Short sequence-paper

Human K-catulin, a novel K-catenin-like molecule with conservedgenomic structure, but deviating alternative splicing1

Barbara Janssens, Katrien Staes, Frans van Roy *Molecular Cell Biology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology

(VIB)-University of Ghent, B-9000 Ghent, Belgium

Received 18 June 1999; received in revised form 26 August 1999; accepted 27 August 1999

Abstract

A new human cDNA was cloned and termed K-catulin, based on sequence similarity with both K-CATenins andvincULIN. The mRNA is present ubiquitously, although low expression levels are found in neural tissues. The genomicorganization of the K-catulin gene CTNNAL1 is closely related to that of the KE-catenin gene CTNNA1, but not at all to thatof the vinculin gene. Alternative splicing of the last exon generates a frameshift, resulting in a truncated protein with a newcarboxy-terminus. ß 1999 Elsevier Science B.V. All rights reserved.

Vinculin and K-catenins, comprising isoforms KE(epithelial) and KN (neural), are functionally andstructurally related proteins. Although the overallsequence similarity is low (approximately 23% atthe protein level), vinculin and K-catenins show anal-ogous functions. Actin binding is a common prop-erty, which renders these proteins important for for-mation of adhesion complexes. In cell^substratecontacts, vinculin proteins are linked via talin to in-tegrins, which are transmembrane receptors for theextracellular matrix [1]. In cell^cell contacts, K-cate-nin binds to either L-catenin or plakoglobin (Q-cate-nin), thus linking the transmembrane cadherin to theactin cytoskeleton [2]. The two protein types have

long been thought to have exclusive positions incell^cell and cell^substrate adhesion complexes; thereis, however, recent evidence for partially overlappinglocalizations [3,4]. The formation of adhesion com-plexes and linkage to the actin cytoskeleton are re-quired for proper tissue di¡erentiation and homeo-stasis, but also for regulation of cell growth, motility,gene expression and apoptosis (reviewed in [5]).

By BLAST analysis [6], using KE-catenin se-quences as a query, a number of overlapping humanEST sequences were found to be similar, but notidentical, to the 3P part of KE-catenin mRNA. Wecloned the corresponding full-length cDNA of 2445bp with an open reading frame (ORF) of 2205 bp(GenBank accession no. U97067), fully in agreementwith a recently reported cDNA [7]. Based on se-quence similarity with both K-CATenin and vinCU-LIN, we termed this cDNA K-catulin. The encodedK-catulin protein shares a sequence similarity of 19%with the human vinculin protein. The similarity toboth human KE-catenin and KN-catenin is 25%.The sequence conservation is especially signi¢cant

0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 7 0 - 0

* Corresponding author. Department of Molecular Biology,VIB-University of Ghent, Ledeganckstraat 35, B-9000 Gent, Bel-gium. Fax: +32-9-264-5331/264-5348;E-mail : f.vanroy@dmb.rug.ac.be

1 The sequence data from this article have been deposited withthe GenBank Data Library under accession numbers U97067,AF135021 and AF134871^AF134888.

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in some putative functional domains (Fig. 1). Theamino-terminal amphiphatic helices are thought toensure an interaction between K-catenin and L-cate-nin [8] and are also present in K-catulin and vinculin.The signature domain for vinculin was determinedwith GCG software (Genetics Computer Group,Madison, WA). The RQQEL sequence is importantfor binding of vinculin to talin [9]. Vinculin containsa unique central domain of 214 amino acids (codons318^532) for which no function has been proposedyet (boxed in Fig. 1). The most striking di¡erencebetween K-catulin and K-catenin/vinculin is a missingcentral domain of about 150 amino acids in K-catulin(starting after codon 532). For this domain, no func-tion in vinculin or K-catenin has been determined sofar, but its absence suggests a unique function ofK-catulin versus K-catenin and vinculin. The pro-line-rich hinge domain, which permits intramolecularinteractions in vinculin, is absent both in K-catulinand in K-catenin. Amphiphatic helices in the C-ter-minal part confer binding of vinculin and K-cateninto the actin cytoskeleton [5].

