Journal of Cell Science 113, 315-324 (2000) 315 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0961

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization

P. C. Baciu*, S. Saoncella, S. H. Lee, F. Denhez, D. Leuthardt and P. F. Goetinck‡ Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA *Present address: Allergan Inc., 2525 Dupont Drive, Irvine CA 92612, USA ‡Author for correspondence (e-mail: [email protected])

Accepted 4 November 1999; published on WWW 13 January 2000

SUMMARY

Syndecan-4 is a cell surface heparan sulfate proteoglycan syndecan family members. The interaction involves both which, in cooperation with integrins, transduces signals for the membrane proximal and variable central regions of the the assembly of focal adhesions and actin stress fibers in cytoplasmic domain. We have named this cDNA and cells plated on fibronectin. The regulation of these cellular encoded protein syndesmos. Syndesmos is ubiquitously events is proposed to occur, in part, through the interaction expressed and can be myristylated. Consistent with its of the cytoplasmic domains of these transmembrane myristylation and syndecan-4 association, syndesmos receptors with intracellular proteins. To identify potential colocalizes with syndecan-4 in the ventral plasma intracellular proteins that interact with the cytoplasmic membranes of cells plated on fibronectin. When domain of syndecan-4, we carried out a yeast two-hybrid overexpressed in NIH 3T3 cells, syndesmos enhances cell screen in which the cytoplasmic domain of syndecan-4 was spreading, actin stress fiber and focal contact formation in used as bait. As a result of this screen, we have identified a a serum-independent manner. novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three Key words: Syndecan-4, Signaling, Cytoskeleton, Focal adhesion

