, L. A. (1996) EMBO J. 15, 810 – 816 W. C., and Lowy, D. R. (1992) Cell 69, 265–273 15. Martin, G. A., Viskochil, D., Bollag, G., McCabe, P. C., Crosier, W. J., 55. White, M. A., Vale, T., Camonis, J. H., Schaefer, E., and Wigler, M. H. (1996) J. Biol. Chem. 271, 16439 –16442 Haubruck, H., Conroy, L., Clark, R., O’Connell, P., Cawthon, R. M., Innis, M. A., and McCormick, F. (1990) Cell 63, 843– 849 56. Tang, Y., Yu, J., and Field, J. (1999) Mol. Cell. Biol. 19, 1881–1891 57. Prendergast, G. C., Khosravi-Far, R., Solski, P. A., Kurzawa, H., Lebowitz, 16. Ohba, Y., Mochizuki, N., Yamashita, S., Chan, A. M., Schrader, J. W., Hattori, S., Nagashima, K., and Matsuda, M. (2000) J. Biol. Chem. 275, P. F., and Der, C. J. (1995) Oncogene 10, 2289 –2296 58. Qiu, R. G., Chen, J., Kirn, D., McCormick, F., and Symons, M. (1995) Nature 20020 –20026 17. Largaespada, D. A., Brannan, C. I., Jenkins, N. A., and Copeland, N. G. (1996) 374, 457– 459 59. Qiu, R. G., Chen, J., McCormick, F., and Symons, M. (1995) Proc. Natl. Acad. Nat. Genet. 12, 137–143 18. Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J., and Sci. U. S. A. 92, 11781–11785 60. Roux, P., Gauthier-Rouviere, C., Doucet-Brutin, S., and Fort, P. (1997) Curr. Stone, J. C. (1998) Science 280, 1082–1086 19. Tognon, C. E., Kirk, H. E., Passmore, L. A., Whitehead, I. P., Der, C. J., and Biol. 7, 629 – 637 61. Coccetti, P. Mauri, I., Alberghina, L., Martegani, E., and Parmeggiani, A. Kay, R. J. (1998) Mol. Cell. Biol. 18, 6995–7008 20. Kedra, D., Seroussi, E., Fransson, I., Trifunovic, J., Clark, M., Lagercrantz, J., (1995) Biochem. Biophys. Res. Commun. 206, 253–259 62. Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Blennow, E., Mehlin, H., and Dumanski, J. (1997) Hum. Genet. 100, 611– 619 Nature 394, 337–343 63. Lammie, G. A., and Peters, G. (1991) Cancer Cells 3, 413– 420 21. Jenkins, N. A., Copeland, N. G., Taylor, B. A., Bedigian, H. G., and Lee, B. K. (1982) J. Virol. 42, 379 –388 64. Mitelman, F., Mertens, F., and Johansson, B. (1997) Nat. Genet. 15 Spec. No., 417– 474 22. Largaespada, D. A., Kaehler, D. A., Mishak, H., Weissinger, E., Potter, M.,

Journal

Journal of Biological Chemistry – Unpaywall

Published: Apr 1, 2001

There are no references for this article.