Proc. Natl. Acad. Sci. USAVol. 89, pp. 11818-11822, December 1992Developmental Biology

RYK, a receptor tyrosine kinase-related molecule with unusualkinase 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 CancerResearch, University of Berne, Tiefenaustrasse 120, 3004, Berne, Switzerland

Communicated by Donald Metcalf, July 6, 1992

ABSTRACT By using the polymerase chain reaction withdegenerate oligonucleotides based on highly conserved motifsheld in common between all members of the protein tyrosinekinase (PTK) family, a PTK-related sequence was isolated frommurine peritoneal macrophage cDNA. Full-length clones havebeen isolated that encompass the entire coding region of themRNA, and the predicted amino acid sequence indicates thatthe protein encoded has the structure of a growth factorreceptor PTK (RTK). We have dubbed this moleculeRYK (forrelated to tyrosine klnase). The RYK-encoded protein bears atransmembrane domain, with a relatively small (183 aminoacid) extrellular domain, containing five potential N-linkedglycosylation sites. The intracellular domain ofRYK is uniqueamong the broader family of RTKs and has several unusualsequence idiosyncrasies in some of the most highly conservedelements of the PTK domain. These sequence differences callinto question the potential catalytic activity of theRYK protein.

The protein tyrosine kinases (PTKs) are a thematic proteinfamily, each with a highly conserved kinase domain capableof phosphorylating protein substrates on tyrosine residues(1). One branch of this family of proteins, the growth factorreceptor tyrosine kinases (RTKs), includes molecules withthe features of growth factor receptors. Typically, RTKs aretransmembrane glycoproteins with an N-terminal extracel-lular ligand binding domain and an intracellular, C-terminallylocated, tyrosine kinase domain. Whereas this type of ar-rangement ofdomains is a feature ofeach ofthe known RTKsfor which a ligand has been found (refs. 2-7, inter alia), it iswidely presumed, by extrapolation, that other RTK se-quences, which are similarly organized with respect to theirstructure, are receptors for as yet unknown ligands (refs.8-11, inter alia).

Previously, we sought new members of the PTK family byapplying the polymerase chain reaction (PCR) (12) in com-bination with degenerate oligonucleotide primers based upontwo highly conserved elements in the catalytic domains ofPTKs (13-15, 18). In this study, we describe the completesequence of a PTK-related molecule, RYK (for related totyrosine kinases),¶ isolated by means ofa modification ofouroriginal approach, using oligonucleotides based on conservedregions of the RTK subfamily, which bears all of the featuresassociated with membership of the growth factor receptorclass ofproteins. TheRYK sequence bears some unusual andidiosyncratic variations on the ordinarily highly conservedelements of this class of protein, which suggests that it mayhave a specialized role to play in signal transduction withinthe cell.

MATERIALS AND METHODSPCR Cloning of PTK-Related Sequences. The amplification

of PTK sequences using the oligonucleotides PTK1 andPTK2 has already been described (12-14). The initial RYKclone was isolated from a similar screen ofa Agtll peritonealmacrophage cDNA library (see below). The targeting ofRTKsequences was achieved by the reamplification of a 10-3dilution of material amplified by PTK1 and PTK2, using theoligonucleotides "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'-GGGAGCTCG-GTACC(T/C)TC(G/C/A)GG(A/G/C/T)GCCATCCA-3']and a 940C (1 min) denaturation, 370C (1 min) annealing, and630C (2 min) extension PCR cycle. The location of theseprimers is shown in Fig. 1. The material in the 4175-base-pair(bp) band generated in this latter PCR was cloned intopBluescript (Stratagene) and the PCR library obtained wassubsequently screened by DNA sequencing (16).

Screening of cDNA Libraries. A total of 13 murine cDNAlibraries was screened with the PCR clone BM13 (see Fig. 1)according to the protocols outlined elsewhere (17). Sequenc-ing was performed using an Erase-a-Base kit or specificoligonucleotide primers. In each case the sequence informa-tion was generated by the dideoxynucleotide chain-termination method (16).

