Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6312-6316, September 1986 Biochemistry pks, a raf-related sequence in humans (oncogene homology/src family/serine/threonine kinase/lymphoid cell) GEORGE E. MARK*, TODD W. SEELEY*, THOMAS B. SHOWSt, AND JOHN D. MOUNTZt *Laboratory of Human Carcinogenesis, National Cancer Institute, and tArthritis and Rheumatism Branch, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892; and tDepartment of Human Genetics, Roswell Park Memorial Institute, Buffalo, NY 14263 Communicated by Thomas F. Anderson, May 9, 1986 ABSTRACT A human fetal liver cDNA library was screened et al. (7) have demonstrated that the v-rafgene product is a at reduced hybridization stringency for v-naf-related sequences. kinase that phosphorylates serine and threonine residues. In addition to the expected c-raf-l cDNA, a second sequence was When used to screen a human fetal liver cDNA library isolated. Comparison of the second gene (pks) to the other under low-stringency conditions, a v-raf-specific probe iden- raf-related sequences revealed nucleotide homologies of 71%. tified two sequences. One was found to represent the c-raf-1 The predicted amino acid sequence of the kinase domain is locus (RAF1 in standard human gene nomenclature), whereas sufficiently similar to that of v-raf to suggest that pks may the other represents a gene, which we call pks, whose encode a polypeptide that exhibits serine/threonine kinase sequence suggests it too may encode a serine/threonine activity. The expression of pks mRNA (2.7 kilobases long) is kinase. elevated in peripheral blood mononuclear cells isolated from two patients with angioimmunoblastic lymphadenopathy with MATERIALS AND METHODS dysproteinemia, a disease in which autoantibodies are pro- Materials. A human fetal liver cDNA library in XgtlO duced following the lymphoproliferative activation of B cells. representing transcripts obtained from a 20-week fetus was Analysis of somatic cell hybrids for segregation of thepks locus kindly provided by E. Fritch (Genetics Institute, Boston, revealed the presence of an additional locus closely related to MA). Radioisotopes and DNA-modifying enzymes were the pks sequence. purchased from Amersham and New England Biolabs, re- spectively. Normal cellular growth appears to be under the control of a Screening of the XgtlO Fetal Liver cDNA Library. About series of interrelated regulatory signals. There is increasing 500,000 plaque-forming units of recombinant phage were evidence that major elements in this system are the normal screened at reduced stringency with a murine v-raf probe counterparts (protooncogenes) of already-described viral [675-base-pair (bp) Xho I-Sst II fragment] representing the oncogenes. The list of protooncogenes expands as viral and kinase domain of this oncogene. Filters lifted off the phage cellular oncogenes are identified; recent additions have been plates (plaque "lifts") were hybridized to the 32P-labeled v-ski (1), L-myc (2), neu (3), and met (4) sequences. Thus, as nick-translated (8) DNA fragment (specific activity 3 x 108 the number of possible targets of transforming lesions in- cpm/,g) for 22 hr at 60°C in 5 x standard saline citrate creases, so does the identity of loci involved in normal (SSC)/1 x Denhardt's solution/10% (wt/vol) dextran sulfate. cellular growth and differentiation. Of the four major classes (lx SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7.4; of oncogenes, the largest class encodes the members of the Denhardt's solution is 0.02% Ficoll/0.02% bovine serum "src family." These proteins, many of which have been albumin/0.02% polyvinylpyrrolidone.) Lifts were washed shown to possess either a tyrosine or serine/threonine kinase five times (15 min per wash) at 55°C in 2x SSC/0.1% activity, are found associated with the cell membrane and are NaDodSO4 and autoradiographed at -70°C with intensifying believed to be components of the cell's receptor system (5). screens. It is the stimulation of one or more of these receptors that DNA Sequencing. DNA sequence was determined by the initiates a cascade of events leading to cellular proliferation. method of Maxam and Gilbert (9) from uniquely labeled Whereas the amino and carboxyl termini of these gene restriction site termini, following isolation by SeaPlaque products are unique to each member, they share a region of (FMC, Rockland, ME) agarose gel electrophoresis as de- approximately 250 amino acids that defines an evolutionarily scribed (10). conserved kinase domain. We describe here the use of a Blot Hybridization Analysis of Poly(A)+ RNA. RNAs were probe corresponding to the sequence encoding this con- isolated from lymphoid tissues by homogenization in served domain to identify another oncogene-related human