Apr 10, 1986 - protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of. 100 amino acids. In contrast to raf, ...
MOLECULAR AND CELLULAR BIOLOGY, JUlY 1986, p. 2655-2662 0270-7306/86/072655-08$02.00/0 Copyright C 1986, American Society for Microbiology
Vol. 6, No. 7
Characterization of Murine A-raf, a New Oncogene Related to the v-raf Oncogene HULEIHEL,l M. GOLDSBOROUGH,' J. CLEVELAND,' M. GUNNELL,2 T. BONNER,3 AND ULF R. RAPP'* Laboratory of Viral Carcinogenesis' and Program Resources, Inc., 2 National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701, and Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 208923
M.
Received 20 December 1985/Accepted 10 April 1986
A 1.6-kilobase cDNA (A-raj) has been isolated from a murine spleen cDNA library which encodes part of a protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of 100 amino acids. In contrast to raf, A-raf shows a highly restricted tissue distribution of expression, with highest levels observed in epididymis, followed by intestine. When incorporated into a retrovirus, the resulting gag-A-raf fusion gene causes transformation in vitro and induces tumors in newborn mice. Thus, A-raf represents a new proto-oncogene. Transformation by A-raf is independent of ras gene function, as is the case for raf and mos but not other oncogenes.
c-raf-1 gene there is a second v-raf homologous gene in humans, the c-raf-1-derived pseudogene c-raf-2 (4).
The v-raf oncogene was originally identified as the transforming gene of the murine retrovirus 3611-MSV (MSV = murine sarcoma virus) (26, 27). A homologous gene, v-mil, was subsequently found in the genome of the avian carcinoma virus MH2 (12, 13, 36), which also carries the oncogene v-myc. The avian homolog of v-raf in MH2 appears to be biologically active (25), although it may not have been maintained as an effective oncogene in this virus (1, 39). In contrast, 3611-MSV efficiently transforms established epithelial (14) and primary as well as established fibroblastic cells in vitro (22a), immortalizes interleukin 3-dependent primary hematopoietic cells (11, 23), and induces predominantly fibrosarcomas and erythroleukemias in newborn mice (25). Combination of v-myc with v-raf in a retrovirus results in the addition of lymphomas and carcinomas to the tumor spectrum of v-raf and a dramatic shortening of the latency period of both v-raf and v-myc specific tumors in mice (25). In humans, the raf homologous gene c-raf-1 (3, 4, 4a) was found to be activated in primary stomach cancer (35) and glioblastoma (9) as determined by DNA transfection into NIH 3T3 cells. 3611-MSV expresses v-raf as part of two differentially modified gag fusion proteins, a myristilated and phosphorylated 79-kilodalton (kDa) protein, p79, and a glycosylated form, p90. However, unlike v-src, which requires myristilation for transforming activity (5, 6, 20, 21, 33), transformation by the normally cytosolic (18, 23) raf protein does not require myristilation. Furthermore, gag-derived functions known to contribute to transformation by other gag-onc fusion proteins (8) are not necessary, since mouse c-raf (18) can be made transforming by promoter insertion (32). The deduced amino acid sequences of v-raf (16) and its avian counterpart v-mil (36) are distantly related to the tyrosine kinase-encoding oncogenes such as src, erbB, fes, abl, and others. However, these viral raf proteins have serine-threonine- rather than tryosine-specific kinase activity (17). Recently the genomic structure of c-raf-1 (3, 4, 4a), the active human homolog of v-raf, has been determined, and the complete amino acid sequence has been deduced from the cDNA sequence (4a). In addition to the active *
