Communicated by Donald Metcalf, July 6, 1992. ABSTRACT. By using the polymerase chain reaction with degenerate oligonucleotides based on highly ...
Proc. Natl. Acad. Sci. USA
Vol. 89, pp. 11818-11822, December 1992 Developmental Biology
RYK, a receptor tyrosine kinase-related molecule with unusual kinase domain motifs (PCR/growth fdtor receptor/kinaswe-related domain)
CHRISTOPHER M. HOVENSt, STEVEN A. STACKERt, ANNE-CATHERINE ANDRESt, AILSA G. HARPURt, ANDREW ZIEMIECKIt, AND ANDREW F. WILKSt§ tLudwig Institute for Cancer Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia; and tInstitute for Clinical and Experimental Cancer Research, University of Berne, Tiefenaustrasse 120, 3004, Berne, Switzerland Communicated by Donald Metcalf, July 6, 1992
ABSTRACT By using the polymerase chain reaction with degenerate oligonucleotides based on highly conserved motifs held in common between all members of the protein tyrosine kinase (PTK) family, a PTK-related sequence was isolated from murine peritoneal macrophage cDNA. Full-length clones have been isolated that encompass the entire coding region of the mRNA, and the predicted amino acid sequence indicates that the protein encoded has the structure of a growth factor receptor PTK (RTK). We have dubbed this molecule RYK (for related to tyrosine klnase). The RYK-encoded protein bears a transmembrane domain, with a relatively small (183 amino acid) extrellular domain, containing five potential N-linked glycosylation sites. The intracellular domain of RYK is unique among the broader family of RTKs and has several unusual sequence idiosyncrasies in some of the most highly conserved elements of the PTK domain. These sequence differences call into question the potential catalytic activity of the RYK protein.
MATERIALS AND METHODS PCR Cloning of PTK-Related Sequences. The amplification of PTK sequences using the oligonucleotides PTK1 and PTK2 has already been described (12-14). The initial RYK clone was isolated from a similar screen of a Agtll peritoneal macrophage cDNA library (see below). The targeting of RTK sequences was achieved by the reamplification of a 10-3 dilution of material amplified by PTK1 and PTK2, using the oligonucleotides "DLAARN" [5'-GGGTCTAGATCGAC-
GA(T/C)CT(A/G/C/T)GC(A/G/C/T)GC(A/G/C/T)(A/ G)C(A/G/C/T)AA-3'] and "WMAPE" [5'-GGGAGCTCGGTACC(T/C)TC(G/C/A)GG(A/G/C/T)GCCATCCA-3'] and a 940C (1 min) denaturation, 370C (1 min) annealing, and 630C (2 min) extension PCR cycle. The location of these primers is shown in Fig. 1. The material in the 4175-base-pair (bp) band generated in this latter PCR was cloned into pBluescript (Stratagene) and the PCR library obtained was subsequently screened by DNA sequencing (16). Screening of cDNA Libraries. A total of 13 murine cDNA libraries was screened with the PCR clone BM13 (see Fig. 1) according to the protocols outlined elsewhere (17). Sequencing was performed using an Erase-a-Base kit or specific oligonucleotide primers. In each case the sequence information was generated by the dideoxynucleotide chaintermination method (16). Northern Analysis; RNA was isolated from tissues and cell lines according to Chowczynski et al. (19) and was selected for mRNA as described (17). Northern blots were hybridized with randomly primed (20) 32P-labeled murine RYK insert. Antibody Reagents and Protein Analysis. Polyclonal rabbit antisera R1 and R2 against keyhole limpet hemocyanincoupled C-terminal peptide (-KFQQLVQCLTEFHAALGAYV-) of mouse and human RYK were raised in rabbits. Immunoprecipitation/in vitro kinase reactions were performed as follows. Mouse NIH 3T3 fibroblasts were lysed in 10 mM Tris HCl, pH 8.0/1.0%o Triton X-100/150 mM NaCl/ 0.1% NaN3/0.2 mM phenylmethylsulfonyl fluoride/10 