Original Paper International Journal of Cell Cloning 10:309-314 (1992)
Molecular Cloning of the cDNA Encoding a Receptor Qrosine KinaseRelated Molecule with a Catalytic Region Homologous to c-met Stephan R. Paul"", David Merberg', Heather Finnerty', George E. Morris', John C. Morris', Simon S. Jones', Ron Kriz', Katherine J. Turner', Clive R. Wood' 'Genetics Institute, Inc., Cambridge, Massachusetts, USA; ?he Dana Farber Cancer Institute and the Childrens' Hospital, Harvard Medical School, Boston, Massachusetts, USA Key Words. Tyrosine kinase
Molecular cloning Polymerase chain reaction
Abstract. Receptor tyrosine kinases mediate a range of growth and differentiation processes in multiple biological systems. In this work, we report the identification of a novel tyrosine kinase-related molecule, nyk-r, and the molecular cloning of its complete cDNA. Its extracellular domain bears no apparent homology with other receptor families, but its intracellular kinase-related region has considerable similarity with members of the insulin-receptor family such as c-mer and trk B. Also, the nyk-r gene is expressed in a wide range of tissues and cell lines.
Introduction Protein kinases have a number of highly conserved amino acid sequences in their catalytic domains [ ll. As a result of this conservation, there have been outstanding successes in the application of the polymerase chain reaction (PCR) using degenerate pools of oligodeoxyribonucleotide primers to isolate novel tyrosine kinase cDNA fragments [2-41. By this approach, novel receptor tyrosine kinases have been discovered in hematopoietic stem cell-enriched populations and in the nervous system [5, 61. In the present work, we have used PCR with degenerate primers to identify a novel putative receptor tyrosine kinase from the cell line T10, a subclone of the murine plasmacytoma T1165 [7]. The complete cDNA coding region of this novel tyrosine kinase, designated nyk-r, has been isolated and is marked by a short extracellular region and a kinase region Correspondence: Dr. Stephan R. Paul, Transplantation Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA.
Received August 26, 1992; accepted for publication August 27, 1992. 0737-1454/92/$2.00/0 OAlphaMed Press
homologous to c-met, the hepatocyte growth factor receptor trk B, and v-sea, but lacking a number of conserved motifs usually observed in these receptors.
Materials and Methods Cell Lines and Mice The derivation of the IL- 11-dependent T 10 subclone of the murine T1165 plasmacytoma has been described [7, 81. Other murine cell lines used in this work include the B9 plasmacytoma [9] and the preadipocyte PA6 [lo]. The rat PC-12 pheochromocytoma cell line has also been described [ 113. Fetal tissue was harvested from C57BL/6J mice at day 14 of gestation (E14). Bone marrow was taken five days after intraperitoneal administration of 150 p g k g of 5-fluorouracil to mice of the same strain.
RNA Isolation and PCR RNA was isolated by the method of Chomczynski and Succhi [12] and poly A+ messenger RNA selected using biotin-conjugated oligo(dT) and streptavidin-paramagneticparticles (Promega, Madison, wr). RNA was separated by size on a 1% (w/v) agarosdformaldehyde gel followed by Northern blot transfer to a nitrocellulose filter. Probes were labeled with CX-~*P dCTP by random hexamer priming as described [13, 141. Reverse transcription of T10 RNA into first strand cDNA was carried out with random hexamer primers and subsequently amplified by PCR with Taq DNA polymerase using GeneAmp RNA PCR reagents and the DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT).PCR-amplified DNA was purified by Centricon-30 microconcentration (AMICON, Danvers, MA) prior to second round reactions. Double-stranded DNA products were ligated into
Paul. et al. 1 1
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2520
Fig. 1. Nucleotide sequence of the murine nyk-r cDNA. The nucleotide sequence of the #8A cDNA clone is shown, along with the predicted amino acid sequence of the nyk-r polypeptide. The putative signal sequence and transmembrane domain are underlined. Potential sites of N-linked glycosylation in the extracellular domain are marked with an arrowhead. Extracellular region cysteine residues are marked with an asterisk. Conserved tyrosine kinase motifs are marked with a double broken line. The amino acid sequence is numbered from +1 at the proposed initiator methionine residue. The sequence nyk-r has been submitted to GenBank and has been assigned accession number L02210.
the pBluescriptIIKS+ plasmid vector (Stratagene, San Diego, CA).
is G, A, T and C; “Y” is C and T; “M’ is A and C; “ R is A and G; “ W is A and T; “S” is C and G; “ K is G and T; and “B” is C, G and T.