Northern Analysis; RNA was isolated from tissues and celllines according to Chowczynski et al. (19) and was selectedformRNA as described (17). Northern blots were hybridizedwith randomly primed (20) 32P-labeled murine RYK insert.

Antibody Reagents and Protein Analysis. Polyclonal rabbitantisera R1 and R2 against keyhole limpet hemocyanin-coupled C-terminal peptide (-KFQQLVQCLTEFHAAL-GAYV-) of mouse and human RYK were raised in rabbits.

Immunoprecipitation/in vitro kinase reactions were per-formed as follows. Mouse NIH 3T3 fibroblasts were lysed in10 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 ofleupeptin per ml/5 mM Na3VO4/0.22 trypsin inhibitor unit ofaprotinin per ml (lysis buffer), and cell extracts were immu-noprecipitated by the addition of 5 1d of preimmune oranti-RYK antiserum, followed by the addition of 20 A1 of a50%6 (wt/vol) solution of protein A-Sepharose (Pharmacia).Immunoprecipitates were washed three times with lysisbuffer 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 minof incubation, samples were eluted and resolved by SDS/PAGE, and radioactively labeled bands were detected by

Abbreviations: LRM, leucine-rich motif; PTK, protein tyrosinekinase; RTK, receptor tyrosine kinase.§To whom reprint requests should be addressed.IThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M98547).

11818

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Nat!. Acad. Sci. USA 89 (1992) 11819

exposure to x-ray film (Kodak XAR-5). For immunoprecip-itation of metabolically labeled NIH 3T3 cells, 1 mCi of[35S]cysteine/methionine (Tran35S-label; ICN) was includedin methionine-depleted growth medium, and the cells weregrown for 4 hr.

Bacterial Fusion Proteins. A bacterial fusion protein of theRYK kinase-related domain was constructed using the FlagBiosystem for protein expression (IBI). In vitro kinase assaysusing aliquots of affinity-purified Flag-RYK fusion proteincombined with poly(Glu, Tyr) (1:1, 5 mg) (Sigma) or acid-denatured enolase (1 mg) as substrates were performed in 10mM Pipes, pH 7.4/10 mM MnCl2/10 mM MgCl2/5 ;LCi of[-32P]ATP and allowed to incubate for 30 min at 37TC. Inother experiments, induction ofthe Flag-RYK fusion proteinwas examined by Western blot analysis using a monoclonalantibody reactive against phosphotyrosine.Computer-Aided Analysis. Hydropathy analysis of the

RYK protein was performed using the method of Kyte andDoolittle (21). Phylogenetic analysis of the RYK kinase-related domain was performed as described by Hanks, Quinnand Hunter (22, 23).

RESULTS AND DISCUSSIONCloning Strategy for Growth Factor Receptor PIKs. Ap-

plication of the PCR to the cloning of members of the PTKfamily has been described elsewhere (13-15, 18). We havedeveloped a modification of this procedure that refines ouroriginal method and narrows the focus of the amplificationprocess so that only growth factor receptors are targeted.This modification of the original protocol was predicated onthe observation that growth factor receptor PTKs bear aparticular sequence idiosyncrasy in one of the highly con-served domains from which PTK-specific oligonucleotideprimers may be developed. In this report we have employedthe nomenclature devised by Hanks, Quinn, and Hunter, (22,23) in describing the conserved elements of the kinase

A

I H m IV v VI vu

FMK1

DLAARN

domain. Thus, in element VIII of the kinase domain, thesequence -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 predominantlyhave a methionine in the starred position in this sequence.Methionine has never been found in this position in membersof the the nonreceptor PTKs. Parenthetically, the only classof PTK receptors that does not have a methionine in thislocation is the "EPH" family ofRTKs (9-11), each of whichcarries a threonine in this location. Thus a nested PCRapproach was conceived, whereby, after amplification ofcDNA populations with the oligonucleotides PTK1 andPTK2 (to amplify the majority of PTK family membersexpressed in a particular cell source), a second round ofPCRwas carried out on a 10-3dilution of the primary amplifica-tion, using two new primers (DLAARN and WMAPE) tar-geted at the receptor-specific motifs (Fig. 1A). This amplifiedmaterial was cloned and members of the PCR library wereisolated and sequenced in an attempt to identify new RTKs.Source material for these amplifications was randomlyprimed cDNA from murine colonic poly(A)+ mRNA.