We now report the isolation of a cDNA representing a new member of the raf gene family, A-raf, which is related to v-raf. The A-raf gene is transcribed with a different tissue distribution than c-raf, the highest levels being observed in epididymis. Incorporation of A-raf cDNA into a retroviral genome generates a transforming virus; thus, A-raf is a new proto-oncogene. Functionally, A-raf is similar to raf and mos in that transformation is independent of ras gene function. MATERIALS AND METHODS Library screening and hybridization. A mouse spleen cDNA library in the vector AgtlO (2) was screened by hybridization to a v-raf-specific probe (the Xho-SstII fragment) in 3x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 60°C followed by washing of filters in 1x SSC at 60°C. A phage containing a 1.6-kilobase (kb) EcoRI insert was purified, subcloned into plasmids pUC12 and pUC13, and characterized by restriction mapping and hybridization to digests of v-raf and the corresponding human gene c-raf-1 (4a). Sequencing. The cDNA was sequenced by the dideoxynucleotide chain termination method (30) on fragments inserted in pUC18 and pUC19. The fragments were either specific restriction fragments or deletions generated by exonuclease III digestion. Construction of transforming A-raf DNA. An analog of 3611-MSV was constructed using a cloned murine leukemia virus, LEUK (22), which is closely related to Moloney leukemia virus, and A-raf cDNA. A plasmid containing the complete, unpermuted LEUK DNA was linearized by cutting with XhoI at a site homologous to the XhoI site of Moloney leukemia virus. The XhoI ends were filled using Escherichia coli DNA polymerase in the presence of dATP, dTTP, dCTP, and dGTP. After treatment with bacterial alkaline phosphatase, these molecules were ligated with the 1.4-kb A-raf NcoI-EcoRI fragment, which had previously been blunt ended by filling the ends using E. coli DNA polymerase in the presence of dATP dCTP, and dTTP, but not dGTP, and treating with S1 nuclease to remove the single unpaired nucleotide from the NcoI site.
Corresponding author. 2655
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HULEIHEL ET AL.
2
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FIG. 1. Southern blot analysis of the human A-raf locus. Highmolecular-weight DNA from the human embryonic cell line HEL 299 (ATCC) was restricted to completion with BglII (lanes 1), SphI (lanes 2), and XbaI (lanes 3), and the fragments were separated electrophoretically on a 1% agarose gel and blotted onto Gene Screen. Blots were hybridized with 32P-labeled, nick-translated c-raf-1 (A) or A-raf (B) cDNA probes in 1.5x SSC at 60°C, washed with 1 x SSC at 60°C, and exposed to Kodak XAR-5 film for 4 days. Sizes of hybridizing fragments were determined from their migration relative to 32P-labeled Hindlll fragments of bacteriophage lambda.
RNA analysis. Total poly(A)+ RNAs from mouse tissues the generous gift of Rolf Muller (European Molecular
were
Biology Laboratory). Polysome-associated RNA from the cell line NFS-60 was isolated as described previously (24), and poly(A)+ RNA was selected by oligo(dT)-cellulose chro-
matography. Equal quantities of poly(A)+ RNA (20 jig) were denatured by glyoxalation (29), transferred to nitrocellulose, and hybridized under stringent conditions to 32P-labeled nick-translated probes as described by Thomas (38). DNA analysis. High-molecular-weight DNA was prepared from isolated nuclei of the human embryonic lung cell line HEL 299 (ATCC) as described previously. DNAs were digested to completion with BglIl, SphI, and XbaI, separated electrophoretically on 1% agarose, and blotted onto Gene Screen (New England Nuclear Corp.). Blots were hybridized with the 2.97-kb c-raf-1 cDNA (4a) and 1.6-kb A-rqf cDNA probes in 1.5x SSC at 60°C, washed extensively with lx SSC at 60°C, and exposed to XAR-5 film. Immunoprecipitation. Cells were labeled and lysed as previously described (3, 32). Clarified lysates were incubated in 1-ml portions with 10 ,ul of antibody and 100 ,ul of protein A-agarose containing 1 mg of bovine serum albumin per ml. Antibodies included rabbit sera raised against p309a9 and against a v-raf C-terminal synthetic peptide, SP63. Incubation was overnight at 4°C with gentle shaking. Immunoprecipitates were washed three times with lysis buffer, and pellets were dissolved in 50 ,ul of sample buffer, heated for 2 min at 100°C, and run on a 5 to 20% sodium dodecyl sulfate-polyacrylamide gel. The gel was fixed in 200 ml of 14% acetic acid-10% methanol for 0.5 h and then transferred
to 200 ml of Amplify (Amersham Corp.) for an additional 0.5 h. The gel was then dried and exposed to SB-5 film (Kodak) overnight. DNA transfection. Transfection of NIH 3T3 cells with cloned DNA was carried out by the calcium phosphate precipitation procedure as previously described (26). Transformed foci were counted 8 days posttransfection.