guanidinium thiocyanate followed by centrifugation through gene. cesium chloride (11, 12); poly(A)+ RNA was enriched by two A previous report (6) described the nucleotide sequence of selections on oligo(dT)-cellulose (13). RNA (10 ,ug), dena- v-raf, the viral oncogene of murine sarcoma virus 3611. tured by 14 mM methylmercury(II) hydroxide, was electro- Comparison of the predicted amino acid sequence of v-raf phoresed in a 1.5% agarose gel and transferred to 0- with other known oncogenes revealed it to be related to diazophenylthioether paper (14). Hybridization to 32P-la- members of the src family. It differed strikingly from most of beled nick-translated cDNA probes in 50% (vol/vol) form- these members within a 32 amino acid region encompassing amide/5x SSC/1x Denhardt's solution/yeast tRNA (250 the tyrosine acceptor site of the tyrosine kinase-encoding ,ug/ml) at 42°C for 12 hr was followed by washes in 0.1 x SSC oncogenes. The substitution in the v-rafsequence of a serine at 65°C and autoradiography at -70°C with an intensifying for the tyrosine commonly present in the phosphate-accept- screen. ing position suggested that v-rafspecifies a serine kinase, not Southern Blot Analysis. Genomic DNA (10 ,ug) was digest- a tyrosine kinase. In keeping with this observation, Moelling ed to completion with restriction endonucleases, electropho- The publication costs of this article were defrayed in part by page charge Abbreviations: AILD, angioimmunoblastic lymphadenopathy with payment. This article must therefore be hereby marked "advertisement" dysproteinemia; SLE, systemic lupus erythematosus; bp, base in accordance with 18 U.S.C. §1734 solely to indicate this fact. pair(s); kb, kilobase(s). 6312 Downloaded by guest on September 29, 2021 Biochemistry: Mark et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6313 resed in a 0.7% agarose gel, and transferred to nitrocellulose TAG paper by the method of Southern (15). The blots were Kpn Nde Rv xba Pvu 11 hybridized overnight to cDNA fragment probes (specific pHB2 I Io I l I I activity 3 x 108 cpm/,ug) at 650C in 5x SSC/5x Denhardt's [R11 Hc 11 Hind 11 Hc 11 (R1] solution/10% (wt/vol) dextran sulfate/50 mM sodium phos- III BgI Apa phate buffer, pH 6.6/salmon sperm DNA (250 ,pg/ml)/0.1% Pvu TAG 100 bp (wt/vol) NaDodSO4. After washing under stringent condi- Nde I Sma Pvu 11 BgI 11/ Nar IT Sac tions (O.lx SSC/0.1% NaDodSO4 at 680C), the blots were pHD2 II I KII II I ii1 IBgI autoradiographed as above. Low-stringency washes were (RI]I Nco' KpnI Hc 11 Apa Barn Hi [RI] performed at 50'C when DNAs from somatic cell hybrids were analyzed. RESULTS FIG. 1. (a) Restriction endonuclease maps of v-raf-related cDNA clones pHB2 and pHD2. EcoRI inserts from positive phage were ram-Related Genes Expressed in Humans. A human fetal subcloned in pBR322. Brackets indicate the site was introduced as a liver cDNA library was screened at reduced stringency with linker. The amber (TAG) translation termination codons (arrows) a v-raf probe representing the kinase domain (a 675-bp Xho were used to align the sequences. (b) Strategy for sequencing pHD2. I-Sst II fragment; ref. 6). Strongly and weakly hybridizing DNA sequence was determined by the method of Maxam and Gilbert plaques were picked, and the structures of their recombinant (9) from labeled restriction site termini (vertical lines). Arrow lengths DNAs were analyzed by restriction mapping. Most of the reflect number of nucleotides sequenced. isolates exhibited restriction maps similar to that of clone pHB2 (Fig. la) and were of the strongly hybridizing variety. responding to those expected for the c-raf-1 locus (16) were When these clones were used as hybridization probes for detected. A sequence (pHD2, Fig. la) whose restriction map restriction endonuclease-digested human DNA, bands cor- seemed unrelated to the c-raf-J gene was isolated from one of KpI KpnI PVU11 Aks AAGAACCTTG GGTACCGGGA CTCAGGCTAT TACTGGGAGG TACCACCCAG TGAGGTGCAG CTGCTGAAGA GGATCGGGAC GGGCTCGTTT GGCACCGTGT 100 c-afl G CCT G AC GA A T A T AA GA G A AT TCC CTC T t A T A T T v-V G CCTG C GA A GA T A AA GGA G AT CTCT CTC T A T C T PVU 11 pk TTCGAGGGCG GTGGCATGGC GATGTGGCCG TGAAGGTGCT CAAGGTGTCC CAGCCCACAG CTGAGCAGGC CCAGGCTTTC AAGAATGAGA TGCAGGTGCT 200 cafat AAAG TAA A C A T A A A C A TGr G C A CC A ATT C G G GCT T v-af ACAAG CAA A T A A A C A GTTG C A TC A ACT T C G C G GC TT Hrec 11 ok CAGGAAGACG CGACATGTCA ACATCTTGCC GTTTATGGGC TTCATGACCC GGCCGGGAGT TGCCATCATC ACACAGTGGT GTGAGGGCTC CAGCCTCTAC 30 c-a-l GC C A A G G TC J T C G A AA A GACAACC G A TG G C C AG v-vaf GC C A A G T C T C G A AA A GACAACC G G TG G T A AG T its CATCACCTGC ATGTGGCCGA CACACGCTTC GACATGGTCC AGCTCATCGA CGTGGCCCGG CAGACCGCCC AGGGCATGGA CTACCTCCAT GCCAAGAACA 0 c-vftl A A CCAG G CAAG c 6 JT A T A T G T