INTRODUCTION and morphology (Bernfield et al., 1992; Carey, 1997; Liu et al., 1998; Woods and Couchman, 1998; Zimmermann and David, Cell morphology, migration, growth, survival and 1999). Recent evidence indicates that the assembly of focal differentiation result from both adhesion dependent and growth adhesions and actin stress fibers in cells plated on fibronectin factor receptor mediated signaling events (Adams and Watt, depends on Rho-dependent cooperative signaling between 1993; Ashkenas et al., 1996; Gumbiner, 1996; Giancotti, 1997; syndecan-4 signals and integrins (Saoncella et al., 1999). Howe et al., 1998). Adhesion dependent signaling events link Syndecan-4 cDNA constructs can also generate the necessary the extracellular matrix with the intracellular cytoskeleton, signaling for the restoration of the assembly of focal adhesions both structurally and biochemically, through the formation of and actin stress fibers in cells that do so poorly (Echtermeyer focal adhesions. The latter are macromolecular complexes et al., 1999; Longley et al., 1999) and unglycanated syndecan- that are made up of transmembrane adhesion receptors, 4 core proteins are sufficient for the generation of these intracellular cytoplasmic structural proteins and signal signals. The syndecan-4 signals result, in part, from the direct transduction molecules (Burridge and Chrzanowska- association of the variable central region of its cytoplasmic Wodnicka, 1996). The structural transmembrane molecules of domain with PKCα and phosphatidyl inositol 4,5-biphosphate focal adhesions are integrins (Clark and Brugge, 1995) and (PIP2) (Oh et al., 1997a,b, 1998), and these interactions syndecan-4 (Woods and Couchman, 1994; Baciu and Goetinck, provide one potential mechanism for the regulation of the 1995). Cytoskeletal proteins that provide structural and/or assembly of these adhesion dependent macromolecular adapter functions include actin, alpha-actinin, paxillin, talin, complexes by syndecan-4. tensin and vinculin. Associated signaling proteins include The cytoplasmic domains of the four syndecan family tyrosine kinases (focal adhesion kinase, src, csk and fyn), the members have a high degree of sequence conservation which, serine-threonine kinase families of PKC and MAPK and based on homology, can be divided into conserved membrane members of the RAS family of small GTP-binding proteins proximal (C1), variable central (V) and conserved C-terminal (Clark and Brugge, 1995; Burridge and Chrzanowska- (C2) subdomains (Woods and Couchman, 1998; Zimmermann Wodnicka, 1996). and David, 1999). The existense of both conserved and variable The syndecan family of cell surface heparan sulfate subdomains in the cytoplasmic domains of the syndecans proteoglycans has been implicated in affecting cell adhesion suggest that interactions may occur with cytoplasmic proteins 316 P. C. Baciu and others that are either restricted to specific members or common to all In vitro transcription-translation assay members of the family. Such interactions would provide either In vitro transcription-translation was carried out using the TNT in common or unique functions for the cytoplasmic domains of the vitro transcription-translation kit from In Vitrogen Inc. (San Diego, syndecan family members. Indeed, in addition to the specific CA, USA). Reactions were carried out as described by the interactions of the V subdomain of syndecan-4 with PKCα and manufacturer. For initial analysis, syndesmos cDNA was subcloned PIP2 (Oh et al., 1997a,b, 1998) several cellular components into pcDNA 3.0 (In Vitrogen, San Diego, CA, USA) and the T7 which associate, either directly or indirectly, with the conserved promoter used for RNA synthesis. Control reactions were carried out subdomains of the cytoplasmic domain of syndecans have been in the presence and absence of vector without insert. Specificity of product was verified by western analysis of in vitro transcription- identified. These include: the PDZ-containing proteins syntenin translation products. For analysis of methionine start sites, individual (Grootjans et al., 1997) and CASK/LIN-2 (Cohen et al., 1998; PCR products were generated using 5′ primers containing T7 Hsueh et al., 1998), which interact with the cytoplasmic promoter sequence and nucleotides 1-30 of syndesmos. To examine domains of all syndecan family members through the highly individual initiation methionines, nucleotides corresponding to the conserved C-terminal EFYA sequence, and a Src-cortactin individual methionine residues were mutated to GTG. 3′ primers complex, which associates with the membrane proximal corresponded to nucleotides 1079-1099 of syndesmos. PCR products domain of syndecan-3 (Kinnunen et al., 1998). were generated using the Extend PCR Polymerase mix (Boehringer, To identify cytoplasmic proteins that may be involved in Mannheim). The resulting PCR products were purified and used in adhesion events unique to syndecan-4, we used the cytoplasmic the in vitro translation-transcription reaction. Control reations were domain of this proteoglycan as bait in a yeast two-hybrid done using no PCR product, or PCR product in which the T7 promoter was linked with a primer corresponding to nucleotides 3′ to the screen. As a result of this screen we identified a novel initiation codons (nucleotides 31-51). cytoplasmic protein, syndesmos. Syndesmos interacts directly and specifically with the cytoplasmic domain of syndecan-4 in Analysis of syndesmos expression in vitro assays. The interaction of syndesmos involves both the Western analysis C1 and the V subdomains of the cytoplasmic domain of Tissues from 8-day chicken embryos were solubilized in 4× SDS-PAGE syndecan-4. Syndesmos and syndecan-4 colocalize to adhesion sample buffer at a concentration of 20 mg wet tissue/50 µl of buffer, complexes in the ventral plasma membranes of cells plated on boiled and frozen. Before use they were thawed and homogenized using fibronectin. When overexpressed in NIH 3T3 cells, syndesmos a 21-gauge needle. 5 µl samples were loaded on a 10% SDS- results in increased cell spreading and actin cytoskeletal polyacrylamide gel, electrophoresed and blotted onto an Immobilon-P organization. membrane. Protein was visualized using affinity-purified anti- syndesmos polyclonal antibodies (see Immunocytochemistry, below) followed by anti-rabbit HRP-conjugated secondary antibodies (Biorad, MATERIALS AND METHODS Hercules, CA, USA) and ECL substrate (NEN Life Science Products, Boston, MA, USA). Identification of a syndecan-4 cytoplasmic domain Northern analysis interactor RNA was isolated from chicken embryonic tissues using the To identify and characterize proteins that might interact with the isothiocyanate method (Sambrook et al., 1989). 10 µg of total RNA cytoplasmic domain of syndecan-4 we used the yeast interactive trap from each tissue were separated on a 1.5% agarose formamide gel and system (Finley and Brent, 1994; Zervos et al., 1994), in which the blotted onto an Immobilon-N membrane (Millipore, Bedford, MA, cytoplasmic domain of syndecan-4 was used as bait. The yeast strain USA). The resulting blot was blocked for 4 hours at 62¡C in 6× SSC, EGY48 MATa trp1 ura3 LEU2:pLexAop6-LEU2 was used as host. 5× Denhardt’s solution containing 0.5% SDS and 100 µg/ml sheared The reporter plasmid pSH18-34 was used for the β-D-galactoside (X- salmon sperm DNA. 50 ng of purified insert from clone LMH4A was gal) assay. The bait plasmid was constructed by fusing the nucleotide randomly labeled with [γ-32P]dCTP using random primers and sequence that encodes amino acids 169-197 of the cytoplasmic domain Klenow fragment (Pharmacia, Uppsala, Sweden). Approximately 106 of chicken syndecan-4 in-frame with the nucleotide sequence of the dpm/ml in a final volume of 10 ml was used to probe the blotted LexA DNA binding domain and C-terminal dimerization domain in mRNA. After hybridization the membranes were washed at high pLexA202+PL2 (LexA-CS4C). The target library for the screen was a stringency according to the manufacturer’s instructions and exposed 4-day chick embryonic limb bud cDNA library made in plasmid pJG to X-Omat film (Kodak, Rochester, NY, USA). Molecular masses 4-5. Control baits for the screen were LexA-n-myc, LexA-R4CK230R, were estimated using RNA standards from Life Technologies LexA-Cyclin C (Zervos et al., 1994; Wang et al., 1994). Plasmids (Gaithersburg, MD, USA). pSH18-34 and LexA-CS4C were introduced in EGY48 and maintained under selection for the URA3 and HIS3 markers. This strain was then Myristylation assay transfected with the 4-day chick embryonic limb bud cDNA library. CEFs (70% confluent) were labeled for 24 hours with 1 mCi of [3H- An estimated 0.5×106 unique yeast clones were selected on Ura− His− 9,10(n)]myristic acid (Amersham) in 5 ml DMEM supplemented with Trp− glucose plates, scraped and pooled and stored at −70¡C. 10% dialyzed fetal bovine serum (FBS). Cells were lysed in RIPA Approximately, 5×106 clones from the amplified yeast stock were buffer and proteins were immunoprecipitated with a rabbit preimmune screened for potential interactors on Ura− His− Trp− Leu− galactose serum, a rabbit anti-syndesmos antibody or a mouse monoclonal anti- plates. Positive colonies were then streaked on Ura− His− Trp− X-gal src antibody (Upstate Biotechnology) as a positive control for galactose and on Ura− His− Trp− X-gal glucose plates. Colonies that myristylation. After immunoprecipitation, the proteins were separated grew on Ura− His− Trp− Leu− galactose and produced blue colonies on by 12% SDS-PAGE, transferred to immobilon-P and examined for Ura− His− Trp− X-gal glucose plates only were isolated and introduced radioactive proteins by autoradiography for 21 days (Mumby and into KC8 cells. Isolated cDNAs were characterized by restriction and Buss, 1990). sequence analysis and tested for specificity of the interaction on control baits mentioned above. Full-length cDNA was obtained by using the Bacterial expression partial cDNA clone, identified in the yeast two-hybrid test, as a probe cDNAs encoding complete or various mutant forms of syndecan-4 or to screen chicken cDNA libraries. syndesmos were cloned in-frame with either glutathione transferase Syndecan-4 interactions with syndesmos 317 using pGEX 5X-2 (Pharmacia, Uppsala, Sweden) or maltose binding (1994) and stained as described earlier (Baciu and Goetinck, 1995). protein using pMAL-C2 (NE Biolabs, Beverly, MA) expression vectors. The polyclonal antibodies used in these studies were directed against Ligated plasmids were initially transfected in Sure cells (Stratagene, La a unique amino acid sequence of the ectodomain of avian