armadillo gene family

Gene, 172 (1996) 155-159 0 1996 Elsevier Science B.V. All rights reserved.

GENE

155

0378-1119/96/$15.00

09759

Identification of a Hydra homologue of the p-catenin/plakoglobinfarmadillo gene family (Cell adhesion;

cnidaria;

molecular

evolution;

cadherin;

development;

tissue morphogenesis)

Engelbert Hobmayef, Masayuki Hattab, Robert FischerC, Toshitaka Thomas W. Holstein” and Tsutomu Sugiyamab

Fujisawab,

“Laboratory of Cell and Developmental Biology, Zoological Institute, University of Frankfurt, 60590 Frankfurt/Main, Germany; bLaboratory of Developmental Genetics, National Institute of Genetics, Mishima 411, Japan. Tel. (81-559) 81-6764; and ‘Division of Cell Biology, German Cancer Research Center, 69120 Heidelberg, Germany. Tel. (49-622) 423-212 Received by T. Sekiya: 21 September

1995; Revised/Accepted:

12 December

1995; Received at publishers:

20 February

1996

SUMMARY

The /I-cateninJplakoglobin/armadillo gene family encodes a group of highly conserved proteins which play important roles in cadherin-mediated cell adhesion and in signal transduction mechanisms involved in regulating development. This gene family previously had been isolated only from higher metazoans. Here, we describe the isolation and characterization of a p-catenin @Ctn) homologue from Hydra magnipapillata, a diploblastic lower metazoan. Comparison of the putative amino acid (aa) sequence of Hydra pCtn, with its homologues in higher metazoans, shows that a repeating 42-aa motif present in its central domain is highly conserved throughout the metazoa. This suggests that pCtn appeared very early in metazoan

evolution,

possibly

when primitive

INTRODUCTION

The P-catenin/plakoglobin/armadillo (PCtn/Pkg/Arm) gene family encodes for a group of highly conserved cytoplasmic proteins. In vertebrates, pCtn is an important part of the cadherin(Cdh)-mediated cell adhesion apparatus. Cdh are cell adhesion molecules, whose extracellular domain mediates homophilic binding with corresponding Cdh (Takeichi, 1991; Shapiro et al., 1995; Overduin et al., 1995). PCtn/Pkg and the structurally unrelated a-catenin Correspondence to: Dr. E. Hobmayer, Laboratory Developmental Biology, Zoological Institute, University Theodor-Stern-Kai 7/7S, 60590 Frankfurt/Main, 6301-7637; Fax (49-69) 6301-7639; e-mail: [email protected]

Germany.

of Cell and of Frankfurt, Tel. (49-69)

Abbreviations: aa, amino acid(s); Arm, armadillo protein; Arm, gene encoding Arm; bp, base pair(s); Cdh, cadherin(s); Ctn, catenin(s); Ctn, gene encoding Ctn; Hm, Hydra magnipapillata; Hv, Hydra vulgaris; kb, kilobase or 1000 bp; nt, nucleotide(s); PCR, polymerase chain reaction; Pkg, plakoglobin(s); wg, wingless. SSDI 0378-1119(96)00162-X

multicellular

animals

started

to form epithelial

cell layers.

(aCtn) bind to the cytoplasmic domain of Cdh, and together serve as linkers to establish a connection between the Cdh-receptor and cytoskeletal actin/intermediate filament fibers in the cytoplasm (for reviews, see Takeichi, 1991, 1993; Koch and Franke, 1994). Ctn are essential for Cdh function. Cells, which express a cryptic Cdh without Ctn-binding site, or express normal Cdh and pCtn but lack aCtn, cannot adhere and establish adherent junctions (Nagafuchi and Takeichi, 1989; Ozawa et al., 1990; Hirano et al., 1992). pCtn has dual function in cell adhesion and signal transduction. Arm, the Drosophila pCtn homologue, is involved in transduction of the wingless (wg) signal during embryogenesis. Reception of wg signal on the cell surface triggers accumulation of Arm protein in the cytoplasm, where it is thought to interact with effector molecules to establish segment polarity (for review, see Kirkpatrick and Peifer, 1995). In vertebrates, expression of pCtn and Pkg is regulated by Wnt-1, a vertebrate homologue of wg, suggesting that PCtn/Pkg/Arm and