RESULTS A-raf is related to v-raf. The 1.6-kb A-raf cDNA was isolated from a mouse spleen cDNA library by using a v-raf-specific probe at moderate stringency. However, its restriction map did not correspond to that of v-raf. In particular, it lacked the SphI site present in v-rafand human c-raf-1 even though it hybridized to portions of v-raf on both sides of the v-raf Sphl site. Moreover, A-raf appeared to be a different gene as judged from a comparison of Southern blots of restricted human genomic DNA (Fig. 1) hybridized with c-raf-1 (Fig. 1A) and A-raf (Fig. 1B) probes. To further characterize the homology of A-raf to v-raf, the cDNA clone was sequenced (Fig. 2). Comparison of the sequences of A-raf and v-raf established that the sequences are related (Fig. 2), showing 77% homology between nucleotides 700 and 980 but less outside this range. The A-raf sequence has an open reading frame of 1,308 nucleotides beginning at the 5' end which encodes a protein that is closely related to the v-raf and v-mil proteins (Fig. 3). However, the A-raf termination codon occurs 6 nucleotides earlier than in viral and human c-raf-1. A 280-nucleotide 3' untranslated region ends with a poly(A) stretch of 14 nucleotides. The 3' untranslated region is thus considerably shorter than the 900-nucleotide 3' untranslated region of the human c-raf-1 message. Since there is no terminator at the 5' end of this open reading frame, the cDNA must be incomplete. Indeed, the stretch of 436 amino acids encoded by the A-raf cDNA is substantially smaller than the 648 amino acids of the complete human raf protein, and the 1.6-kb size of the A-raf cDNA is substantially less than the 2.4-kb A-raf mRNA (see below). Comparison of the deduced amino acid sequence of murine A-raf with those of murine v-raf and human c-raf-1 indicates that the amino acid homology is as high as 85% for amino acids 187 to 287 but decreases to 70 to 75% at the carboxy terminus and between amino acids 130 and 187 (Fig. 3). Before amino acid 130 there is almost no homology of murine A-raf to human c-raf-1 and avian v-mil, although c-raf-1 and avian v-mil have substantial homology to each other in this region. Indeed, this region showed no significant homology with any other protein in the National Biomedical Research Foundation protein data bank. The one exception is amino acids 40 to 61, which match the c-raf-1 sequence at 19 of 22 amino acids if a gap of 4 amino acids is introduced elsewhere in the A-raf sequence. This homology region coincides with the 5' end of v-mil. Thus, the homology between A-raf and raf appears to be restricted to the kinase domain as defined by homology to other kinase genes as well as the position (amino acids 146 to 151) of the apparent nucleotide binding site. Based on the sequence comparison, the observation that A-raf hybridizes to different restriction fragments in human DNA than does v-raf (Fig. 1), and the fact that these fragments map to different chromosomes, 7 and X (K. Huebner, A. ar-Rushdi, C. A. Griffin, M. Isobe, C. Kozak, B. S. Emmanuel, L. Nagarajan, J. L. Cleveland, M. A. Gunnell, M.D. Goldsborough, C. M. Croce, and U. R. Rapp, Proc. Natl. Acad. Sci. USA, in press) rather than 3