Northern hybridization of various cell lines withfull-length K-catulin cDNA as a probe revealed spe-

ci¢c signals for an apparently single mRNA moleculeexpressed in most human cell lines (data not shown).Dot-blot analysis of 50 di¡erent RNA samples(Clontech, Palo Alto, CA) revealed a quasi ubiqui-tous expression of K-catulin, despite a generally lowexpression level in all neural tissues tested (Fig. 2).The highest expression was observed in the adrenalgland.

Two partially overlapping genomic PAC clones(63P9 and 183G16) with speci¢city for the humanK-catulin gene CTNNAL1 were isolated from anRPCI1 PAC library (UK-HGMP Resource Centre,Hinxton, UK), using PCR with the primers 5P-GATGGGTCTCAGTTACAAATA-3P and 5P-TTT-AGCAATTACCAAGACATT-3P. These PAC cloneswere used for FISH analysis, which con¢rmed thepreviously reported gene location on chromosome9q31^32 [7]. It is noteworthy that none of the othermembers of the vinculin/K-catenin gene family is lo-cated on chromosome 9 [10^12]. Subcloning, long-range PCR with ExTaq polymerase (TaKaRa, Shu-zo, Shiga, Japan) and direct sequencing of the PACclone DNA allowed us to determine the genomicstructure of the CTNNAL1 gene. The gene consists

Fig. 2. Dot-blot expression analysis of human K-catulin mRNA. RNA from 50 di¡erent tissues (Clontech) was hybridized with thefull-length K-catulin cDNA as probe.

Fig. 1. Alignment of the predicted human K-catulin, KE-catenin and vinculin protein sequences, using the CLUSTAL W program[19]. Identical residues are boxed in black, similar residues in gray. Some putative functional domains are annotated and discussed inthe text.6

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of 19 exons with sizes varying between 37 and 298 bp(GenBank accession nos. AF134871^AF134888). All36 splice donor and splice acceptor sequences are inaccordance with the rules for exon^intron boundaries

[13]. Comparison of the exon composition of the K-catulin ORF with that of the KE-catenin ORF re-vealed that 13 out of 18 exon^exon boundaries areshared by both molecules (Fig. 3). The lack of co-

Fig. 3. Alignment of the predicted human K-catulin and KE-catenin protein sequences, using the CLUSTAL W program [19]. Exon^exon boundaries are indicated. Identical or similar residues are shown in bold. Exon^exon boundaries are shown by boxed aminoacids and are based on the genomic structures as determined for K-catulin (this report) and published for KE-catenin [20]. An addi-tional exon^exon boundary in the KE-catenin gene was determined by Dr. F. Nollet (personal communication).

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incidence of junctions was mainly apparent in the 3P-region of the gene, downstream of exon 12, where agap of 150 codons is observed in the sequence align-ment. On the other hand, there is hardly any simi-larity of the exon^exon boundary positions betweenthe human K-catulin and vinculin genes [14] (data notshown). This suggests a closer evolutionary relation-ship with KE-catenin than with vinculin, which mightresult in a more analogous function.

By BLAST analysis of the K-catulin sequence, twoEST clones (EST 392018 and EST 256942) werefound to show an additional insert of 36 bp at posi-tion 2183 in the 3P-part of the cDNA. Comparison ofthese EST sequences with our genomic data identi-¢ed the 36-bp insert as part of intron 18 and sug-gested that this region was prone to alternative splic-ing (Fig. 4). Such an event should generate aframeshift and a truncated gene product, designated

Fig. 4. Alternatively spliced human K-catulin mRNA encodes the K2-catulin isoform. (A) Schematic overview of the alternative spliceacceptor sequence within intron 18, the resulting frameshift and the shortened ORF in alternative exon 19alt. (B) Schematic represen-tation of RT-PCR reactions used to con¢rm expression of the alternative splice form: forward primer Ex19altF1 (5P-AGTGAAAC-CAGATACTAATT-3P) or Ex15F1 (5P-GAAAACTTCCCAGGATTTA-3P) in combination with a reverse primer Ex19R2 (5P-CCAT-GATGGCTCTCTTAGTC-3P) are splice-speci¢c or splice-£anking, respectively. Predicted sizes of the RT-PCR products are asindicated.