Several putative growth factor receptors have been uncov-ered in this screen. Many of the sequences shown in Fig. 1Bare derived from acknowledged growth factor receptor-related proteins [e.g., bFGF-R (18, 24), bek (25), FGFR4 (26,27), MET (28), PDGFA-R (7), and IGF1-R (29)]; the remain-der have been characterized in this laboratory (R. B. Oel-richs, A. S. Runting, S.A.S., C.M.H., and A.F.W., unpub-lished data) and are also clearly members ofthe growth factorreceptor family of proteins. RYK was a frequently isolatedRTK sequence in this screen. Our original isolate ofRYKwasderived from a PCR screen of a murine macrophage cDNAlibrary using PTK1 and PTK2. This PCR isolate was selectedfor further characterization.

Isolation and Sequence of the Murine RYK cDNA. Thirteenmouse cDNA libraries were screened as described in Mate-rials and Methods to isolate a full-length cDNA clone. The

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Z9EDLLAXVLLAQGKI GLARDIMSYVSKGST ...FzVKWAPZ3LhawRNcAvAF.TVKDrRDIYETDYYRKGGKG....LLPVIOEILAILLVENY A LRGQ.. ..VVETMG .. .RLPVRWAPZIL&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. SubdomainsI-XI ofPTKs as described by Hanks et al. (22, 23) are represented. (B) Predicted amino acid sequences ofPCR amplified products isolated froma mouse peritoneal macrophage cDNA library (BM13 only) or murine colon cDNA. The 10 sequences are aligned according to the highlyconserved "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, basicfibroblast 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.

Developmental Biology: Hovens et aL

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11820 Developmental Biology: Hovens et a!.

two RYK mRNAs are 3.5 and 2.8 kilobases (kb). One clone,XZ, represented an almost full-length copy of the 2.8-kbmRNA. The sequence ofthe RYKmRNA is presented in Fig.2. The 5' end of this clone was extremely (G+C)-rich (88% inthe region 5' of the putative initiation codon), a feature thatperhaps explains the relative rarity of clones bearing thisregion of the RYK mRNA in any of the libraries screened.This type of element has been described by Kozak (30) as afeature characteristic of the mRNAs of a number of proteinsthat regulate cell growth control, including many growthfactor 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 likelycandidate for the start codon of RYK on the basis of thefollowing observations: (i) there is a better candidate leaderpeptide following this methionine; (ii) it is positioned imme-diately after the highly (G+C)-rich region ofthe XZ clone (andnot buried within it, as is the case for the methionine at position-27), a feature typical of many of the initiation codons ofgrowth factor receptors (30); (iii) comparison ofthe mouse andhuman RYK coding sequences shows a degeneration of theotherwise extremely high degree of homology between thesetwo sequences immediately prior to this methionine (S.A.S.,C.M.H., and A.F.W., unpublished data). However, the pu-tative methionine is surrounded by a relatively poor "Kozak"(31) consensus sequence, and it still remains formally possiblethat the alternative ATG, at position -27, could be utilized.Initiation at position 1 would lead to an open reading frame of1698 bases. The predicted RYK protein is therefore 566 aminoacids long, with a predicted molecular mass of63,598 daltons.

xonofRYKmRNA. The mouseRYKgene is expressedwidely as two mRNAs of2.8 and 3.5 kb (Fig. 3), in mouse tissue

taagcttgatatcgaattcCC _ G G c _ c_-27M R A G R G G V P G S G G L___T~~~~~~~~~~~~~~~~~~~