VOL. 6, 1986
THE raf ONCOGENE FAMILY
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0 1 1 K1 41 50 ~~~~~~~~1LUeTr'c1G yProSerProPheTtrGirGi rArs 0nb sreSrr iVc.asP Fxv :A~4AG::C4'11T70TB0~GCT6T44CCCCC 4BBICCC4CCCCT TACC 4CA CGIGB0CC 0GC4TTCTC01T-1-TGCCC3T6C>.4TCC-Cb0CTS ~r, I0170T0 L TTCCAG A CTGTTAT6TCTCAG A ATAT C A CT C6CCTT ACCTTTA AC CAGT CT A1 446G T TCTCC -ra4l 140
130
150
160
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2150
2160
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270
290
200
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300
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320
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3040
350
BlyAsp61yAlaProArQBlySerProSerProAlaSerValSerSerGl Ar LysSerProHisSerLysLeuProSer6luGlnArq6luArqLysSerLeuAlaAsp6luLysLys 6616GATGGAGCCCCTCG 66 AGCCCTAGCCCAGCTAGTGTGT CTTCAGG 646 AAGTCCCCACATTCCAAGTTACCTTCA6AACAGCG6 646C6G 46 TCCTTIG6CAGAT 644446444 Araf A CvriO .---------------------- T 4ACC 6 C A AATCTG CCCAA AGGCT TCAC C 644 ACCC 6T1CC 6 ACA 466G CA CAGTA T66 A CC 6 6 4 C cra4l TGTCCA 4 44 64414 I A
.----C TT GTCT
AACATG
6 T
GGCT TCTC
CCC
ACGC AGTCCC 6 CC464666
C
A
A4CC CCGGAA
C 6 6 A
380 390 400 410 440 450 370 420 430 460 470 LyslalLsAsnLeu6l ylrArqAspSer6l TlyrTyrTrp6luValProProSer6luVal6lnLeuLeuLysAr qlle6I yThrGlySerPhe6lyThrValPheAr q6l LeuTrp A4AGTGA46AACCTGGGG61 CC6664 ~CA1C66 CTATTACTGGG6AGGTGCCACCCAGTGAGGTACAGCTGTTGAGAG6AT1C6A CAG6CTCTUTG66ACT6BTUTTCG 666 CTTTGG 44 64A6 641 I I C A T 6CCT6GT CGAA G64 ACAA CAAG CTICIC I I T 144 A T 6CCT6GT AC 6AA T A A 641 A CC CTC AAAA 6A 1444TAA T ACAAA CAAA A T 6CCTI6T AC AAATTA1141A44 6464 C ACCT TCTCC A A T T TA A
490
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530
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veil
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crafi veil
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HisBIyAspValAlaValLysValLeuLysValAlaGInProThrAla6lu6lnAlaGInAlaPheLysAsn6luMETGInValLeuAr gLysThrAr HisValAsnlleLeuLeuPhe Araf CATG6C6T6AGCTT6A AGTGCTCAA66TGGCCCAACCTACCGCTGAGCA6GCCCAGGCCTTCAA GAATGAGATGCAGGTGCTCAG 6AAGACAC6G6CATBTCAACATITT6CT6TTC 4 I 6 C 66GCT TT6C CA CC vraf T A4AGA4C4 TTGSCAITCA ACIT 6 GCI 1 6C C A 6 C AlT C A T A A GA C A T1T6 C A C A 6 T crafl 6
4
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6ACAG6 GCTAT A
1
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C
C
veil