syndecan- Jolla, CA) and clones were verified by sequence analysis and analyzed 4 (CS-4-E) and against syndesmos. The generation and affinity for expression by SDS-PAGE. For protein expression cDNAs, purification of CS-4-E has been described (Baciu et al., 1994). transfected into Sure cells for propagation, were transfected into BL-21 Affinity-purified CS-4-E was used at a concentration of 20 µg/ml. cells (Novagen, Madison, WI, USA). Protein expression was Anti-syndesmos antibodies were generated in rabbits using a pMAL- accomplished by growing the transfected cells to an OD600 of 0.6 in syndesmos fusion protein as immunogen. Anti-syndesmos antibodies high growth medium followed by a 1-2 hour induction period using 1 were affinity-purified initially by passing the immune serum over a mM IPTG (Sigma, St Louis, MO, USA). At the end of the incubation pMAL-syndesmos-CL-4B affinity column. Bound antibodies were period cells were pelleted and resuspended in lysis buffer pH 7.5 (15 eluted with 0.1 M glycine, pH 2.5 and neutralized with 1/10 volume mM sodium phosphate, 30 mM NaCl, 0.25% Tween 20, 10 mM EDTA, 1 M Tris, pH 8.0. Antibodies that recognize the maltose binding 10mM EGTA, 2 µg/ml lysozyme and protease inhibitors cocktail protein were eliminated by chromatography on a maltose binding (CompleteTM, Boehringer Mannheim). Cells were lysed by sonication protein-CL-4B affinity column. The specificity of the reactivity of the for 1 minute on ice using a microtip probe at a setting of 5 at 50% resulting antibodies was verified by western blot analysis of GST- duration (Branson). Insoluble material was removed by centrifugation syndesmos and pMAL-syndecan fusion proteins. at 14,000 g for 10 minutes. Expressed proteins were either passed over FITC-phalloidin (Molecular Probes, Eugene, OR, USA) was used a maltose affinity column (NE Biolabs, Beverly, MA, USA) for pMAL for actin staining according to the manufacturer’s instructions. fusion proteins or over a glutathione affinity column for GST fusion Hemagglutinin (HA)-tagged syndesmos was visualized with an anti- proteins. After binding and in preparation for elution, the columns were HA mAb, which was a generous gift of Dr A. Molnar. The secondary extensively washed with lysis buffer, followed by a lysis buffer wash antibodies used are FITC-conjugated anti-rabbit and TRITC- that contained 0.5 M NaCl, and equilibrated in phosphate buffer. pMAL conjugated anti-mouse antibodies (Pierce, Rockford, IL, USA), used fusion proteins were eluted in lysis buffer containing 10 mM maltose. at a 1:100 dilution. After washing three times with PBS, coverslips GST-syndesmos fusion proteins remained bound to the GST affinity were mounted using Fluoromount G (Biomedia, Foster City, CA, matrix for protein binding assays. USA). Immunocytochemical analysis was carried out using a Leica In vitro binding assay Confocal microscope. Of the three emission channels used during 15 µl of a 50% slurry of GST-syndesmos bound to GST beads were multiple labeling experiments, excitation levels and gain were set to incubated with 10 µg of soluble pMAL-syndecan-4 in 0.5 ml of lysis eliminate bleed-through from one channel to the other as outlined by buffer that contained 1% bovine serum albumin (BSA). After the manufacturer. This was verified experimentally. Non-specific incubation for 30 minutes at room temperature with constant mixing, staining was determined by use of secondary antibody alone. the reaction mixtures were placed on ice and washed 3× with lysis buffer followed by 3× with lysis buffer containing 1 M NaCl. Protein Analysis of ventral plasma membranes bound to GST-syndesmos beads was solubilized in SDS-PAGE buffer, Xylosyltransferase-deficient CHO cells (CHO-745) (Esko et al., 1985; electrophoresed, transferred to an Immobilon-P membrane and LeBaron et al., 1988) stably transfected with a cDNA that encodes the analyzed for bound syndecan-4 core protein using the avian-specific core protein of chicken syndecan-4 (Echtermeyer et al., 1999) were syndecan-4 polyclonal antibody CS4E (Baciu et al., 1994). transiently transfected with a pcDNA-3 construct that harbors the cDNA for syndesmos with an in-frame HA-tag at its 3′ end. 2 days Cell culture after the syndesmos cDNA transfection, the cells were trypsinized and Primary chicken embryo fibroblasts (CEF) were obtained from dorsal plated on fibronectin for 2 hours. Ventral plasma membranes were skins of eight day old embryos as described in (Baciu et al., 1994). prepared by subjecting the cells to a hypotonic solution before fixation NIH 3T3 cells were obtained from ATCC (Catalog number CRL (Echtermeyer et al., 1999). The ventral plasma membranes were 1658). Both cell types were maintained at subconfluent densities in examined for syndecan-4 with the polyclonal antibody CS-4-E and Dulbecco’s Modified Eagles Medium (DMEM) supplemented with for HA-syndesmos with an anti-HA mAb. 10% FBS, streptomycin (250 µg/ml) and penicillin (250 i.u./ml) (Life Technologies, Gaithersburg, MD, USA) in a humidified atmosphere Quantitation of cell spreading at 37¡C. For serum deprivation experiments, cells were cultured for Images of actin-stained NIH 3T3 cells were captured using a Nikon 16-18 hours in medium containing only DMEM and antibiotics prior E800 microscope using epifluorescence and a Spot digital camera to trypsinization and replating. (Diagnostic Instruments, Sterling Heights, Michigan). Quantitation of For transfection experiments cells were plated in 6-well tissue cell surface area was measured using the ImagePro Plus image culture plates (Falcon, Bedford, MA, USA) at 2.5×105 cells per well analysis system (Mediacybernetics, Silver Spring, Maryland). Values and cultured overnight in complete growth medium. The cells were are means ± s.d. from the analysis of 90 cells obtained at random from then washed once in serum-free DMEM and transfected with 2 µg of each sample. circular plasmid DNA using 5 µl of Lipofectin (Life Technologies, Gaithersburg, MD, USA) in l ml of serum-free DMEM. After 5 hours the transfection medium was replaced with complete growth medium. RESULTS To obtain stable NIH 3T3 transformants cDNAs were cloned into the pcDNA 3.0 vector (In Vitrogen, Carlsbad, CA, USA). 48 hours after Identification of a novel protein that interacts with transfection, the cells were plated into three 150 mm dishes and the cytoplasmic domain of syndecan-4 µ selected for G418-resistant growth (700 g/ml) for 10 days. At this Sequence analysis of clones which specifically interacted with point individual colonies were cloned with cloning rings (Specialty the cytoplasmic domain of syndecan-4 in the yeast two-hybrid Media Inc., Lavalette, NJ, USA), expanded, and frozen stocks were prepared. Clones expressing the transgene products were screen identified a partial cDNA that encoded a novel polypeptide characterized by western analysis and immunocytochemistry. sequence. Using the partial cDNA to screen an LMH (Baciu et al., 1994) and a chicken embryonic cDNA library (Clontech, Palo Immunocytochemistry Alto, CA, USA), several full-length clones were isolated and Cells were fixed according to the procedure of Woods and Couchman sequenced. All clones shared a common large open reading frame 318 P. C. Baciu and others initiated at a cluster of four methionine residues. The encoded A polypeptide sequence of the C-terminal region in the full-length -12 ggcggcgagg cg -1 clones was identical to that of the initial partial cDNA isolated in 1 atggcggcca tgggagccat gggagtgatg gcggccgtag gagcgttgcc 50 the two-hybrid screen. The nucleotide and amino acid sequences 51 ggcgggcgcg gggtccctcc cgccgctgcc gacgctgggg gtgcccggcg 100 are shown in Fig. 1A,B, respectively. We have named this cDNA 101 tgcccgagct gaagccgctg acgcggtacg aggccatgcg gctgggcccg 150 and its encoded protein, syndesmos. In vitro transcription-translation analyses using a full-length 151 ggctggagcc actcgtgcca cgccatgctg tacgcgccca acccgggcat 200 clone subcloned into pcDNA 3.0 (LMH4A) identified a 40 kDa 201 gctgttcggc cgcatcccgc tgcgctacgc cgtgctgatg cagatgcgct 250 band as the principal translation product (Fig. 2A). Deletion of 251 ttgacggcct actgggcttc cccggggggt tcgtggaccg ccggtactgg 300 the 5′ nucleotides from −12 to +30 blocked all protein 301 tccctggagg acggtctgaa ccgggtgctg ggcctgggcc tgggctgcgt 350 synthesis, implicating the cluster of methionine residues encoded by nucleotides +1 through +30 (Fig. 1A) as the 351 gcgcctgacg gaggccgact acctgtgctc gcacctgacg gacgggccgc 400 initiation site. The 40 kDa polypeptide obtained in in vitro 401 atcgcgtggt ggctcactta tacgcccggc agctgaccct ggaggagctg 450 transcription-translation experiments was recognized by an 451 cacaccatcg agatcagcgc ggtgcactcc cgagaccacg ggctggaggt 500 anti-syndesmos polyclonal antibody and corresponds in molecular mass to a band identified in tissue extracts from 501 gatgggcatg gtccgtgtcc ccctctacac ccagaaagat cgcatgggtg 550 chicken embryonic tissues (Fig. 2B). 551 ggctgccaaa cttcctggcc aactccttcg ttggaactgc caaattccag 600 To determine the initiation methionine in the cluster of four 601 ctgctctttg ctctgaagat cttgaacatg gtgccggagg agaagctggc 650 potential initiation sites encoded by nucleotides +1 through 651 cgaggcggtg gctgccacgc agaagccgaa gaagccggcg atcgaccacg 700 +30, we coupled site-directed mutational and in vitro transcription-translation analyses. Mutations in which only one 701 cggctgtggc agcagctaag caggcgaacg agctggcggc ggccgccaga 750 of the four potential initiation codons is left intact or in which 751 gcaggcaatg aatacgcaga tagcggagag aaccaggcag ctgcgcacgc 800 individual initiation codons are abolished, indicate that 801 tgcggccgag ctggcagagc agcaggcggc cgggctggag agccaggctg 850 translation can be initiated in vitro at any of the four 851 tgctggagca tctggcggcc gtgccggggg ctgaggccgt ggtggcggag 900 methionine residues (data not shown). The methionine residues encoded by the second and third potential initiation codons are 901 ctgcacgcgc agcccggggc agacgctgtg ctggagcagc cggtggctga 950 followed by a glycine residue, which provides the structural 951 ggccatggag tgatgccccc gtgtttgtaa ttgattaaaa gtgggtgagg 1000 requirements for myristylation of syndesmos (Towler et al., 1001 agactagaga ctttcttcta acttcccaac cagttgctgg ctgcgagatt 1050 1987). The determination that syndesmos can be myristylated (see below) indicates that intiation in vivo can occur at 1051 ccgctgtgta gccaggaggg tttggaattg tctgaagcag gggaaagcta 1100 either the second or third methionine residue. However, 1101 tgtattttta tggccattaa actctagcga gcttcccaga tcaatctgcg 1150 the second site shows the greatest homology to the 1151 cagccccctg cagagagttc atgtgcttat gtgaaggtgc tgaagcctta 1200