PCR Oligonucleotides Oligonucleotides included: N 1:SdCGGAATTCGGNGCNTTYGGNMARGT3’, N2:S‘dAGGAATTCCRWAGGACCASACRTC3’, N3:S’dGTGAATTCACMGRGAYKTRGCAGCBMG3’, and N4:SdCACGGAATTCNGGNGCCATCCA3’,where “ N
cDNA Cloning and Nucleotide Sequence Analysis First strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and oligo(dT),,,, primer, followed by second strand replacement synthesis with ribonuclease H, DNA
A
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polymerase I and Escherichia coli DNA ligase [ 15, 161. The resulting cDNA was ligated to EcoR1 adapters and cloned into hZAPII digested with EcoR 1 and dephosphorylated with calf intestinal alkaline phosphatase (Stratagene, San Diego, CA). The ligation products were packaged in vitro and used to infect E. coli BB4. Plasmid subclones of isolated bacteriophages were obtained by the UAPII excision process. The cDNAs were sequenced as described [ 171 for both strands by using a Ba131 nuclease deletion series [181 and synthetic oligonucleotide primers with Sequenase version 2.0 (US Biochemical, Cleveland, OH). All computer programs for sequence analysis were from the GCG software package [19]. Pairwise sequence comparisons were performed using the method of Needleman and Wunsch [20], and multiple sequence alignments were generated by the method of Higgins and Sharp [21]. Putative signal peptides were identified using the algorithm of von Heijne [22]. Transmembrane domains were predicted using the method of Klein, Kanehisa, and DeLisi [23].
Results We adopted a PCR strategy designed to amplify receptor tyrosine kinase sequences. The first PCR used primers N1 and N2 that prime at the “GXGXXGXV” nucleotide binding site motif in subdomain I, and the “DVWS” motif in subdomain IX, respectively [ 11. The reaction products were then purified by Centricon 30 microconcentration, and a portion was amplified with a second pair of primers in a nested reaction. The second pair were N3 and N4, corresponding to the “HRDLAAR” motif of subdomain VI and the “WMAPES” motif of
Novel Tyrosine Kinase-Related Molecule: nyk-r
subdomain VIII, respectively. A significant bias in favor of receptor tyrosine kinases is introduced by primers N1, N3 and N4. Also, EcoR1 sites were included in the primer sequences to facilitate rapid cloning of the resulting fragments. This nested set of PCRs was carried out with first strand cDNA synthesized from RNA isolated from the T10 plasmacytoma cell line. The approximately 150 bp PCR products were digested with EcoRl, eluted from a 1% (w/v) low-melt agarose TAE gel and ligated into the EcoR1 site of pBluescriptIIKS+. The plasmid clones were then screened by nucleotide sequencing. One PCR clone, called 4T-6, had several of the expected sequence similarities to tyrosine kinases, and the predicted amino acid sequence of the PCR fragment was most similar to trk B [24, 251. AT10 cDNA library was constructed in U A P U and screened with 4T-6 sequences. Four recombinant bacteriophages were isolated and sequenced. All contained the 4T-6 PCR fragment sequence. The nucleotide sequence of one of these clones, #8A, is shown in Figure 1. This clone contained the nucleotide sequence of 4T-6, in an open reading frame of 1784 bp with a predicted amino acid sequence of 594 residues. This novel sequence was found to be related to the receptor tyrosine kinases. and was named nyk-r. The ATG codon at position 112 is followed by a hydrophobic region typical of signal sequences, giving a predicted cleavage site between positions 34 and 35. In addition, there is an ATG codon at position 193, but the nucleotide sequence context differs from the consensus in four out of nine residues. The ATG codon at position 112 has a nucleotide sequence context similar to the consensus initiation ATG requirement [26]. An additional hydrophobic region is found from position 210 to 240 and is assumed to be a trans-
Table I. Needleman-Wunsch alignment of nyk-r with other tyrosine kinase receptors Alignment Scores
Kinase
Ratio
8 Similarity
FGF-R [34] EGF-R [35]
0.69 0.70 0.67 0.70 0.71 0.79 0.80
57.1 60.1 57.8 57.1 59.4 63.9 63.9
eph P61 ir [37] trk B [25] mer [27. 281 v-sea [29]
Needleman-Wunsch sequence alignment of the kinase regions of the murine insulin receptor, murine c-met. murine trk B, murine FGF-R (bek), human eph and human EGF receptor with nyk-r was performed as described. The ratio and percentage similarity values are shown. Sequences are from references in the table.