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GAGGGCCCGCCGCCGGCCTGCTGCTGC TGCT TCCCCGGCCC

G R A P A G P S V S L Y L S E D 1 V R R L L G LTGGCCGCGCTCCCC GAcCACGCcCCTGGTCT

D A E L Y Y V R N D L S 8 H T A L 8 F A L L V PTGATGCAGAGCTTTACTAT JTGAGAAATGACCTQCATCGTCACTACGCTCTGTCCTTTAACCTGCTAGTGCC

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S E T N F L H F T N H A K S K V F. Y K L G F 0 V +83CAGTGAGACAAACTTCCTGCACTTCACTTGGCATGCAAAGTCCAAGGTTGAATATAAGCTGGGATTCCAAGT 432

N N F V A M G M P Q V 1 I S A Q GIt G P R T L SGAACAACTTTGTGGCTATGGGCATGCCCCAGCTCAATATTTCT-CTCA-CATC

V F R V E L S C T G K V D S I V M I SN SL1 LAGTGTTTrTCGAOCTTTGC CSGCA&C GTGATTCTAQA:YACTCAATCT

T V N S S K N I T V LNAK K PM C K K L EGACAGTGAATTCCTCAAAAATTTTACAGAAAACAAAAAACTTGA

E V K T S A L D K 1 T 8 R T I Y D P V H A A P TAGAAGTAAAAACTTCAGCCTTGG A CCACTATTTATG^CCCTGTcCATC

T S T R V ST Z5 v a vOa v Zr & VAL Is LGACTTCCACGCGTGTTTACATCU;TTA _ G I _TSC COG TM!I!T

A v 1.31Z3a a* I I L D D 3 I1 A 38 8 QAGCCGT5 =_ ~ a

G L 8 Q P 82 0Q If Q Y L R A D T P 11 N A e P IGGGGCTGTSCTCAGCCGTrCAC IO GQ SC T

T 8 8 G Y P T L R IV K N D L R 8 V T L LE ACACCAGCTCCTCAGGTTATCCTACCTTGCGGATAGAGRAGAACGACTTGCOAAGTGTCACTCTTCTGGAAGC

+107504

+131576

+155648

+179720

+203792

+227864

+251936

+2751008

Proc. Nat!. Acad. Sci. USA 89 (1992)

mRNAs and in vitro cell lines. Highest levels ofRYK are foundin ovary, lung, and placenta poly(A)+ RNA,al all of thetissues examined express detectable RYK mRNA. The prove-nance of the two transcripts is currently not understood, al-though different poly(A) sites are the most likely source of thevariation. It is noteworthy that RYK mRNA is also expressedin the human mammary carcinoma cell line A431 but that onlythe larger mRNA species appears to be present.Structal Featurs oftheRYK Protein. We have designated

RYK as being related to RTK because ofthe clear presence ofa highly hydrophobic transmembrane domain (data notshown), a feature held in common with all other members ofthe RTK family, and a kinase domain possessing all of theconserved elements defined by Hanks, Quinn, and Hunter (22,23). However, the predictedRYK protein is unusual-in almostevery other respect when compared to all other members ofthe RTK family. For example, the putative extracellulardomain of the RYK protein is exceedingly short, being a mere183 amino acids long [compare the extraceilular domains oftheepidermal growth factor receptor (621 amino acids; ref. 2) orthe platelet-derived growth factor receptor (524 amino acids;ref. 3)]. The extracellular domain ofRYK appears to be largelydevoid offeatures characteristic ofother RTKs families, suchas the immunoglobulin-like domains (32), fibronectin type IIIrepeats (33), or cysteine-rich domains (2, 6). Recently a seriesof LRMs were uncovered in the extracellular domain of theneurogenic RTKs trk and trkB (34). This class of motif hasbeen detected in proteins as diverse as yeast adenylate cyclase(35) and ribonuclease/angiogenin inhibitor (35) as well as in anumber of cell adhesion proteins (36). Two candidate LRMsare located in the RYK extracellular domain (Figs. 2 and 4).These elements appear to be implicated in highly specificprotein/protein interactions (34, 35) as well as in cell adhesion

+2991080

+3231152

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+3711296

+3951368

+4191440

+4431512

+4671584

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+5151728

+5391800

+5631872

+566194420162065

IK A K V K D I G I S R 1 R I T *L K D V L QQL OS

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III G R S F H G I L V D E K R P N IC Z Q T F V K