GCCGCC(A/G)CCAUGG Kozak consensus sequence (Kozak, 1201 gaattacgct ttgatttcag ggacacactg ctgctttcag gtcccggttt 1250 1987) relative to the third initiation site, suggesting that in vivo initiation at the second methionine is likely to be favored. 1251 catttttaag gaacaaaact tactttgaga tgccctacgt tgactcggat 1300 A comparison of the syndesmos sequence to those in the 1301 gcagctgtgt tttgcagtgg gcacggctga ggaataaggc ttctggtaaa 1350 GenBank database identifies syndesmos as a novel protein 1351 gcgcctttgc cgccgtgggc agatgggctc tgctctgctg gcagcagtga 1400 and the predicted polypeptide identifies no large regions of 1401 ggccgggctc cctttggaat gccggtgccc ggggtgacgt gcactgaatt 1450 homology with polypeptide sequences present in that database. However, a motif search revealed several subdomains in the 1451 ccctctgggg cccggggaca aactcatgtg ggtttctatc tggagatctg 1500 protein that show homology to known protein motifs. As 1501 tgggagcagg aaagtggaat aaatggcttt gctg 1534 indicated above, following the second and third methionine residues are glycine residues that conform to the myristylation B M A A M G A M G V M A A V G A L P A G A 20 motif (Towler et al., 1987). The PPLP sequence (amino acids G S L P P L P T L G V P G V P E L K P L 40 24-27) in the N-terminal domain satisfies the requirement for an T R Y E A M R L G P G W S H S C H A M L 60 Y A P N P G M L F G R I P L R Y A V L M 80 SH3 domain binding site motif (Feng et al., 1994). Within the Q M R F D G L L G F P G G F V D R R Y W 100 S L E D G L N R V L G L G L G C V R L T 120 central portion of syndesmos, amino acids 190-199 show limited E A D Y L C S H L T D G P H R V V A H L 140 homology with the activation loop of MEK-1 (Hardie and Y A R Q L T L E E L H T E I S A V H S R 160 D H G L E V M G M V R V P L Y T Q K D R 180 Hanks, 1995). Additional homology is seen for relative positions M G G L P N F L A N S F V G T A K F Q L 200 of amino acids DHG (residues 162-164) and PE (residues 212- L F A L K I L N M V P E E K L A E A V A 220 A T Q K P K K P A I H A A V A A A K Q A 240 213) with DFG in subdomain VII and PE in subdomain VIII of N E L A A A A R A G N E Y A D S G E N Q 260 MEK-1. No additional homology to either MEK or other A A A H A A A E L A E Q Q A A G L E S Q 280 A V L E H L A A V P G A E A V V A E L H 300 eukaryotic kinases was found. Syndesmos also contains three A Q P G A D A V L E Q P V A E A M E * 318 threonine residues (residues 177, 196, 223) that fit the consensus for phosphorylation by PKC (Kishimoto et al., 1985). Fig. 1. cDNA and amino acid sequence of syndesmos. (A) cDNA nucleotide sequence of syndesmos. The GenBank accession number Syndesmos is widely expressed in embryonic is AF095446. (B) Deduced amino acid sequence of syndesmos. tissues Western blot analysis with anti-syndesmos polyclonal tibia and skin (Fig. 2B). Northern analysis indicates the antibodies reveals a single 40 kDa polypeptide in 8-day chick presence of three mRNAs of 2.1, 1.7 and 1.5 kb (Fig. 3). The embryonic brain, eyes, gizzard, heart, intestine, kidney, liver, smaller transcript corresponds to alternate poly(A) addition Syndecan-4 interactions with syndesmos 319