700 610 620 630 640 6'r0 680 690 650 660 710 METGi PheMlETThrAr 4Pro61 yPheAlIlleIleThrGlnTr pCys6lu6l ySerSerLeuTyrHisHisLeuHisValAlaAspThrAr PheAZpETVal6lnLeulleAspVal 416661 TICATG6ACTC6 6CC666 611GC CATCATCACACABGTB TGTGAGGGTTCCAGCCT CTACCACCACCTACATGTGGCTGA CAC6C6MC114 AT66TCCA6CICATT64166 Araf 64A 44 A CA T vraf AAA 6ACAACC6 6 16TG6 1 A CAB T 6 CCA6 6 CAAA CC 6 I A CAB 44 6 CAITcrafi C 64A CCAGBGCAAG CB I AAA6GACAACC6GA TGGC CA T veil TB A CA6 BTGT A CA AAA6ATAACC 6 BGC TCAA 6CAAG6CCA I
770 730 740 780 790 800 8310 750 820 210O 760 AlaAr qBInThrAlaBIn6l yMETAsp TyrLeuHisAlaLysAsnllelleHisArqAspLeuLysSerAsnAsnhIePheLeuHis6lu6l yLeuThrValLysIle6l yAspPhe6l y CAlT I GACC TAA4GTCCAACAATATCTTCTTACATBA6GGGGCTCACAGTCAAGAITT6IT6ACTTC66C Araf GCCC6 CAGACTGCCCAGGGCA41664ACTACCTCCACGCCAAGAACAl TCATC 11614 A T A A A C CCA 464A 4TCC 664A ATTTIlYrif AB A CTA TIBI CA A 64A 61T A TC C AlTl Tcrafil TA I 116 I A I 46 A 64 C A C CA4 A TCIT 6 A A 4 A B T T vail T 92 870 880 890 940 850 860 9I)0 950 910 a3 LeuAIaThrValLvsThrAr T rpSerBl yAlaiGnProLeuGluGlnFroSerGi ySerVa1LeuTr~META1Ala41BuVa1IieAr8fMETIBnAs8ProAsnProTvrSerPhe6ln CTGBCCACAGTGA4BACArGTG TBBGGBBGCACABCCTGGCGC CCT446463 A4GBGCB T:GTCTATG 0416634 BCTIGAGGTGBTCCBG ATGCA4664A CCGAACCCCT CAGCTTCCAG IA I 3 41 6 B A 111~~~~~~~~T 4614 61T AG 3C: TGAC I C AT I A A I A C TAAC ITT CC4 AGGBT A A4 TA41TT 3 413 4 1 C4 61T AI4 6 A T ABC 34116T A 41AT6 AAT 4666G
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1070 1060 970 1000 1030 1050 980 990 10)4 0 1I11) I0 21' SerAsp Va1TvrA1aTyr6lyVa1Va1LeuTyrG1uLeumETThr6lySerLeuProTvrSerHis1le6lvSerAr AspGlnIlelleFheMETVaIBlyArqBlvTyrLeuSerPra TrBB GGABGCTATBCCTATB6BTGTTTGCTT4BGAGCTC'AT6AC3'GGCTCACT6CC3'74C4G rC4C4TTGGCAGCCGT BA CACAT41 TCT3T' TGGT66G66CC6166C 141 CI6CTCC6 Araf B A 34444 3C' GCTBC CA C 66166T C C66GCT C Tvri4 BC GGG T CAA 41ACT44 GCC CAcrafl T66646 CA TTIC7 CT AG GA A vail A CI 444 4T6 ICT A 44646 4 A444 14 S CT C
1)90 11)0 1111 120 1131 ~~~~ 115 0 II 0 Is8 1190 1,170 ~~11A6 ~ ~~~~ 4s LeuSerLvs1'ieFhecer4c,C~sProLvsAiaMETAr Arq LeULed1'r4'SPC0SLeuLvsPheB1nArqGluG!uAr4ProLeuPhePro61nh1eLeuA1aThrI1e61uLeu Tr4GCr4AA44C'3GT4ATTE3: 34A4i33' GCC6C -TGCTG4Tr 3C46TC63 ATSCBCC AITCCBTCTGGCCACGATCBAGCTA SAC: T B C TITTC 'B A AAA6TCAAAA 4 411T6 A A AGT 6 G SEC A463r T 443 64G C ITITTC I C44rA G T1 B T66 44461444 4 4 3' 6A 464 446 CB 464 11 4446114 B A A T1TT C T CATS6 T6 AA444 lB
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FIG. 2. Nucleotide sequence of A-raf cDNA in comparison with sequences of v-raf, c-raf-1, and v-mil. Dashed lines indicate gaps introduced to align sequences and the lack of corresponding sequence in v-raf and v-mil.
2658
MOL. CELL. BIOL.
HULEIHEL ET AL. 30 40 60 10 20 50 QKAPSNLSVNELLTPQGPSPFTQQRDQEHFSFPAPANPPLQRIRSTSTPNVHMVSTTAPMDSSLMQFTAQ
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FIG. 3. Amino acid sequence of A-raf in comparison with other raf family members. Dashed lines indicate gaps introduced to align sequences, and solid lines demarcate the end of corresponding sequence in v-raf and v-mil.