156 Wnt/wg

signaling

(Bradley

et al., 1993; Hinck et al., 1994; Peifer et al., 1994a;

systems

are

functionally

connected

Funayama et al., 1995). Competition for binding pCtn by Cdh in the cell membrane and by effector molecules in the cytoplasm cell adhesion 1994a). Recently,

appears and

to be a possible

signal

transduction

link between (Peifer

et al., 240

Cdh

and

&tn

have

been

isolated

from

Drosophila (Oda et al., 1993, 1994), suggesting that Cdh/ Ctn-based cell adhesion occurs widely in higher metazoans. In the present communication and characterization gene family

from

of a member Hydra,

we report the isolation of the PCtn/Pkg/Arm

one of the lowest

420

metazoans

possessing well-established epithelial cell layers with defined junctional complexes. Our results indicate that a cell adhesion throughout mechanism evolution

mechanism

based on Cdh/Ctn

is conserved

the metazoa. Acquisition of this cell adhesion might have been a crucial step in metazoan from a simple ‘cell mass’ organization

to epithe-

lial tissue organization. 660

EXPERIMENTAL

720

AND DISCUSSION

780

(a) Isolation and sequencing of cDNA clones Using degenerate primers and random primed first strand cDNA from Hydra magnipapillatu (Hm) as a template, DNA fragments of expected size were amplified by PCR. Subcloning and sequencing of these amplification products revealed a 280-bp fragment homologous to vertebrate fiCtn/Pkg and Drosophila Arm. Using the 280-bp fragment as a probe, cDNA libraries made from Hm (150 000 plaques) and Hydra vulgaris (Hv) (200 000 plaques) were screened, and nine positive clones were isolated. One Hm clone contained an insert covering the full length sequence. Overlapping nt sequences were determined from this clone. (b) Sequence analysis of Hydra j3Ctn and comparison with homologues in higher metazoa The deduced aa sequence of the Hydra cDNA clone and its relationship to homologues in echiurans (Urechis), sea urchins (Tripneustes), insects (Drosophila) and vertebrates (Homo) is shown in Fig. 1. All these molecules share a repeating 42-aa (referred to as Arm motif; Riggleman et al., 1989; Peifer et al., 1994b). They contain 12 copies of the Arm motif in their central domain, with a smaller insertion between repeat 10 and 11. Identity of the Hydra molecule is extremely high throughout the entire repeat domain, whereas the N- and C-terminal ends are more divergent (Fig. 1). Similarity of the Hydra sequence is closer with pCtn than with Pkg. Hence, the molecule will be referred to as Hydra pCtn.

Fig. 1. Deduced

aa sequence

of Hydra

BCtn and its relationship

to

homologues in echiurans, sea urchins, insects and vertebrates. Alignment was done using the CLUSTAL V software (Higgins and Sharp, 1989). Symbols (*) represent identical aa in all proteins, (.) conserved substitutions. Gaps were included to optimize identity. Arm repeats are indicated by arrows and numbered. Sequences were obtained from the GenBank and SWISS-PROT database with NCBI Entrez-software. Proteins and accession Nos.: (ire&is PCtn (P35224); Tripneustes j3Ctn (P35223); Drosophila Arm (P18824); Homo PCtn Homo Pkg (P14923). Methods: Standard procedures as (P35222); described by Sambrook et al. (1989) were used for PCR, cDNA cloning and sequence analysis. T-GAATTCARAAYTGYYTNTGGAC and