TTTTGGGCGTATTTTcCATGGGTTTTAGTAATGAAAAAAGSCCAAATAAAGAGAAGCAAACATTTGTAAAIII

T V S D Q A S 3 V Q V T N N L T 3 S C K L R G LAACAGTTAAATCTCCTCGAGTTGCTCGACGAGCTCAAGCT A =TTGAGGTCT

IVH H R N L L P I T H V C I Z E G IK P N V V L P

GCACACARAcT T7CATTACTCATGGGAA=GAATGGWAAGCTGGGTATT6CCV

Y M NN 0 N L K L F L R Q C K L V Z A N N P Q AATACATGAATTGAGGGATCTRTAG CAACCACAGGC

VIaI S Q Q D L V H M A I Q I A C O K S Y L A R R E

AATTTCCCAGCAAGATCTGGTCCATATGGCTATTCAGATTGCCTGCGGTGAGCTACCTGGCGAGGAGAGSVIb VII

V I 1 St D L AA RE C V I D D T L Q V K I T D NAGTGATCCATAGG$CTGTCT7AGGRTTTAcGcRATCRGCARCCAGACA

VIIIA L S R D L F P N D NH C L G D N Z N R P V Ra

TGCCCTCRRCTGTCCTiATGGChCACTGCCAGGACATGAGCCAGCCAGGAGATGIX

M A L X 8 L V N N I F S S A 8 D VEW Aa F V T LGATGGC TTACTGG T T AGTGCTAGTGACGTGTGG=CCTTTGGAGTGACGCT

XN1 L N T L G Q T P T V D I D P F E N A A Y L KGTGGGCTCTACCTTTcCGAcGcCAcTGGRCA~GcCCGTARTCTTACCTGAA

X XID B Y R I A Q l I N C P D I L F A V N A C C W A

AGRTGGTTACCGR&AOCCAGCCR&CA&CGITRTCTGTTCTGTGTOC= TGCTG_

L D P E E R' P K F Q Q LI V Q C L T E F H A A L G

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TTAACGAQARG!ARTAAcGSAGMTGT YTCYAOAG!=&cCTOTAT

FIG. 2. Nucleotide and predicted amino acid sequences of the mouse RYKcDNA clone. The putative initiation methionine (ATG) is locatedat position 1 and is immediately followed by a possible signal peptide sequence (amino acids +1 to +26). A second potential initiation codonat position -27 of the amino acid sequence is also indicated. The transmembrane domain is shaded and extends from residue 184 to residue211. In the cytoplasmic domain, the subdomains of the conserved PTKs are indicated over the sequence by Roman numerals (I-XI) and aelocated 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). Apotential proteolytic cleavage site "KRRK" is located at positions 145-148. Serine- and threonine-rich regons of the juxta-membrane domainare 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.

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Proc. Nati. Acad. Sci. USA 89 (1992) 11821

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FIG. 3. Northern analysis of RYK mRNA expression in tissuesand cell lines. Poly(A)+ RNA was isolated from the following adultmouse tissues and in vitro cell lines: A431 (human epidermoidcarcinoma cell line), K562 (human erythroid leukaemia cell line),NIH 3T3 (mouse), 13-day embryo, placenta, ovary, thymus, liver,lung, kidney, spleen, brain, salivary gland, heart, skeletal muscle.Five micrograms of poly(A)+ RNA was analyzed per lane on a 1%agarose/formaldehyde gel and the RNA was transferred to nitrocel-lulose. The transferred RNA was hybridized with a 2.0-kb fragmentofthe RYK cDNA and the filter was washed and processed. The 28Sand 18S ribosomal standards are indicated.