Fig. 2. In vitro and in vivo translation products of syndesmos. (A) In vitro transcription-translation reaction identifies a 40 kDa protein as the principal translation product encoded by syndesmos cDNA. Full-length cDNA (LMH4A) and deletion mutant ∆ (LMH4A M), in which nucleotides kDa −12 through +30 were deleted, were kDa cloned into pcDNA 3.0 and the resulting plasmid was used in in vitro transcription-translation reactions. Controls were no plasmid DNA (Control), or pcDNA 3.0 without insert (Vector). (B) Western blot analysis of syndesmos expression in day-8 embryonic avian tissues. The indicated tissues were dissected and homogenized in reducing SDS-PAGE sample buffer. 2 mg (wet mass) from each homogenized tissue were electrophoresed, transferred to an Immobilon-P membrane and probed with affinity-purified anti-syndesmos polyclonal antibodies. A single 40 kDa band was observed in every tissue examined. sites as determined by sequence analysis. All three species of (CS4E), anti-syndesmos polyclonal antibodies or pre-immune mRNA are observed in aorta, heart, brain, gizzard, kidney, IgG. Equivalent amounts of total protein lysates from CEFs liver, proventriculus and skin from 10-day-old embryonic were immunoprecipitated using each of the indicated tissues and in intestine and muscle from 15-day-old embryos antibodies. Immunoprecipitates were separated by 10% (Fig. 3). The tissues in which syndesmos is expressed also reducing SDS-PAGE and western blotted using affinity- express syndecan-4 (Baciu et al., 1994), although the levels of purified anti-syndesmos polyclonal antibodies. The presence of syndecan-4 mRNA may be developmentally more variable. a 40 kDa reactive band in the whole cell lysate, anti-syndesmos and anti-syndecan-4 immunoprecipitations, and not from the Syndesmos can be myristylated pre-immune IgG, indicates an in vivo association of syndesmos The second and the third methionine residues in the cluster of with syndecan-4 (Fig. 5). the four potential initiation sites are followed by glycine residues. If translation were initiated at these methionine Syndesmos interacts directly with the cytoplasmic residues, the glycine residues that follow them could be domain of syndecan-4 myristylated. Direct tests of the possible myristylation of Whereas the co-immunoprecipitations demonstrate in vivo syndesmos were carried out in CEF cultures. Subconfluent associations, they do not demonstrate a direct association of CEFs were labeled in vivo with [3H]myristic acid and cell syndesmos with the cytoplasmic domain of syndecan-4, nor do extract prepared and immunoprecipitated with anti-syndesmos they indicate a specificity of syndesmos for syndecan-4. To antibodies. The resulting immunoprecipitates were address both the direct interaction of syndesmos with syndecan- electrophoresed on SDS-PAGE, transferred to Immobilon-P and examined for 3H- labeled syndesmos by autoradiography. Src, a known myristylated protein, was immunoprecipitated from the same extract with anti-src antibodies and analyzed in the same manner as syndesmos as a positive control. After autoradiography a single band with the predicted molecular mass of kb 40 kDa for syndesmos was found in the syndesmos immunoprecipitate. A single band was also identified in the src immunoprecipitate with the predicted molecular mass of 60 kDa of this protein (Fig. 4). Syndesmos interacts with syndecan-4 in vivo Fig. 3. Northern blot analysis of syndesmos mRNA expression. Total mRNA (10 mg) from To examine the in vivo association of the indicated tissues was separated on a 1% agarose formaldehyde gel and blotted onto an syndesmos with syndecan-4, an Immobilon-N membrane. The resulting blot was probed with randomly primed syndesmos immunoprecipitation assay was carried out cDNA insert as a template. Three mRNA species of syndesmos are seen in all tissues using affinity-purified anti-syndecan-4 examined. 320 P. C. Baciu and others