5’-GAATTCARRTGNCGNARNGCRCA oligos were used as primers under the following amplification conditions: denaturation 95’C/l.O min; annealing 50”/1.5 min; elongation 72”,‘2.0 min; 30 cycles. PCR fragments were inserted library [oligo(dT)-primed

into pUC18. 150000 plaques of a Hm cDNA in hZAP II] and 200000 plaques of a Hu

cDNA library [oligo(dT)and random primed in hZAP II] were screened, and positive clones purified by in vivo excision as described in the supplier’s manual. PCR products and cDNA clones were sequenced by the chain-termination method (Sanger et al., 1977). Note: The GenBank accession No. for the Hydra BCtn nt sequence reported here is U36781.

An alignment of the Hydra pCtn Arm repeats is given in Fig. 2. Specific aa are preferred at many positions within the repeats, and they are expressed as a consensus sequence beneath the alignment. Individual repeats and the insertion show 32-52% identity with the consensus sequence, conservative substitutions included. The Hydra

157 sequence

Repeat

pCtn mRNAs in

1 2 3 4 5 6 7 8

KNNVIDLINYQDETDVALRAVPELARLLCNS-DAQTIHQASI-M VNQLTKEEASCYAVMNNTNIVAALVGVTATSND-GETIRNVVQA LHNMSHBRQGLM-AIFKCSQIPALVKLLGHR-I-EAVVFYAITT LHNLLLEQEGAKMAVRLALQLQKMVSLLQRP-NV-KFLAIVTDC LQILAYGNQESKLIILSSQQPAELVRIMR-SYTYEKLLYTTCRV LKVLSV-CSSNKPAIVEAQQMQALA~YLSHQ-STR-LVQNCLWT LRNLS-D-VATKQDGLE-QLLQMLVQLLS-SNDIN-VVTCVSGI ISNLTCNNPRBIQVVFQVQQIEALVRTIINAGDR~EITEPAVCA

9 10

LRHLTSRHPDAEHAENGVRL%LV~LLNPP-SRWPLIKAVVGL IRNLGLCPS-NUTPIRDQQQLPKLVQLLMKSYQ-D-IQRRGPGA QNMQDGVRMEEIVEGTVQA LBILA-REALNRSIIRDLNCIPTFVQLLY-S-EVENIVRVAAQV LCELAQDKEGAD-AIEREQATTILTELLH-SRN-DGIAAYARAV

sequence assigned,

of Arm

repeats

from

Hydra

are much more abundant of

Rosenthal,

adults

in embryos

(Riggleman

et

al.,

than 1989;

significance

Our results demonstrate the presence of pCtn in Hydra, one of the most ancient metazoans. By using Hydra as an outgroup, our analysis offers a better understanding

A

PCtn.

the

1993).

(c) Evolutionary

LoNLx-o---NK-AI+.+QG+PILVPLL--S-D-E-IVO_ + 0

Fig. 2. Alignment

tissues

+

A consensus

is given beneath the repeats. Consensus matches when ~30% of the repeats were identical. (0) indicates

were K, R

and H; (x) indicates T and S; (+) indicates V, L and I; (0) indicates D and E. Consensus matches including their conservative substitutions are shown in bold type. The small letters YGI in repeat 9 are aa looped out to maximize alignment.

of the evolutionary relationship of pCtn in higher metazoans. Fig. 4 shows a phylogram, generated with maximum

likelihood

housefly

sequences

here. The branch

estimates.