pg of leupeptin per ml/5 mM Na3VO4/0.22 trypsin inhibitor unit of aprotinin per ml (lysis buffer), and cell extracts were immunoprecipitated by the addition of 5 1d of preimmune or anti-RYK antiserum, followed by the addition of 20 A1 of a 50%6 (wt/vol) solution of protein A-Sepharose (Pharmacia). Immunoprecipitates were washed three times with lysis buffer at 40C and finally resuspended in 50 pL1 of kinase buffer (10 mM Tris-HCl, pH 7.5/10 mM MgCl2/10 mM MnCl2) containing 5 jCi of [y32P]ATP (1 Ci = 37 GBq). After 10 min of incubation, samples were eluted and resolved by SDS/ PAGE, and radioactively labeled bands were detected by
The protein tyrosine kinases (PTKs) are a thematic protein family, each with a highly conserved kinase domain capable of phosphorylating protein substrates on tyrosine residues (1). One branch of this family of proteins, the growth factor receptor tyrosine kinases (RTKs), includes molecules with the features of growth factor receptors. Typically, RTKs are transmembrane glycoproteins with an N-terminal extracellular ligand binding domain and an intracellular, C-terminally located, tyrosine kinase domain. Whereas this type of arrangement of domains is a feature of each of the known RTKs for which a ligand has been found (refs. 2-7, inter alia), it is widely presumed, by extrapolation, that other RTK sequences, which are similarly organized with respect to their structure, are receptors for as yet unknown ligands (refs. 8-11, inter alia). Previously, we sought new members of the PTK family by applying the polymerase chain reaction (PCR) (12) in combination with degenerate oligonucleotide primers based upon two highly conserved elements in the catalytic domains of PTKs (13-15, 18). In this study, we describe the complete sequence of a PTK-related molecule, RYK (for related to tyrosine kinases),¶ isolated by means of a modification of our original approach, using oligonucleotides based on conserved regions of the RTK subfamily, which bears all of the features associated with membership of the growth factor receptor class of proteins. The R YK sequence bears some unusual and idiosyncratic variations on the ordinarily highly conserved elements of this class of protein, which suggests that it may have a specialized role to play in signal transduction within the cell.
Abbreviations: LRM, leucine-rich motif; PTK, protein tyrosine kinase; RTK, receptor tyrosine kinase. §To whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M98547).
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11818
Proc. Nat!. Acad. Sci. USA 89 (1992)
Developmental Biology: Hovens et aL exposure to x-ray film (Kodak XAR-5). For immunoprecipitation of metabolically labeled NIH 3T3 cells, 1 mCi of [35S]cysteine/methionine (Tran35S-label; ICN) was included in methionine-depleted growth medium, and the cells were grown for 4 hr. Bacterial Fusion Proteins. A bacterial fusion protein of the RYK kinase-related domain was constructed using the Flag Biosystem for protein expression (IBI). In vitro kinase assays using aliquots of affinity-purified Flag-RYK fusion protein combined with poly(Glu, Tyr) (1:1, 5 mg) (Sigma) or aciddenatured enolase (1 mg) as substrates were performed in 10 mM Pipes, pH 7.4/10 mM MnCl2/10 mM MgCl2/5 ;LCi of [-32P]ATP and allowed to incubate for 30 min at 37TC. In other experiments, induction of the Flag-RYK fusion protein was examined by Western blot analysis using a monoclonal antibody reactive against phosphotyrosine. Computer-Aided Analysis. Hydropathy analysis of the RYK protein was performed using the method of Kyte and Doolittle (21). Phylogenetic analysis of the RYK kinaserelated domain was performed as described by Hanks, Quinn and Hunter (22, 23).