kDa

kDa

Fig. 4. Myristylation of syndesmos. Extracts from cells incubated with 3H-myristate were immunoprecipitated with pre-immune serum, anti-syndesmos or anti-src antibodies. Immobilon-P membranes, to which the samples had been transferred after electrophoresis, were Fig. 5. Syndesmos interacts with syndecan-4 in vivo. analyzed by autoradiography. Exposure was for 21 days. Radioactive Immunoprecipitations were carried out on CEF extracts using bands of 40 kDa and 60 kDa were detected in the anti-syndesmos affinity-purified anti-syndesmos, anti-syndecan-4 (CS-4-E) and anti-Src immunoprecipitates, respectively. antibodies or affinity-purified pre-immune IgG. The western blot of the cell extract reveals a single 40 kDa band with anti-syndesmos 4 and its specificity, an in vitro binding assay was performed in antibodies (lane 1). Both the immunoprecipitates with anti- which bacterially expressed GST-syndesmos fusion protein was syndesmos antibodies (lane 2) and with anti-syndecan-4 antibodies (lane 3) reveal a 40 kDa band when challenged with anti-syndesmos immobilized on glutathionine-agarose beads and assayed for its antibodies. The immunoprecipitates with pre-immune IgG were ability to bind soluble purified pMAL-syndecan-4 fusion negative when challenged with anti-syndesmos antibodies (lane 4). proteins. Bound syndecan-4 core protein was visualized by elution with SDS-PAGE sample buffer followed by western blotting using the avian syndecan-4 ectodomain antibody. Full- syndecan-4 with GST alone (Fig. 6B, lane 1). These analyses length syndecan-4 core protein (Fig. 6A, S-4), but not a indicate that the interaction of syndecan-4 core protein with cytoplasmic deletion mutant (Fig. 6A, Tailless), binds syndesmos results from a direct interaction with the syndesmos. That the interaction is between syndesmos and not cytoplasmic domain. GST is evident from the extremely low binding of full-length We next examined the specificity of the interaction for the

Fig. 6. In vitro assays of syndecan-4 core protein interactions with syndesmos. Soluble bacterially expressed pMAL-syndecan-4 fusion proteins were tested for interaction with immobilized GST-syndesmos. After SDS- PAGE, bound pMAL-syndecan-4 was visualized by western blot analysis with anti-syndecan-4 antibodies (CS4E). (A) Syndesmos binds specifically to the cytoplasmic domain of syndecan-4. Full-length syndecan-4 core protein (S-4) but not syndecan-4 lacking the cytoplasmic domain (Tailless) binds syndesmos. No interactions could be detected between syndesmos and chimeric core proteins in which the cytoplasmic domain of either syndecan-1 (S-1), syndecan-2 (S-2) or syndecan-3 (S-3) was fused in-frame with the extracellular and transmembrane domains of syndecan-4. (B) Analysis of amino acid deletions on the interactions between the syndecan-4 cytoplasmic domain and syndesmos. Lane 1, no binding is observed between full-length syndecan-4 and GST alone; lanes 2-9, interaction of syndecan-4 core protein constructs with syndesmos. Binding of full-length syndecan-4 core protein (lane 2). Diminished binding is observed with cytoplasmic domain deletions ∆194-197, ∆189-197, ∆183-197, ∆180-197 (lanes 3-6). Complete deletion (∆172-197) of the cytoplasmic domain (lane 7) or internal deletions of the central variable domain (∆180- 193, lane 8) or the membrane proximal domain (∆172-179, lane 9) abolish binding. Syndecan-4 interactions with syndesmos 321