The

not presented

Xenopus,

mouse,

and

in Fig. 1 are included

links in this figure are proportional

the degree of relatedness

between

different

proteins,

to but

not to their genetic distance. This analysis shows ~ with a high degree of certainty - that pCtn from echinoderms, and echiurans form a distinct cluster. insects, Echinoderms comprise together with the vertebrates the

pCtn

consensus

sequence

itself is highly

similar

to the

consensus sequences of other Arm motif-containing peptides (Peifer et al., 1994b). Expression of Hydra pCtn was analyzed in a Northern blot using mRNA extracted from intact, total Hydra (Fig. 3). The 280-bp PCR fragment used to isolate cDNA clones hybridized to a mRNA of about 3.4 kb size, which is similar

to the mRNA

size of Drosophila

main phyla of a well defined monophyletic group, the deuterostomia. This has been shown by morphological data and by recent sequence data on 18s RNA (Lake, 1990) and 28s RNA (Christen et al., 1991). Since pCtn

Arm (3.2 kb;

Riggleman et al., 1989) and Xenopus j3Ctn (3.5 kb; McCrea et al., 1991). Expression was moderate, resembling the conditions found in other organisms, where

100

r-J

r-

Xenopus I

,oo

plakoglobin Mus plakoglobin

-Homo

62

plakoglobin

--i

I

-Xenopus

p-catenin

28S-

I

Tripneustes

p-catenin

18s -

-Drosophila -Hydra

Fig. 3. Detection of/3Ctn mRNA in Hm. Methods: 0.1 Fg poly(A)+RNA from intact Hm polyps were fractionated by electrophoresis in a 2.2 M formaldehyde/l% agarose gel and transferred to nylon membrane. Total Hydra RNA was included to indicate 28s and 18s size. The Northern blot was hybridized with a [32P]dCTP-1abe1ed 280-bp fragment according to standard procedures described in Sambrook et al. (1989).

arinadillo

pcatenin

Fig. 4. Phylogram of BCtn, Pkg and Arm proteins. This figure was generated using multiple-aligned sequences (CLUSTAL V) with programs from the Phylip software package (Felsenstein, 1989, 1993): the SEQBOOT (bootstrap analysis), PROTDIST (distance measure for protein sequences using maximum likelihood estimates based on the Dayhoff PAM matrix), NEIGHBOR (neigbor joining distance matrix), and CONSENSE (consensus-tree by the majority-rule consensus method). The numbers above the nodes are bootstrap values calculated from 100 bootstrap replicates.

158 from echinoderms clusters with other invertebrates (Fig. 4), we suggest that Pkg and BCtn in vertebrates arose from a common vertebrate ancestor, probably at the base of the vertebrates or chordates. A common invertebrate ancestor (Rosenthal, 1993) seems to be unlikely. The characteristic motifs of the pCtn molecule - Arm repeats - have been found in a variety of other molecules, i.e., p120”“” and plakophilin 1 (Reynolds et al., 1992; Schafer et al., 1993; Heid et al., 1994; Hatzfeld et al., 1994), the APC tumor suppressor gene (Kinzler et al., 1991; Groden et al., 1991), the smgGDS exchange factor for Ras-related small G-proteins (Kikuchi et al., 1992), importin (Gorlich et al., 1994), importin-like tumor suppressor gene oho31 (T&ok et al., 1995), and the yeast SRPl suppressor of RNA polymerase I mutations (Yano et al., 1992). However, individual repeats of Hydra pCtn exhibit particularly high similarity to corresponding repeats in BCtn of higher organisms (Fig. 1). Peifer and coworkers (1994b) argued that an initially diverging repeat pattern became fixed during evolution due to the conservation of specific interactions with target proteins. Since corresponding repeats are highly conserved from Hydra throughout the metazoa, a functional pCtn molecule must have been invented very early in metazoan evolution. In additional experiments, we found that aggregation of Hydra epithelial cells closely resembles Cdh-mediated aggregation of vertebrate cells. It depends on the presence of calcium, and calcium is able to protect aggregation against inactivation by trypsin treatment (E.H., P. Snyder, D. Alt and T.W.H., in preparation). Hence, not only the BCtn molecule, but the entire Cdh/Ctn-cell adhesion complex might have been established at the base of metazoan evolution.

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J.: PHYLIP

(Phylogeny

Distributed

by

University

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