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K2-catulin (GenBank accession no. AF135021). Atthe C-terminus of the encoded protein a shorterstretch of 5 amino acids substitutes for the standardsequence of 21 amino acids. When splice scores [15]were calculated for the two splice acceptor sites, val-ues of 89.7 and 95.7 were obtained for the standardand the alternative splice acceptor, respectively. Tocon¢rm the general occurrence of the alternativesplicing event, RT-PCR analysis with primers speci¢cfor the alternative insert (Fig. 4B) was performed onmRNAs from various human tissues and cell lines.Fragments of the expected size were obtained (Fig.5A), showing ubiquitous expression of the alternativeK2-catulin. When primers speci¢c for the standardexons 15 and 19 were combined in an RT-PCR ex-periment, only the ampli¢cation product of thestandard K1-catulin splice form was readily visible(Fig. 5B). This points at far less abundant expressionof the alternative K2-catulin splice form.

Vinculin as well as both K-catenins can be ex-pressed as isoforms with in-frame inserts nearby theC-terminal ends. In KE-catenin, alternative splicing isobserved between exons 16 and 17, resulting in theaddition of 24 amino acids in-frame on position 812[16]. At the same position in KN-catenin, an insert of48 amino acids is present [12] and is supposed to be

the result of alternative splicing. Although bothsplice forms can be observed in mouse cDNA [17],only the longer form has been cloned for the humanKN-catenin [12,16]. In the case of vinculin, alterna-tive splicing occurs further upstream from the C-ter-minus, at position 915 of the amino acid sequence.The so-called meta-vinculin protein contains an in-frame insert of 68 amino acids, which possibly in£u-ences binding of vinculin to actin [18]. However,truncation of the C-terminus as reported here forK2-catulin was not seen in any of these cases of alter-native splicing.

In conclusion, the limited, but signi¢cant, sequencehomology between K-catulin and K-catenin or vincu-lin suggests that K-catulin may bind to L-cateninand/or talin, and also to the actin cytoskeleton.The conserved genomic structure suggests that theK-catulin gene CTNNAL1 is derived from the sameancestor gene as the KE-catenin gene CTNNA1.Nevertheless, this widely expressed K-catulin is sig-ni¢cantly di¡erent from the other K-catenin/vinculinfamily members. A central protein domain is com-pletely lacking in K-catulin and an alternative spliceform clearly deviates from splice forms of K-cateninsand vinculin. In this context, it should be consideredthat the well-studied picture of the classical E-cad-

Fig. 5. Con¢rmation of expression of the alternative splice product by RT-PCR. The speci¢city of PCR products was con¢rmed bysequence analysis. (A) Using primer Ex19altF1 with Ex19R2, the 246-bp product speci¢c for alternative splicing is ampli¢ed ubiqui-tously. (B) When primer Ex15F1 is used with primer Ex19R2, the normal splice form is preferentially ampli¢ed (478 bp) as comparedto the alternative splice form (514 bp). (C) The quality of all mRNA templates was veri¢ed by ampli¢cation of L-actin cDNA. M,100-bp (A, B) or VBstEII (C) size markers. MCF7 is a breast carcinoma cell line, GLC34 and GLC8 are small-cell lung carcinomacell lines, A431 is an epidermoid carcinoma cell line and PC-3 is a prostate carcinoma cell line.

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herin^catenin complex may need re¢nement and ex-tension with new molecules, such as the presentlyreported K-catulin. Therefore, generation of speci¢cantibodies and detailed functional analysis of K-ca-tulin may provide important clues to the understand-ing of formation and regulation of cell adhesion.

We thank Dr. P. Kools and Dr. J. van Hengel forsupporting this project. We are grateful to C. Eich-perger and E. Vanden Eynde for technical assistance.This work was supported by the Fonds voor Weten-schappelijk Onderzoek-Vlaanderen, the Geconcer-teerde Onderzoeksacties, the Sportvereniging tegenKanker, the Centrum voor Studie en Behandelingvan Gezwelziekten and the Algemene Spaar- en Lijf-rentekas. B.J. is supported by the Vlaams Instituutvoor de Bevordering van het Wetenschappelijk-tech-nologisch Onderzoek in de Industrie. F.v.R. is a Re-search Director with the Fonds voor Wetenschappe-lijk Onderzoek-Vlaanderen.

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