Table 1. Summary of binding between GST-syndesmos of other family members, suggest that both the membrane and pMAL-syndecan-4 with various cytoplasmic domain proximal and central variable region of the cytoplasmic domain deletions are involved in the interaction with syndesmos. Relative Cytoplasmic domain sequence Name binding Syndesmos co-localizes with syndecan-4 in ventral plasma membrane adhesion plaques RMKKKDEGSYDLGKKPIYKKAPTNEFYA 170-197 +++ Syndecan-4 localizes to focal adhesions (Woods and RMKKKDEGSYDLGKKPIYKKAPTN ∆ 194-197 ++ Couchman, 1994; Baciu and Goetinck, 1995) and we have shown in Fig. 5 that syndecan-4 and syndesmos can be co- ∆ RMKKKDEGSYDLGKKPIYK 189-197 ++ immunoprecipitated. We therefore wished to test if syndesmos RMKKKDEGSYDLG ∆ 183-197 ++ and syndecan-4 colocalize to focal adhesions. To examine this possibility, we used xylosyltransferase-deficient CHO cells RMKKKDEGSY ∆ 180-197 ++ (CHO-745) (Esko et al., 1985; LeBaron et al., 1988) stably transfected with a cDNA that encodes the core protein of RM ∆ 172-197 − chicken syndecan-4. Syndecan-4 core protein colocalizes with RMKKKDEGSY EFYA ∆ 180-193 − vinculin in the focal adhesions of the ventral plasma membranes of such transfected mutant cells (Echtermeyer et RM DLGKKPIYKKAPTNEFYA ∆ 172-179 − al., 1999). Upon transiently transfecting such syndecan-4 core protein expressing CHO-745 cells with a pcDNA-3 construct cytoplasmic domain of syndecan-4. For this analysis we that harbors the cDNA for syndesmos with an in-frame constructed chimeric proteins in which the cytoplasmic domain hemagglutinin-tag at its 3′ end we could demonstrate that of syndecan-4 was replaced with the cytoplasmic domains of syndecan-4 and syndesmos colocalize in the adhesion either syndecan-1, -2 or -3 in the full-length syndecan-4 pMAL complexes of such cells plated on fibronectin (Fig. 7). construct. As shown in Fig. 6A, syndesmos interacts specifically with the cytoplasmic domain of syndecan-4 (S-4) Overexpression of syndesmos in NIH 3T3 cells and not with any of the chimeric core proteins in which the results in increased cell spreading and accelerated cytoplasmic domain of syndecan-4 had been replaced with that cytoskeletal organization of syndecan-1 (S-1), syndecan-2 (S-2) or syndecan-3 (S-3). NIH 3T3 cells were transfected with either the pcDNA 3.0 These results indicate that the interaction of syndesmos is expression vector, as a control, or with the pcDNA 3.0 vector specific for the cytoplasmic domain of syndecan-4 and they that harbors the chicken syndesmos cDNA. Expression of suggest that amino acids unique to the cytoplasmic domain of syndesmos was verified by western blot analysis (Fig. 8A). The syndecan-4 mediate that interaction. control transfectant clone (Fig. 8A, lane 1), which shows no expression of chicken syndesmos, was compared with three Syndesmos interacts with both the variable central transfected clones that express different levels of syndesmos and conserved membrane proximal amino acids of (lanes 2-4). When serum-deprived cells were allowed to adhere the cytoplasmic domain of syndecan-4 and spread on fibronectin-coated slides in the absence of To identify amino acids in the cytoplasmic domain of serum, the syndesmos expressing cells showed a clear increase syndecan-4 that mediate the interaction with syndesmos, we in cell surface area and in actin stress fiber formation (Fig. 8B). generated a full-length pMAL-syndecan-4 and various The control and the three syndesmos expressing clones shown cytoplasmic deletion mutants and tested them for their ability in Fig. 8A, lanes 1, 2, 3 and 4 had an average surface area of to interact with immobilized GST-syndesmos. No significant 358±172, 635±265, 581±205 and 589±236 µm2 (means ± s.d.), binding between full-length syndecan-4 and GST could be respectively. This represents an increase of 77, 62 and 65 % in detected (Fig. 6B, lane 1) relative to the binding of full-length the total cell surface area of the syndesmos expressing clones, syndecan-4 with GST-syndesmos (Fig. 6B, lane 2). Removal respectively (Fig. 8B, panels 2, 3, 4) relative to the control of amino acids 197-180 from the carboxy terminus through the clone (Fig. 8B, panel 1). Furthermore, an enhancement in central domain of the cytoplasmic domain diminished, but did filopodia formation was observed in the two clones that not abolish, binding of syndecan-4 to syndesmos (Fig. 6B, expressed the higher levels of syndesmos (Fig. 8B, panels 2 lanes 3-6; Table 1). The removal of the final membrane and 3). The effect of syndesmos expression on cell spreading proximal amino acids abolished almost all binding (Fig. 6B, and on actin stress fiber formation was greatly diminished lane 7). These results suggest that the conserved membrane when cells were plated in the presence of serum or if they were proximal region is essential for binding and that the variable not serum-starved before trypsinization. This observation central and conserved C-terminal regions affect the degree of suggests an adhesion-dependent regulation of cell spreading interaction. To test the involvement of both the conserved and actin stress fiber formation by syndesmos. membrane proximal region and the variable central regions of the cytoplasmic domain in the binding to syndesmos, we generated internal deletions for these subdomains. In both DISCUSSION instances, the internal deletion mutant constructs had no specific interaction with GST-syndesmos in the binding assays Cell surface heparan sulfate proteoglycans of the syndecan (Fig. 6B, lanes 8,9). The data from the deletion constructs, family have been implicated in the regulation of growth factor coupled with the observation that syndesmos interacts only signaling, cell migration, attachment and morphology with the cytoplasmic domain of syndecan-4 and not with that (Bernfield et al., 1992; Carey et al., 1994; Woods and 322 P. C. Baciu and others

Fig. 7. Syndesmos colocalizes with syndecan-4 in ventral plasma membranes. Xylosyltransferase mutant CHO cells that express chicken syndecan-4 core protein were transiently transfected with a pcDNA3.0 vector that harbors the coding sequence for syndesmos. Ventral plasma membranes were prepared from these cells 2 hours after plating on fibronectin and stained for syndecan-4 with the rabbit polyclonal antiserum CS-4-E (A) and HA-syndesmos with a mouse anti-HA mAb (B). The merged picture (C) clearly indicates the colocalization for syndecan-4 core protein and syndesmos.

Couchman, 1998; Zimmermann and David, 1999). In these 1997). The transmembrane and cytoplasmic domains, on the functions the syndecans are viewed as coreceptors for primary other hand, are highly conserved among the four members. The receptors such as growth factor receptor kinases or integrins. cytoplasmic domains of the syndecans are short (28-34 amino Cell surface HSPGs and heparin binding sites of ECM acids) and have no intrinsic catalytic activity that could explain molecules play an important role in the assembly of focal their signaling role when cells interact with the ECM through adhesions and actin stress fibers during cell matrix interactions this transmembrane receptor. This suggests that signaling in vitro (Woods et al., 1986, 1993; Bloom et al., 1999). pathways downstream of syndecan function may involve Recently, we have shown that syndecan-4 is an HSPG that interactions with kinases or other signaling molecules. signals cooperatively with integrins in the assembly of focal In the present study we searched for syndecan-4 cytoplasmic adhesions and actin stress fibers in fibroblasts plated on domain interactors by using this domain as bait in the yeast fibronectin (Saoncella et al., 1999). This function of syndecan- two-hybrid system and we report on the finding of a novel 4 is consistent with the observations that this proteoglycan is protein, syndesmos. The interaction of syndesmos with the a transmembrane receptor found in focal adhesions (Woods cytoplasmic domain of syndecan-4 is direct and specific and and Couchman, 1994; Baciu and Goetinck, 1995). involves both the membrane proximal (C1) and variable central Syndecan-4 is one of four syndecan family members that (V) regions of this domain. Although the conserved membrane have heparan sulfate as the major GAG chain attached to very proximal domain of the cytoplasmic domain of syndecan-4 divergent extracellular domains (Bernfield et al., 1992; Carey, alone is able to bind syndesmos, the central variable domain is