Paul, et al.
312 -. ..
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IDPFEMAAYL LSNNEVIECI Fig. 2. Kinase region alignment of nyk-r and related kinases. The kinase-related region of nyk-r is aligned with residues 67 to 312 of v-sea: residues 1083 to 1328 of murine c-met; and residues 544 to 799 of rnurine trk B. Highly conserved residues that are invariant in all four proteins are shown in a stippled box. Highly conserved kinase catalytic region residues that are not conserved in nyk-r are shown in bold print.
membrane domain. Potential sites for N-linked glycosylation are found at residues 123, 158, 162, 166 and 193. The mature extracellular region from positions 35 to 21U does not contain any immunoglobulin domains or have cysteine-rich repeats typically found in some receptors. However, the intracellular region of nyk-r is similar to several receptor tyrosine kinases. Upon searching the PIR, Swissprot and GenPept databases, using the Fasta program, this putative receptor kinase was found to have greatest similarity to the insulin receptor subfamily of tyrosine kinases, especially c-met, the receptor for hepatocyte growth factor, and v-sea [27-291. As shown in Table I, a Needleman-Wunsch alignment was camed out comparing nyk-r to these receptors and other representative receptor kinases. The highest score was found with murine c-met and avian v-sea. The substantial similarity of nyk-r sequence with other kinases is shown in Figure 2. However, it should be noted that the consensus “GXGXXG motif is not perfect, and the almost invariant “DFG’ motif is present as a “DNA” sequence. Three additional‘cDNA clones (#6A, #18A and #19B) were isolated from the T10 cDNA library. Each of these three clones aligns with the 5‘ ,end of #8A. The 3‘ end of the #19B clone is approximately 300 bp shorter than #8A. The #6A and #8A clones have an identical 3’ terminus, including a poly A sequence. However, only a poor consensus poly A addition sequence is found at this 3’ end.
Northern blot analysis was used to examine the expression of the nyk-r gene. The result is shown in Figure 3. It is apparent that there are two mRNA species that hybridize with a probe to the 3’ end of the sequence. Expression of both mRNAs was found in fetal liver and fetal brain as well as a number of cell lines including the B9 plasmacytoma, NIH 3T3 and rat PC-12 cells. Expression was also observed in murine bone marrow harvested five days after 5fluorouracil administration, which enriched for hematopoietic stem cells and progenitors.
Discussion In this report, we describe the complete cDNA cloning of a novel tyrosine kinase-related molecule, nyk-r. Kari Alitalo and colleagues have also reported the PCR cloning of an approximately 150 bp sequence from human K562 leukemia cells that is identical to our nyk-r PCR fragment [30]. The nyk-r polypeptide is proposed to have an unusually short predicted mature extracellular region of 175 amino acids with no apparent homology to other receptor tyrosine kinases. Short extracellular domains have been reported for other receptors. For example, the extracellular region of the murine interleukin 2p (IL-2p) receptor is 214 amino acids, and that of the keratinocyte growth factor receptor, a tyrosine kinase, is 242 amino acids [31, 321. The intracellular region is similar to the catalytic domain
A Novel Tyrosine Kinase-Related Molecule: nyk-r
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in the 3‘ untranslated region. However, additional cDNA clones are required to c o n fm this possibility. The nyk-r gene product has an intriguing structure. It is critical to explore the function of this unusual tyrosine kinase region and identify the secreted or cell-associated ligand for the extracellular domain.