and 4 for raf (4), we conclude that A-raf is a new member of the raf gene family. Since v-mil is more closely related to raf than is A-raf, it appears that the A-raf and raf genes were derived from a gene duplication event which substantially predates the evolutionary divergene of birds and mammals. Tissue-specific expression of A-raf. Expression of A-raf was investigated in a series of mouse tissues (Fig. 4) and in whole mouse embryos at two stages of development. c-raf was expressed as a 3.1-kb RNA at high levels in 14-day embryo, heart, lung, testes, epididymis, brain, and intestine; lower levels were detectable in spleen, liver, placenta, and 18-day embryo (Fig. 4A). In contrast to c-raf, expression of A-rafrelated RNA was remarkably tissue specific. A 2.4-kb transcript was detected at high levels in epididymis and at lower levels in liver and intestine (Fig. 4B). A-raf is capable of transformation. To test whether A-raf has transforming potential, an analog of 3611-MSV, the virus carrying v-raf, was constructed by cloning the A-raf cDNA in-frame into the gag-p30 region of the LEUK strain of Moloney leukemia virus (22). This new recombinant retrovirus should express a gag-A-raf fusion protein of 75,302 calculated molecular weight, slightly smaller than the gagv-raf fusion protein of 3611-MSV, which has a calculated molecular weight of 79,652. When tested for transforming ability on NIH 3T3 cells, the A-raf recombinant (designated
9IV A-raf MSV) transformed fibroblasts with an efficiency comparable to 3611-MSV (see Table 1). In contrast, a construct containing the A-raf insert in the opposite orientation (8IV) failed to induce any foci. The foci were made up of crisscrossing cells that were less refractile than 3611MSV-transformed NIH 3T3 cells and were more dense than foci induced by Jl virus (25), a variant of 3611-MSV carrying a raflmil hybrid gene. Infected cells grew readily in soft agar at a rate intermediate between Jl and 3611-MSV. Moreover, high-titer stocks of 9IV A-raf MSV have been prepared and found to be tumorigenic in NFS/N mice injected intraperitoneally as newborns. Two different helper viruses were used for rescue of 91V A-raf MSV from nonproducer-transformed, soft agar-derived NIH 3T3 cells. Both pseudotype stocks, one with the LEUK strain of Moloney leukemia virus and the other with Cas Br-M murine leukemia virus (R. Yetter, unpublished data), induced rapid fibrosarcoma and erythroid hyperplasia after a latency of 5 to 10 weeks. This tumor type was not induced by either of the helper viruses alone. Nonproducer cell clones transformed by the 9IV A-raf recombinant virus were isolated from soft agar and tested for the expression of A-raf by Northern blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern blots using A-raf and murine leukemia virus long terminal repeat probes demonstrated a genome-size RNA of
VOL. 6, 1986
THE raf ONCOGENE FAMILY
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-2.4
FIG. 4. Northern hybridization analysis of c-raf-1 (A) and A-raf (B) transcripts expressed in mouse tissues. Total poly(A)+ RNA (20 ,ug) was glyoxalated, blotted, and hybridized with 32P-labeled, nick-translated c-raf-1 (A) and A-raf (B) cDNA probes. The sizes of the RNAs were determined from their migration relative to 32P-labeled, denatured HindlIl fragments of bacteriophage lambda. The blots were exposed for 1 week.
9.6 kb, the predicted size of this virus (data not shown). As was observed with v-raf and c-raf-1, and unlike the situation with v-myc versus c-myc (24), expression of the cellular A-rafgene was not suppressed in cells expressing A-raffrom the viral genome. When we looked for expression of the predicted gag-A-raf fusion protein, using p30-specific antiserum, we observed the expected polypeptide as 75 kDa (Fig. SB, lane 1); the 85-kDa polypeptide in the same lane is presumably a glycosylated version of gag-A-raf, analogous to the glycosylated form of gag-v-raf of 90 kDa (Fig. SA). Immunoprecipitations were also performed with antibody SP63, with specificity for the 12 C-terminal amino acids of c-raf-1 (32). As expected from the sequence (Fig. 5), this antibody recognized the gag-v-rafpolypeptides present in J2 virus (25)-transformed cells (Fig. 5A, lane 3) and the endogenous cellular c-raf-1 protein of 72 kDa, but failed to detect gag-A-raf proteins. Transformation by A-raf is independent of ras gene function. Cellular ras proteins are required for growth of NIH
3T3 fibroblast cells in serum-containing medium (19). However, the cells continue to replicate in the absence of active ras proteins if they express v-raf or v-mos. In contrast, cells transformed by v-sis, v-fms, v-src, and v-fes do not replicate in the absence of active ras proteins (35a). We therefore determined whether expression of A-raf from the viral genome might also overcome the dependence of NIH 3T3 cells on cellular ras proteins by injecting anti-ras antibody 259 (19) into NIH 3T3 cells before and after transformation by either v-raf (3611-MSV) or mouse A-raf (virus construct 91V), in the presence of helper virus (LEUK strain of Moloney leukemia virus or 4070A). Determination of tritiated thymidine incorporation by cells after antibody injection (Table 2) showed that transformation of cells either by v-raf or by virus containing A-raf obviated the cellular dependence on ras proteins for growth in standard medium. We conclude that the N-terminally truncated portion of A-raf present in virus construct 9IV can substitute for v-raf in this assay.