(36). The detection of two candidate LRMs in the putativeextracellular domain of the RYK RTK may suggest that it isalso a member of the LRM superfamily.A hydrophobic transmembrane domain is located between

amino acid residues 187 and 215 in the protein predicted bythe RYK coding sequence. This is clearly perceptible in ahydrophobicity plot, based on the algorithm of Kyte andDoolittle (21) (data not shown). The transmembrane domainis 28 amino acids long and is followed by a "stop transfer"motif, composed ofa lysine/arginine doublet (-KR-) (see Fig.2). The presence within this transmembrane domain ofa pairof cysteine residues at amino acid locations 195 and 1%, is,however, highly unusual.The juxtamembrane region of the RYK protein is consider-

ably longer than most other RTKs [84 amino acids versus 50amino acids for the epidermal growth factor receptor (2) and49 amino acids for the platelet-derived growth factor receptor(3)]. This region ofthe protein is high in serine residues, therebeing, in particular, two clusters ofserines, 12 amino acids and43 amino acids, C-terminal of the transmembrane region. Theepidermal growth factor receptor bears a threonine residue ina similar location that is a target for protein kinase C phos-phorylation (37, 38) and that may play a role in the modulationof the signal transduced by this receptor.RYK Has an Unusual PTK Domain. The catalytic domain

of all known PTKs appear to be composed of some 11 or 12highly conserved motifs. These are defined in Fig. 1 (see alsorefs. 22 and 23). The location and primary amino acidsequence of each of these motifs appear to be almost invari-ant from PTK to PTK. Three elements are usually scrutinizedto determine whether a particular kinase domain might havetyrosine specificity: these are elements VIb and VIII definedby Hanks and co-workers, (22, 23) coupled with the presenceof a tyrosine residue located between elements VII and VIII.For example, for element VI, tyrosine kinases bear thesequence -HRDLAARN- or -HRDLRAAN-, whereas ser-ine/threonine kinases usually have -HRDLKPEN-. Simi-larly, for element VII, PTKs have -KWMAPE-, -KWTAPE-,or -KWYAPE-, whereas serine/threonine kinases have -RW-YRAPE- or -RFKGPE-. Examination of each of these ele-ments in the murine RYK sequence clearly predicts tyrosinespecificity for this kinase-related domain. However, theRYKcatalytic domain has a number of idiosyncratic differencesbetween itself and "classical" PTK domains. The mostunusual is the presence in element VII ofthe sequence -DNA-in lieu of the universally encountered -DFG- (22, 23). TheRYK molecule is almost unique among the family ofPTKs inthis respect, there being a single other exception, kig (39),

47126 M6893117 t6893 to117 t670 1694 t716 tO.1

ZSMHLSFN.LLVPST ... .NFITWQLNL. .8SNTV... LNIKRMK

LTZLYZ QLEL... aLLGLPWTZVS.GLUFAP... .DMITPLSRKLSFW.ALESLSN ... KTVGGLSLTZLZANQKRLZEZN.. .DDVE&YVLNLTZVD8.QZAY ... AIW SLRHNITRN. KT8LSR .. .VrM.Wl.. .. .RL8 ..ANTPGYZVTSLUlg.NLT8SDV ... .DQL.PTNLTHLDZXWN.ElQMU1IATVMWVRTM

CotSSUS$ . . ... . xxxxxxx

FIG. 4. Alignment of the predicted RYK amino acid sequencewith "leucine-rich" consensus sequences found in trk, trkB, and toll(34). Sequences are aligned to the lefthand leucine residue in theconsensus sequence (shown at the bottom). The lefthand columnindicates the amino acid number of the first leucine in the consensussequence. Amino acids in bold type indicate conserved residues withthe consensus sequence. The single-letter amino acid code has beenused. Sequences of trk, trkB, and toll have been taken from Schnei-der and Schweiger (34).

which bears the sequence -ALS-, in lieu of-DFG-, and whichalteration results in an inactive PTK domain. The aspartate(D) residue in the equivalent -DFG- motif is located in thebody of the catalytic domain of the cyclic AMP-dependentprotein tyrosine kinase (40), and, although the homologousmotif in the RYK catalytic domain retains this amino acid, theimpact of the variation of the rest of this highly conservedmotif on the catalytic activity of the RYK protein remains tobe determined.