Acknowledgments
The authors gratefully acknowledge the support and helpful suggestions of Dr. S. C. Clark. They are also indebted to Ms. K. Campbell and Mr. G. Pedneault of the Laboratory Animal Resources Group and MI:Hemchand Sookdeo of the D N A Synthesis Group at the Genetics Institute for invaluable expertise and assistance. References
Fig. 3. Northern analysis of the expression of nyk-r. RNA was transferred to nitrocellulose and hybridized with a probe containing nucleotides 2061 to 3543 of nyk-r. The hybridized filter was washed in 0.2 x SSC, 0.1% (w/v) SDS at 65°C. The migration position of 28s and 18s RNA was recorded and is shown on the figure. The filter was later hybridized with a p-actin probe, and the result is shown in the lower box. For PC-12, PA6 and NIH 3T3 lanes, 15 up. total RNA was loaded. One M of poly A+ RNA from ;urine bone marrow harvesteddays-afier 5fluorouracil administration (BM 5-FLJ)and 3 pg poly A+ RNA from the B9 cell line was loaded. Six .M_of -poly- A+ RNA was loaded for each of the following: placenta (P 14); fetal brain (FB 14) from E14); fetal liver mice at day 14 of gestation; and adult liver (LIV).
1 Hanks SK,Quinn M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. In: Hunter T, Sefton BM, eds. Methods in Enzymology 1991;200:38-62. Wilks AF, Kurban RR, CM, Ralph sJ.The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family. Gene 1989;85:67-74. 3 Wilks AF. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:1603-1607. 4 Raz V, Kelman Z, Avivi A. Neufeld G,Givol D, Yarden Y.PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors. Oncogene 1991;6:753-760, 5 Matthews W, Jordan CT, Wiegard GW, Pardoll D,
of receptor tyrosine kinases, particularly c-mer and v-sea. The consensus nucleotide binding site is not perfectly conserved in nyk-r. Thus, it will be important to test the in vitro kinase activity of this protein. In addition, the highly conserved “DFG’ kinase motif has been replaced with “DNA” sequence. Interestingly, the DFG motif is also not conserved in the polypeptide encoded by D trk, a Drosophila melanogaster gene that is homologous to the trk family and encodes a tyrosine kinase [33]. The nyk-r gene is expressed in a range of tissues and cell lines as two mRNA species. Four independent cDNA clones from the TI0 cell line have almost identical 5’ ends and common coding region sequences. It is likely that the difference in size between the two nyk-r mRNAs is due to differences
Lemischka IR. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991;65:1143-1152. 6 Lai C, Lemke G. An extended family of proteintyrosine kinase genes differentially expressed in the vertebrate nervous system. Neuron 1991;6:691-704.
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monal responsiveness of a preadipose cell line derived from newborn mouse calvaria. J Cell Phys 1982;112:83-88. 11 Greene LA, Tischler AF. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 1976;73:2424-2428. 12 Chomczynski P. Sacchi N. Single step method of RNA isolation by acid guanidinium-thiocyanatephenol-chloroform extraction. A d 1 Biochem 1987; 162:156-159. 13 Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13. 14 Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonucleasefragments to high specific activity. Anal Biochem 1984;137:266-267. 15 Okayama H, Berg P. High efficiency cloning of full
length cDNA. Mol Cell Biol 1982;2:161-167. 16 Gubler U, Hoffman RJ. A simple and very efficient method for generating cDNA libraries. Gene 1983; 25:263-273. 17 Sanger F, Nicklen S , Coulson AR. DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 1977;74:5463-5467. 18 Poncz M, Solowiejczyk D. Ballantine M, Schwartz E, Surrey S . Nonrandom DNA sequence analysis in bacteriophage M13 by the dideoxy chain termination method. Proc Natl Acad Sci USA 1982;79:4298-4302. 19 Devereaux J, Haeberli P, Smithies 0. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 1984;12:387-395. 20 Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 1970;48: 443-453. 21 Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 1988;73:237-244. 22 von Heijne G. Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit. New York Academic Press, 1987. 23 Klein P. Kanehisa M, DeLisi C. The detection and classification of membrane spanning proteins. Biochim Biophys Acta 1985;815:468-476. 24 Klein R. Parada LF, Coulier F, Barbacid M. rrk B, a novel tyrosine protein kinase receptor expressed during mouse neural development. EMBO J 1989;8: 3701-3709. 25 Middlemas DS, Lindberg RA, Hunter T. rrk B, a neural receptor proteintyrosine kinase: evidence for a full-length and two truncated receptors. Mol Cell Biol 1991;11: 143-153.