TABLE 1. Transforming efficiency of v-raf and A-raf
DISCUSSION By characterizing a 1.6-kb cDNA derived from mouse spleen, we have identified a new proto-oncogene, which we have called A-raf in recognition of its relatedness to the raf oncogene. The protein encoded by the cDNA has amino acid homology as high as 85% with the kinase region of c-raf-1 but very little similarity elsewhere. In this sense it is similar to the neu gene, which has recently been shown to be related to the erbB oncogene and the EGF receptor. The neu oncogene (31) has been shown by hybridization to be related only to the kinase region of erbB, and the apparently identical DNA fragment MAC117 (31) has been shown by sequence analysis to have 85% amino acid homology to a
Transformation
Transfected DNA a
efficiency DNA)
LEUK (helper) ....................................... PM1 + helper ........................................ 3611-MSV + helper .................................. 91V + helper ........................................ 81V + helper ........................................
0
12,000 4,000 6,000 0
a PM1, A plasmid containing an active v-mos gene. 91V, A hybrid of the LEUK strain of Moloney leukemia virus (XhoI) with the NcoI-EcoRI fragment of A-raf. 81V, A reverse orientation of the insert (A-raf) in hybrid 91V. b FFU, Focus-forming units.
2660
MOL. CELL. BIOL.
HULEIHEL ET AL.
1
M
A
2
3
B
200K-
1
2
3
M
-200K _,* P90
98K69K-
-\
P79
46K-
98K
P85~.
P75 P72
-
69K
-
46K
-
30K
In
30KK 12K- _dd"a.
I*1-_
FIG. 5. raf proteins expressed in fibroblasts transformed by v-raf (J2) and A-raf (9IV) recombinant retroviruses. Lysates of [35S]methionine-labeled J2 virus-transformed NIH 3T3 (A) and 91V-transformed FRE (B) cells were immunoprecipitated with antisera to p30gag (A and B, lanes 1) and SP63 (33) with (lanes 2) and without (lanes 3) competing SP63 peptide. Immunoprecipitates were then analyzed on 5 to 20% sodium dodecyl sulfate-polyacrylamide gels, and the gels were exposed to SB-5 film (Kodak) overnight. M, '4C-labeled molecular weight standards (New England Nuclear Corp.).
portion of the kinase region. Other genes, N-myc and r-fos, which are less closely related to the previously known oncogenes myc and fos, have been described (7, 15, 10, 34, 37). These results suggest that a number of oncogenes belong to small families with members having related yet distinct functions. In the case of A-raf this supposition is supported by the observed differences in tissue distribution of RNA expression relative to raf. Although the A-raf and raf proteins are closely related, we have shown that they can be distinguished by using antibodies directed against a synthetic peptide corresponding to the C-terminal 12 amino acids of raf. Presumably antibodies against the C-terminal peptide of A-raf will allow the detection of cellular A-raf without cross-reaction with c-raf. Incorporation of A-raf cDNA into the genome of murine TABLE 2. Labeling efficiences of NIH 3T3 cells transformed by various oncogenesa Labeling efficiencyb Oncogene
Cell line
None v-raf
NIH 3T3 NIH F4-3611-LEUK NIH F4-3611-4070A
v-raf A-raf
NIH-9IV-LEUK
Avg % (-SE)
No.