RESULTS AND DISCUSSION Cloning Strategy for Growth Factor Receptor PIKs. Ap-
plication of the PCR to the cloning of members of the PTK family has been described elsewhere (13-15, 18). We have developed a modification of this procedure that refines our original method and narrows the focus of the amplification process so that only growth factor receptors are targeted. This modification of the original protocol was predicated on the observation that growth factor receptor PTKs bear a particular sequence idiosyncrasy in one of the highly conserved domains from which PTK-specific oligonucleotide primers may be developed. In this report we have employed the nomenclature devised by Hanks, Quinn, and Hunter, (22, 23) in describing the conserved elements of the kinase A
11819
domain. Thus, in element VIII of the kinase domain, the sequence -LYS-TRP-*-ALA-PRO-GLU- (rendered as -KW*APE- in one-letter amino acid code in Fig. 1) is found. Members of the growth factor receptor family predominantly have a methionine in the starred position in this sequence. Methionine has never been found in this position in members of the the nonreceptor PTKs. Parenthetically, the only class of PTK receptors that does not have a methionine in this location is the "EPH" family of RTKs (9-11), each of which carries a threonine in this location. Thus a nested PCR approach was conceived, whereby, after amplification of cDNA populations with the oligonucleotides PTK1 and PTK2 (to amplify the majority of PTK family members expressed in a particular cell source), a second round of PCR was carried out on a 10-3dilution of the primary amplification, using two new primers (DLAARN and WMAPE) targeted at the receptor-specific motifs (Fig. 1A). This amplified material was cloned and members of the PCR library were isolated and sequenced in an attempt to identify new RTKs. Source material for these amplifications was randomly primed cDNA from murine colonic poly(A)+ mRNA. Several putative growth factor receptors have been uncovered in this screen. Many of the sequences shown in Fig. 1B are derived from acknowledged growth factor receptorrelated proteins [e.g., bFGF-R (18, 24), bek (25), FGFR4 (26, 27), MET (28), PDGFA-R (7), and IGF1-R (29)]; the remainder have been characterized in this laboratory (R. B. Oelrichs, A. S. Runting, S.A.S., C.M.H., and A.F.W., unpublished data) and are also clearly members of the growth factor receptor family of proteins. RYK was a frequently isolated RTK sequence in this screen. Our original isolate of R YK was derived from a PCR screen of a murine macrophage cDNA library using PTK1 and PTK2. This PCR isolate was selected for further characterization. Isolation and Sequence of the Murine RYK cDNA. Thirteen mouse cDNA libraries were screened as described in Materials and Methods to isolate a full-length cDNA clone. The Vm
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Z9EDLLAXVLLAQGKI GLARDIMSYVSKGST ... FzVKWAPZ 3LhawRNcAvAF.TVKDrRDIYETDYYRKGGKG....LLPVIOE A LRGQ.. ..VVETMG .. . RLPVRWAPZ ILAILLVENY NEX IL&NIILLSENNVVICDNGLAIDIYKNPDYVRRGDT ... RLPLKUAPZ FIG. 1. Strategy for construction of oligonucleotide probes to "nested" conserved regions of RTKs. (A) Pairs of oligonucleotide primers (PTK1/PTK2; DLAARN/WMAP) were made to sequences of the conserved catalytic domain of PTKs and RTKs, respectively. Subdomains I-XI of PTKs as described by Hanks et al. (22, 23) are represented. (B) Predicted amino acid sequences of PCR amplified products isolated from a mouse peritoneal macrophage cDNA library (BM13 only) or murine colon cDNA. The 10 sequences are aligned according to the highly conserved "IHRDLAARN" and "WMAP" sequences. Amino acids in bold type indicate highly conserved residues in the PTK catalytic domain. Previously isolated sequences are identified using accepted nomenclature. Asterisks indicate previously unreported sequences. bFGF-R, basic fibroblast growth factor receptor; FGFR-4, fibroblast growth factor 4 receptor; PDGF-A, platelet-derived growth factor A receptor; IGF1R, insulin-like growth factor 1 receptor. PDGF-A IGF1R JIL
11820
Developmental Biology: Hovens et a!.