314 26 Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 1986;44:283-292. 27 Park M, Dean M. Kaul K, Braun MJ, Gonda MA, Vande Wonde GF. Sequence of met proto-oncogene cDNA has features characteristic of the tyrosine kinase family. Proc Natl Acad Sci USA 1987;84: 6379-6383. 28 Bottaro DP. Rubin JS, Faletto DL, Chan AM-L, Krniecik TE, Vande Wonde GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-mef proto-oncogene product. Science 1991; 2511802-804. 29 Smith DR, Vogt PK, Hayman MJ. The v-sea oncogene of avian erythroblastosisretrovirus S13: another member of the protein-tyrosine kinase gene family. Proc Natl Acad Sci USA 1989;86:5291-5295. 30 Partanen J. Makela TP, Alitalo R, Lehvaslaiho H, Alitalo K. Putative tyrosine kinases expressed in K-562 human leukemia cells. Proc Natl Acad Sci USA 1990;87:8913-8917. 31 Miki T, Fleming TP, Bottaro DP, Rubin JS. Ron D, Aaronson SA. Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop. Science 1991;251:72-75. 32 Kono T, Doi T, Vamada G, Hatakeyama M, Minimoto s, Tsudo M,Miyasaki M, Miyata T, Taniguchi T. Murine interleukin 2 receptor p chain: dysregulated gene expression in lymphoma line EL-4 caused by a promoter insertion. Proc Natl Acad Sci USA 199O;87:1806-1810. 33 Pulido D, Campuzano S, Koda T, Modolell J, Barbacid M. D trk, a Drosophila gene related to the rrk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J 1992;111391-404. 34 Safran A, Avivi A, Orr-Urtereger A, Neufeld G, Lonai P, Givol D, Yarden Y. The murinef2g gene encodes a receptor for fibroblast growth factor. Oncogene 1990;5:635-643. 35 Ullrich A, Coussens L, Hayflick JS, Dull TJ, Gray A, Tam AW, Lee J, Yarden Y, Libermann TA, Schlessinger J, Downward J, Mayes EL, Whittle N, Waterfield MD, Seeburg PH. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 1984;309:418-425. 36 Hirai H, Maru Y, Hagiwara K, Nishida J. Takaku F. A novel putative tyrosine kinase receptor encoded by the eph gene. Science 1987;238:1717-1720. 37 Flores-Riveros JR, Sibley E, Kastelic T, Lane MD. Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit. J Biol Chem 1989;264:21557-21572.
Molecular Cloning of the cDNA Encoding a Receptor Qrosine KinaseRelated Molecule with a Catalytic Region Homologous to c-met Stephan R. Paul"", David Merberg', Heather Finnerty', George E. Morris', John C. Morris', Simon S. Jones', Ron Kriz', Katherine J. Turner', Clive R. Wood' 'Genetics Institute, Inc., Cambridge, Massachusetts, USA; ?he Dana Farber Cancer Institute and the Childrens' Hospital, Harvard Medical School, Boston, Massachusetts, USA Key Words. Tyrosine kinase
Molecular cloning Polymerase chain reaction
Abstract. Receptor tyrosine kinases mediate a range of growth and differentiation processes in multiple biological systems. In this work, we report the identification of a novel tyrosine kinase-related molecule, nyk-r, and the molecular cloning of its complete cDNA. Its extracellular domain bears no apparent homology with other receptor families, but its intracellular kinase-related region has considerable similarity with members of the insulin-receptor family such as c-mer and trk B. Also, the nyk-r gene is expressed in a wide range of tissues and cell lines.
Introduction Protein kinases have a number of highly conserved amino acid sequences in their catalytic domains [ ll. As a result of this conservation, there have been outstanding successes in the application of the polymerase chain reaction (PCR) using degenerate pools of oligodeoxyribonucleotide primers to isolate novel tyrosine kinase cDNA fragments [2-41. By this approach, novel receptor tyrosine kinases have been discovered in hematopoietic stem cell-enriched populations and in the nervous system [5, 61. In the present work, we have used PCR with degenerate primers to identify a novel putative receptor tyrosine kinase from the cell line T10, a subclone of the murine plasmacytoma T1165 [7]. The complete cDNA coding region of this novel tyrosine kinase, designated nyk-r, has been isolated and is marked by a short extracellular region and a kinase region Correspondence: Dr. Stephan R. Paul, Transplantation Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA.
Received August 26, 1992; accepted for publication August 27, 1992. 0737-1454/92/$2.00/0 OAlphaMed Press
homologous to c-met, the hepatocyte growth factor receptor trk B, and v-sea, but lacking a number of conserved motifs usually observed in these receptors.