Similarly unusual sequence variations are found in con-served elements I and II, which constitute, in conjunction withelement VII (-DFG-), the putative ATP binding site of thekinase domain (22, 23). Here the glycine-rich motif(element I)and the downstream lysine (element II) are significantly dif-ferent from the typical consensus sequence. This may well bea compensation for the alteration of element VII noted pre-viously. Thus the ATP binding site of RYK is likely to bestructurally different from those found in other PTKs. Theconsequences of these changes are yet to be established.The unusual nature ofthe putative PITK domain oftheRYK

protein begs the question of the precise nature ofthe catalyticactivity it possesses. To resolve this issue, several approacheswere taken. First, rabbit antiserum was raised against theC-terminal 20 amino acids, coupled to keyhole limpet hemo-cyanin as carrier. Western blot analysis of bacterial fusionproteins (see below) demonstrated that this antiserum reactedwith the C-terminal peptide in the context ofthe whole protein(data not shown). Immunoprecipitation of Triton X-100 ex-tracts of 35S-labeled cellular extracts of mouse NIH 3T3fibroblasts revealed that the RYK protein was approximately-90 kDa (Fig. 5A). Immunoprecipitation of unlabeled ex-tracts, followed by an in vitro kinase reaction, demonstrWdthe ability of the immunoprecipitated RYK to phosphorylate(among others) a protein of the same size as the 35S-labeledimmunoprecipitates (Fig. 5B). This phosphorylation was an-ticipated to be on tyrosine residues by virtue of its resistanceto hydrolysis by potassium hydroxide; however, phosphoami-no acid analysis of radiolabeled protein from the 90-kDa banddemonstrated the presence of phosphoserine and phospho-threonine, with little or no phosphotyrosine present (data notshown). To examine the potential kinase activity of the RYKkinase-related domain, a bacterial fusion protein was con-structed using the immunoreactive epitope "Flag" (see Ma-terials andMethods) coupled to the entire intracellular domainof mouse RYK. In vitro kinase assays were performed usingaliquots of affinity-purified Flag-RYK fusion protein com-bined with poly(Glu, Tyr) or acid-denatured enolase as sub-

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- 30 -

- 30

FIG. 5. Immunoprecipitation of the RYK protein. (A) 35S-labeledextracts of murine NIH 3T3 cells were immunoprecipitated usingeither immune (anti-RYK) or preimmune antiserum. Immunoprecip-itates were electrophoresed on SDS/PAGE gels in the presence (5mM; reduced) or absence (nonreduced) of 2-mercaptoethanol as areducing agent. Autoradiography of the gels was for48 hr. (B) In vitrophosphorylation of the RYK protein. Immunoprecipitate/in vitrokinase assays of the mouseR YK protein were performed using eitherimmune (anti-RYK) or preimmune antiserum. The material was thenloaded onto an SDS/PAGE gel under reducing (5 mM 2-mercapto-ethanol) conditions and treated with 1 M KOH at 550C for 2 hr;phosphorylation of the RYK protein was revealed by autoradiogra-phy. Molecular masses are indicated in kDa.

strate. In none of these experiments was any significant levelof kinase activity detected. Moreover, in other experiments,induction of the Flag-RYK fusion protein was examined byWestern blot analysis using a monoclonal antibody againstphosphotyrosine. In these latter experiments no phosphoty-rosine was detected in whole bacterial cell lysates. We con-clude from these experiments that there is a possibility that thephosphorylation detected in in vitro kinase assays of immu-noprecipitates ofRYK may not derive from the RYK moleculeitself but may be a product of an associated protein. Whetherthis associated protein is a genuine component of a complexcontaining RYK or an artifact of the immunoprecipitationprocedure remains to be determined. A further possibility isthe absence of a suitable ligand with which to stimulate thekinase activity. Finally, the RYKcatalytic domain may requirea specific substrate to manifest its catalytic activity. RYK isthus a widely expressed member of the PTK family, with allof the features of a growth factor receptor.

We thank Prof. Tony Burgess and Dr. Anastasia Gabriel for theircritical reading of the manuscript. C.M.H. was the recipient of anAustralian Commonwealth Postgraduate Scholarship.

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