Fig. 8. Overexpression of syndesmos in NIH 3T3 cells influences cell spreading and cytoskeletal architecture. NIH 3T3 cells were transfected with either pcDNA3.0 vector alone or with pcDNA3.0 containing syndesmos cDNA. (A) Western blot analysis of a vector control (lane 1) and three syndesmos expressing clones (lanes 2-4). (B) Vector control (panel 1) and syndesmos overexpressing cells (panels 2-4) were serum- starved overnight, plated on fibronectin-coated glass coverslips and allowed to adhere for 2 hours before fixation and staining for actin stress fibers with FITC-labeled phalloidin. Cells overexpressing syndesmos (panels 2-4) show a significant increase in cell spreading and actin stress fiber formation relative to vector control cells (panel 1). Syndecan-4 interactions with syndesmos 323 also involved and may confer specificty to the interaction. The attachment is via the myristylation-palmitylation and the involvement of the central domain as a mediator of this second through an interaction of basic amino acids close to the specificity is indicated from three observations. First, sites of myristylation-palmitylation (Resh, 1994). The syndesmos does not bind to the cytoplasmic domains of either membrane anchoring effect of the basic amino acids results syndecan-1, 2 or 3, even though these cytoplasmic domains from an interaction with the acidic membrane phospholipids. contain membrane proximal (C1) and C-terminal (C2) Syndesmos does not have the basic amino acids immediately subdomains that are virtually identical to the corresponding following either of the two potentially myristylated glycine subdomains of the cytoplasmic domain of syndecan-4; second, residues for interaction with membrane phospholipids. the membrane proximal domain alone shows diminished However, since syndesmos interacts with the cytoplasmic binding relative to the intact cytoplasmic domain; and finally, domain of syndecan-4 it is possible that the second point of an internal deletion of the central variable domain abolishes membrane attachment for syndesmos is syndecan-4. binding. The interactions of syndesmos with the cytoplasmic domain The observation that the specific interaction of syndesmos of syndecan-4 and the effect of syndesmos on cell spreading with the cytoplasmic domain of syndecan-4 involves both the and actin reorganization are consistent with the demonstration membrane proximal portion that is shared by all syndecan that syndecan-4 acts cooperatively with integrins in the family members and the central variable region that is unique assembly of focal adhesions and actin stress fibers (Saoncella for this family member, suggests that analogous interactions et al., 1999). The effect of overexpressing syndesmos on cell may occur between the cytoplasmic domains of syndecan-1, - spreading was more pronounced during the initial stages of cell 2 and -3 and proteins that may make up a family of syndesmos- adhesion and spreading than in cultures which had been plated related proteins. Interaction with individual syndesmos family for 24 hours. Plating cells in the absence of serum further members would occur through their ability to recognise the enhanced the phenotype. These observations suggest that the proximal amino acids while adjacent amino acids in the central regulation of cell morphology by syndesmos is independent of variable domain would confer specificity of binding. A serum and requires an adhesion dependent signal. This signal possible example of such binding may be found in the may be reflected in the formation of filipodia, which were interactions of the cytoplasmic domain of syndecan-3 with a frequently observed in cells that overexpressed syndesmos. protein complex that contains Src family kinases and the The mechanism by which syndesmos affects cell spreading is substrate cortactin as well as a 30 kDa protein (Kinnunen et not known; however, the ability of syndesmos to influence actin al., 1998). These interactions could be competed with a stress fiber formation suggests a function parallel to or synthetic peptide with the sequence of the conserved downstream from serum dependent pathways. The localization membrane proximal region of the cytoplasmic domain. It was of syndesmos to focal contacts in ventral plasma membranes not reported if the binding of the protein complex was specific suggests that syndesmos may function as an adapter molecule for the cytoplasmic domain of syndecan-3 or if it could also between syndecan-4 and the cytoskeleton. bind the cytoplasmic domains of syndecan-1, -2 and -4. If the interaction were specific for the cytoplasmic domain of The authors thank Antonis Zervos for his valuable help with the syndecan-3, one might expect an involvement of the central yeast two-hybrid screen, Cliff Tabin and Debbie Goff for the 4-day variable region in a manner analogous to the one reported here chick embryonic limb bud cDNA library made in plasmid pJG 4-5, for syndesmos and the cytoplasmic domain of syndecan-4. A Janice Buss for advice on the myristylation assays and Yimin Ge for confocal microscopy. We also thank John Gallagher and Allison general interaction of proteins with all syndecan cytoplasmic Fleetwood for providing us with a cDNA for syndecan-2 and Robert domains has been reported between the highly conserved C- Kosher for a syndecan-3 cDNA. This work was supported by grants terminal amino acids, EFYA, and the PDZ-domain containing HD- 22016 and HD-37490 of the National Institutes of Health and proteins, syntenin (Grootjans et al., 1997) and CASK/LIN-2 from the Cutaneous Biology Research Center through the (Cohen et al., 1998; Hsueh et al., 1998). Thus, both general MGH/Shiseido Company Ltd agreement. and specific interactions between the cytoplasmic domains of the syndecan family members and various cytoplasmic proteins can be envisaged, depending on whether the binding involves REFERENCES the highly conserved, the variable, or a combination of conserved and variable regions. How each of these proteins Adams, J. C. and Watt, F. M. (1993). Regulation of development and interacts with a short cytoplasmic domain is not clear. Multiple differentiation by the extracellular matrix. Development 117, 1183-1198. interactions may be possible as a result of the oligomerization Ashkenas, J., Muschler, J. and Bissell, M. J. (1996). 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