Materials and Methods Cell Lines and Mice The derivation of the IL- 11-dependent T 10 subclone of the murine T1165 plasmacytoma has been described [7, 81. Other murine cell lines used in this work include the B9 plasmacytoma [9] and the preadipocyte PA6 [lo]. The rat PC-12 pheochromocytoma cell line has also been described [ 113. Fetal tissue was harvested from C57BL/6J mice at day 14 of gestation (E14). Bone marrow was taken five days after intraperitoneal administration of 150 p g k g of 5-fluorouracil to mice of the same strain.
RNA Isolation and PCR RNA was isolated by the method of Chomczynski and Succhi [12] and poly A+ messenger RNA selected using biotin-conjugated oligo(dT) and streptavidin-paramagneticparticles (Promega, Madison, wr). RNA was separated by size on a 1% (w/v) agarosdformaldehyde gel followed by Northern blot transfer to a nitrocellulose filter. Probes were labeled with CX-~*P dCTP by random hexamer priming as described [13, 141. Reverse transcription of T10 RNA into first strand cDNA was carried out with random hexamer primers and subsequently amplified by PCR with Taq DNA polymerase using GeneAmp RNA PCR reagents and the DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT).PCR-amplified DNA was purified by Centricon-30 microconcentration (AMICON, Danvers, MA) prior to second round reactions. Double-stranded DNA products were ligated into
Paul. et al. 1 1
310
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2520
Fig. 1. Nucleotide sequence of the murine nyk-r cDNA. The nucleotide sequence of the #8A cDNA clone is shown, along with the predicted amino acid sequence of the nyk-r polypeptide. The putative signal sequence and transmembrane domain are underlined. Potential sites of N-linked glycosylation in the extracellular domain are marked with an arrowhead. Extracellular region cysteine residues are marked with an asterisk. Conserved tyrosine kinase motifs are marked with a double broken line. The amino acid sequence is numbered from +1 at the proposed initiator methionine residue. The sequence nyk-r has been submitted to GenBank and has been assigned accession number L02210.
the pBluescriptIIKS+ plasmid vector (Stratagene, San Diego, CA).
is G, A, T and C; “Y” is C and T; “M’ is A and C; “ R is A and G; “ W is A and T; “S” is C and G; “ K is G and T; and “B” is C, G and T.
PCR Oligonucleotides Oligonucleotides included: N 1:SdCGGAATTCGGNGCNTTYGGNMARGT3’, N2:S‘dAGGAATTCCRWAGGACCASACRTC3’, N3:S’dGTGAATTCACMGRGAYKTRGCAGCBMG3’, and N4:SdCACGGAATTCNGGNGCCATCCA3’,where “ N
cDNA Cloning and Nucleotide Sequence Analysis First strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and oligo(dT),,,, primer, followed by second strand replacement synthesis with ribonuclease H, DNA
A
311
polymerase I and Escherichia coli DNA ligase [ 15, 161. The resulting cDNA was ligated to EcoR1 adapters and cloned into hZAPII digested with EcoR 1 and dephosphorylated with calf intestinal alkaline phosphatase (Stratagene, San Diego, CA). The ligation products were packaged in vitro and used to infect E. coli BB4. Plasmid subclones of isolated bacteriophages were obtained by the UAPII excision process. The cDNAs were sequenced as described [ 171 for both strands by using a Ba131 nuclease deletion series [181 and synthetic oligonucleotide primers with Sequenase version 2.0 (US Biochemical, Cleveland, OH). All computer programs for sequence analysis were from the GCG software package [19]. Pairwise sequence comparisons were performed using the method of Needleman and Wunsch [20], and multiple sequence alignments were generated by the method of Higgins and Sharp [21]. Putative signal peptides were identified using the algorithm of von Heijne [22]. Transmembrane domains were predicted using the method of Klein, Kanehisa, and DeLisi [23].