Proc. Nat!. Acad. Sci. USA 89 (1992)
two RYK mRNAs are 3.5 and 2.8 kilobases (kb). One clone, XZ, represented an almost full-length copy of the 2.8-kb mRNA. The sequence ofthe R YK mRNA is presented in Fig. 2. The 5' end of this clone was extremely (G+C)-rich (88% in the region 5' of the putative initiation codon), a feature that perhaps explains the relative rarity of clones bearing this region of the R YK mRNA in any of the libraries screened. This type of element has been described by Kozak (30) as a feature characteristic of the mRNAs of a number of proteins that regulate cell growth control, including many growth factor receptors. There are two potential initiation methionine codons at the 5' end of the XZ clone (located at positions 1 and -27 in Fig. 2). We anticipate that the ATG at position 1 is the most likely candidate for the start codon of RYK on the basis of the following observations: (i) there is a better candidate leader peptide following this methionine; (ii) it is positioned immediately after the highly (G+C)-rich region of the XZ clone (and not buried within it, as is the case for the methionine at position -27), a feature typical of many of the initiation codons of growth factor receptors (30); (iii) comparison of the mouse and human R YK coding sequences shows a degeneration of the otherwise extremely high degree of homology between these two sequences immediately prior to this methionine (S.A.S., C.M.H., and A.F.W., unpublished data). However, the putative methionine is surrounded by a relatively poor "Kozak" (31) consensus sequence, and it still remains formally possible that the alternative ATG, at position -27, could be utilized. Initiation at position 1 would lead to an open reading frame of 1698 bases. The predicted RYK protein is therefore 566 amino acids long, with a predicted molecular mass of 63,598 daltons. xonofRYKmRNA. The mouseRYKgene is expressed widely as two mRNAs of 2.8 and 3.5 kb (Fig. 3), in mouse tissue taagcttgatatcgaattcCC
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mRNAs and in vitro cell lines. Highest levels of RYK are found in ovary, lung, and placenta poly(A)+ RNA, al all of the tissues examined express detectable RYK mRNA. The provenance of the two transcripts is currently not understood, although different poly(A) sites are the most likely source of the variation. It is noteworthy that RYK mRNA is also expressed in the human mammary carcinoma cell line A431 but that only the larger mRNA species appears to be present. Structal Featurs of the RYK Protein. We have designated RYK as being related to RTK because ofthe clear presence of a highly hydrophobic transmembrane domain (data not shown), a feature held in common with all other members of the RTK family, and a kinase domain possessing all of the conserved elements defined by Hanks, Quinn, and Hunter (22, 23). However, the predicted RYK protein is unusual-in almost every other respect when compared to all other members of the RTK family. For example, the putative extracellular domain of the RYK protein is exceedingly short, being a mere 183 amino acids long [compare the extraceilular domains of the epidermal growth factor receptor (621 amino acids; ref. 2) or the platelet-derived growth factor receptor (524 amino acids; ref. 3)]. The extracellular domain of RYK appears to be largely devoid of features characteristic of other RTKs families, such as the immunoglobulin-like domains (32), fibronectin type III repeats (33), or cysteine-rich domains (2, 6). Recently a series of LRMs were uncovered in the extracellular domain of the neurogenic RTKs trk and trkB (34). This class of motif has been detected in proteins as diverse as yeast adenylate cyclase (35) and ribonuclease/angiogenin inhibitor (35) as well as in a number of cell adhesion proteins (36). Two candidate LRMs are located in the RYK extracellular domain (Figs. 2 and 4). These elements appear to be implicated in highly specific protein/protein interactions (34, 35) as well as in cell adhesion
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FIG. 2. Nucleotide and predicted amino acid sequences of the mouse RYK cDNA clone. The putative initiation methionine (ATG) is located at position 1 and is immediately followed by a possible signal peptide sequence (amino acids +1 to +26). A second potential initiation codon at position -27 of the amino acid sequence is also indicated. The transmembrane domain is shaded and extends from residue 184 to residue 211. In the cytoplasmic domain, the subdomains of the conserved PTKs are indicated over the sequence by Roman numerals (I-XI) and ae located between the two arrows (residues 291-551). Putative N-linked glycosylation sites are indicated with a star. Leucine-rich motifs (LRMs) are indicated in the putative extracellular domain by arrows (LI lies between amino acids 51 and 65; L2 lies between residues 126 and 144). A potential proteolytic cleavage site "KRRK" is located at positions 145-148. Serine- and threonine-rich regons of the juxta-membrane domain are indicated by bold lettering and an inverted triangle symbol above the amino acid. Cysteine residues in the extracellular domain are circled. Nucleotide residues 1-19 are in lowercase letters and are derived from pBluescript. The single-letter amino acid code has been used.
Proc. Nati. Acad. Sci. USA 89 (1992)
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