10 (1.3) 93 (4.0) 91 (2.3) 92 (3.1)
7 10 6 4
Antibody 259 was microinjected into the cytoplasm of the cell types listed. A 3-h pulse of [3 Hithymidine between 18 and 24 h after injection was followed by methanol fixation and fluorescent-antibody staining to identify injected cells (14). Injections with nonneutralizing antibody 238 have also been performed in these cell lines. In all cases of antibody 238 injections, average labeling efficiencies varied from 95 to 105% (35a). bLabeling efficiencies were obtained by first determining the percentage of injected cells which had incorporated thymidine. This percentage was then divided by the percentage of uninjected cells on the same plate which were labeled. SE, Standard error value. cNumber of determinations, each of which averaged between 150 and 200 injected cells. a
leukemia virus, such that it was expressed as a gag fusion protein, revealed a strong potential transforming activity of the A-raf proto-oncogene. Similar constructions with the human homolog of mouse A-raf extend this observation to include the human A-raf gene (M. Huleihel and U. R. Rapp, unpublished data). We conclude that point mutations within the tested coding segment of the gene are not required for oncogenic activation. In analogy to raf, we suspect that linkage to viral gag sequences and the concomitant Nterminal myristilation of the fusion protein are probably not critical for oncogenic activity, and we consider aminoterminal truncation as the most likely contributing factor to the transforming activity of A-raf. Comparison between c-raf-1 and A-raf shows an abrupt loss of homology 5' of the kinase domain of the A-raf protein, suggesting a functional separation of the molecule into catalytic and regulatory domains. NH2-terminal truncation might thus release the catalytic portion of the protein from its normal regulation and trigger constitutive enzymatic activity. However, more constructions with the complete A-raf cDNA as well as additional deleted forms will have to be tested to evaluate this possibility. The expression of c-raf-1 in many normal tissues suggests some common regulatory function for this gene. In contrast, A-raf appears to be more exclusively employed due to differential transcription of the gene in a select set of tissues. Nevertheless, both genes can substitute for each other when expressed in growth-restricted fibroblast cells. This finding comes from experiments, reported here for A-raf and elsewhere for other transformants (35a), in which a functional classification of oncogenes was attempted according to their dependence on intact ras gene function for replication of fibroblast cells in culture. They involve injection of ras antibody into the cytoplasm of normal and oncogenetransformed cells. Whereas normal cells respond with growth arrest to antibody injection when grown in serum-
VOL. 6, 1986
THE raf ONCOGENE FAMILY
containing medium (19), cells transformed by v-raf, A-raf, and v-mos (35a) continue to replicate. In contrast, oncogenes v-src, v-fms, v-src, and v-fes do not overcome this growth arrest (35a). v-raf and v-mos have other similarities besides their ability to complement inactive ras proteins; they are both cytoplasmic proteins and have associated serine-threonine-specific protein kinase activity. These shared properties suggest a common position of raf, mos, and A-raf in the transduction of mitogenic signals. Whether their observed independence on the functioning of ras genes is due to a more downstream position of ras and mos in signal transduction pathways involving ras or indicates the existence of independent pathway(s) remains to be determined. Use of anti-raf antibodies injected into rastransformed cells should help clarify this issue. ACKNOWLEDGMENTS We thank Patricia Borhert for her excellent technical assistance and M. R. Smith and D. W. Stacey for the microinjection work with anti-ras antibody. We thank Pramod Sutrave for help with the sequence. This project was funded at least in part by Federal funds from the Department of Health and Human Services under contract no. NO1-CO-23910 with Program Resources, Inc. LITERATURE CITED 1. Bechade, C., G. Calothy, B. Ressac, P. Martin, T. Coll, F. Denher, S. Saule, J. Ghysdael, and D. Stehelin. 1985. Induction of proliferation or transformation of neuroretina cells by the mil and myc viral oncogenes. Nature (London) 316:559-562. 2. Benoist, C. O., P. D. Mathis, M. R. Kantes, V. E. Williams, and H. 0. McDevitt. 1983. The murine Iaa chains, Ea and A, show a surprising degree of sequence homology. Proc. Natl. Acad. Sci. USA 80:534-538. 3. Bonner, T. I., S. B. Kerby, P. Sutrave, M. A. Gunnell, G. Mark, and U. R. Rapp. 1985. Structure and biological activity of human homologs of the raflmil oncogene. Mol. Cell. Biol.
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