Results We adopted a PCR strategy designed to amplify receptor tyrosine kinase sequences. The first PCR used primers N1 and N2 that prime at the “GXGXXGXV” nucleotide binding site motif in subdomain I, and the “DVWS” motif in subdomain IX, respectively [ 11. The reaction products were then purified by Centricon 30 microconcentration, and a portion was amplified with a second pair of primers in a nested reaction. The second pair were N3 and N4, corresponding to the “HRDLAAR” motif of subdomain VI and the “WMAPES” motif of
Novel Tyrosine Kinase-Related Molecule: nyk-r
subdomain VIII, respectively. A significant bias in favor of receptor tyrosine kinases is introduced by primers N1, N3 and N4. Also, EcoR1 sites were included in the primer sequences to facilitate rapid cloning of the resulting fragments. This nested set of PCRs was carried out with first strand cDNA synthesized from RNA isolated from the T10 plasmacytoma cell line. The approximately 150 bp PCR products were digested with EcoRl, eluted from a 1% (w/v) low-melt agarose TAE gel and ligated into the EcoR1 site of pBluescriptIIKS+. The plasmid clones were then screened by nucleotide sequencing. One PCR clone, called 4T-6, had several of the expected sequence similarities to tyrosine kinases, and the predicted amino acid sequence of the PCR fragment was most similar to trk B [24, 251. AT10 cDNA library was constructed in U A P U and screened with 4T-6 sequences. Four recombinant bacteriophages were isolated and sequenced. All contained the 4T-6 PCR fragment sequence. The nucleotide sequence of one of these clones, #8A, is shown in Figure 1. This clone contained the nucleotide sequence of 4T-6, in an open reading frame of 1784 bp with a predicted amino acid sequence of 594 residues. This novel sequence was found to be related to the receptor tyrosine kinases. and was named nyk-r. The ATG codon at position 112 is followed by a hydrophobic region typical of signal sequences, giving a predicted cleavage site between positions 34 and 35. In addition, there is an ATG codon at position 193, but the nucleotide sequence context differs from the consensus in four out of nine residues. The ATG codon at position 112 has a nucleotide sequence context similar to the consensus initiation ATG requirement [26]. An additional hydrophobic region is found from position 210 to 240 and is assumed to be a trans-
Table I. Needleman-Wunsch alignment of nyk-r with other tyrosine kinase receptors Alignment Scores
Kinase
Ratio
8 Similarity
FGF-R [34] EGF-R [35]
0.69 0.70 0.67 0.70 0.71 0.79 0.80
57.1 60.1 57.8 57.1 59.4 63.9 63.9
eph P61 ir [37] trk B [25] mer [27. 281 v-sea [29]
Needleman-Wunsch sequence alignment of the kinase regions of the murine insulin receptor, murine c-met. murine trk B, murine FGF-R (bek), human eph and human EGF receptor with nyk-r was performed as described. The ratio and percentage similarity values are shown. Sequences are from references in the table.
Paul, et al.
312 -. ..
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met
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..........AQERSPTVKE
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IDPFEMAAYL LSNNEVIECI Fig. 2. Kinase region alignment of nyk-r and related kinases. The kinase-related region of nyk-r is aligned with residues 67 to 312 of v-sea: residues 1083 to 1328 of murine c-met; and residues 544 to 799 of rnurine trk B. Highly conserved residues that are invariant in all four proteins are shown in a stippled box. Highly conserved kinase catalytic region residues that are not conserved in nyk-r are shown in bold print.
membrane domain. Potential sites for N-linked glycosylation are found at residues 123, 158, 162, 166 and 193. The mature extracellular region from positions 35 to 21U does not contain any immunoglobulin domains or have cysteine-rich repeats typically found in some receptors. However, the intracellular region of nyk-r is similar to several receptor tyrosine kinases. Upon searching the PIR, Swissprot and GenPept databases, using the Fasta program, this putative receptor kinase was found to have greatest similarity to the insulin receptor subfamily of tyrosine kinases, especially c-met, the receptor for hepatocyte growth factor, and v-sea [27-291. As shown in Table I, a Needleman-Wunsch alignment was camed out comparing nyk-r to these receptors and other representative receptor kinases. The highest score was found with murine c-met and avian v-sea. The substantial similarity of nyk-r sequence with other kinases is shown in Figure 2. However, it should be noted that the consensus “GXGXXG motif is not perfect, and the almost invariant “DFG’ motif is present as a “DNA” sequence. Three additional‘cDNA clones (#6A, #18A and #19B) were isolated from the T10 cDNA library. Each of these three clones aligns with the 5‘ ,end of #8A. The 3‘ end of the #19B clone is approximately 300 bp shorter than #8A. The #6A and #8A clones have an identical 3’ terminus, including a poly A sequence. However, only a poor consensus poly A addition sequence is found at this 3’ end.
Northern blot analysis was used to examine the expression of the nyk-r gene. The result is shown in Figure 3. It is apparent that there are two mRNA species that hybridize with a probe to the 3’ end of the sequence. Expression of both mRNAs was found in fetal liver and fetal brain as well as a number of cell lines including the B9 plasmacytoma, NIH 3T3 and rat PC-12 cells. Expression was also observed in murine bone marrow harvested five days after 5fluorouracil administration, which enriched for hematopoietic stem cells and progenitors.
Discussion In this report, we describe the complete cDNA cloning of a novel tyrosine kinase-related molecule, nyk-r. Kari Alitalo and colleagues have also reported the PCR cloning of an approximately 150 bp sequence from human K562 leukemia cells that is identical to our nyk-r PCR fragment [30]. The nyk-r polypeptide is proposed to have an unusually short predicted mature extracellular region of 175 amino acids with no apparent homology to other receptor tyrosine kinases. Short extracellular domains have been reported for other receptors. For example, the extracellular region of the murine interleukin 2p (IL-2p) receptor is 214 amino acids, and that of the keratinocyte growth factor receptor, a tyrosine kinase, is 242 amino acids [31, 321. The intracellular region is similar to the catalytic domain
A Novel Tyrosine Kinase-Related Molecule: nyk-r
313
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in the 3‘ untranslated region. However, additional cDNA clones are required to c o n fm this possibility. The nyk-r gene product has an intriguing structure. It is critical to explore the function of this unusual tyrosine kinase region and identify the secreted or cell-associated ligand for the extracellular domain.
Acknowledgments
The authors gratefully acknowledge the support and helpful suggestions of Dr. S. C. Clark. They are also indebted to Ms. K. Campbell and Mr. G. Pedneault of the Laboratory Animal Resources Group and MI:Hemchand Sookdeo of the D N A Synthesis Group at the Genetics Institute for invaluable expertise and assistance. References
Fig. 3. Northern analysis of the expression of nyk-r. RNA was transferred to nitrocellulose and hybridized with a probe containing nucleotides 2061 to 3543 of nyk-r. The hybridized filter was washed in 0.2 x SSC, 0.1% (w/v) SDS at 65°C. The migration position of 28s and 18s RNA was recorded and is shown on the figure. The filter was later hybridized with a p-actin probe, and the result is shown in the lower box. For PC-12, PA6 and NIH 3T3 lanes, 15 up. total RNA was loaded. One M of poly A+ RNA from ;urine bone marrow harvesteddays-afier 5fluorouracil administration (BM 5-FLJ)and 3 pg poly A+ RNA from the B9 cell line was loaded. Six .M_of -poly- A+ RNA was loaded for each of the following: placenta (P 14); fetal brain (FB 14) from E14); fetal liver mice at day 14 of gestation; and adult liver (LIV).
1 Hanks SK,Quinn M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. In: Hunter T, Sefton BM, eds. Methods in Enzymology 1991;200:38-62. Wilks AF, Kurban RR, CM, Ralph sJ.The application of the polymerase chain reaction to cloning members of the protein tyrosine kinase family. Gene 1989;85:67-74. 3 Wilks AF. Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:1603-1607. 4 Raz V, Kelman Z, Avivi A. Neufeld G,Givol D, Yarden Y.PCR-based identification of new receptors: molecular cloning of a receptor for fibroblast growth factors. Oncogene 1991;6:753-760, 5 Matthews W, Jordan CT, Wiegard GW, Pardoll D,
of receptor tyrosine kinases, particularly c-mer and v-sea. The consensus nucleotide binding site is not perfectly conserved in nyk-r. Thus, it will be important to test the in vitro kinase activity of this protein. In addition, the highly conserved “DFG’ kinase motif has been replaced with “DNA” sequence. Interestingly, the DFG motif is also not conserved in the polypeptide encoded by D trk, a Drosophila melanogaster gene that is homologous to the trk family and encodes a tyrosine kinase [33]. The nyk-r gene is expressed in a range of tissues and cell lines as two mRNA species. Four independent cDNA clones from the TI0 cell line have almost identical 5’ ends and common coding region sequences. It is likely that the difference in size between the two nyk-r mRNAs is due to differences
Lemischka IR. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991;65:1143-1152. 6 Lai C, Lemke G. An extended family of proteintyrosine kinase genes differentially expressed in the vertebrate nervous system. Neuron 1991;6:691-704.
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