(2004) 23, 8688–8694 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc SHORT REPORTS The human Hugl-1 substitutes for Drosophila Lethal giant larvae tumour suppressor function in vivo

Daniela Grifoni*,1,4, Flavio Garoia1,4, Christoph C Schimanski2,Go¨ sta Schmitz2, Elisa Laurenti1, Peter R Galle2, Annalisa Pession3, Sandro Cavicchi1 and Dennis Strand2

1Alma Mater Studiorum, Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, 40126 Bologna, Italy; 2First Department of Internal Medicine, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55131 Mainz, Germany; 3Alma Mater Studiorum, Dipartimento di Oncologia, Sezione di Patologia, Ospedale Bellaria, Via Altura 3, 40139 Bologna, Italy

Drosophila lethal giant larvae (lgl), discs large (dlg) 2002); indeed, during the growth of epithelial tumours, and scribble () are tumour suppressor acting cells at the invasive front undergo an epithelial-to- in a common pathway, whose loss of function leads to mesenchymal transition, exhibiting a less polarized disruption of and tissue architecture, structure, becoming less adhesive and prone to migra- uncontrolled proliferation and growth of neoplastic tion (Thiery, 2002). Understanding the regulation of cell lesions. Mammalian homologues of these genes are highly polarity and how it is linked to is conserved and evidence is emerging concerning their role therefore fundamental to biology. In Drosophila, in cell proliferation control and tumorigenesis in humans. Lethal giant larvae (Lgl), Discs large (Dlg) and Scribble Here we investigate the functional conservation between (Scrib) are evolutionarily conserved molecules that Drosophila lethal giant larvae and its human homologue function in a common genetic pathway linking cell Hugl-1(Llgl1). We first showthat Hugl-1 is lost in human polarity regulation to cell proliferation control in the solid malignancies, supporting its role as a tumour epithelial and nervous tissues (Bilder et al., 2000). These suppressor in humans. Hugl-1 expression in homozygous establish the basolateral identity of the cell, by lgl Drosophila mutants is able to rescue larval lethality; recruiting several other proteins and transcripts to these imaginal tissues do not showany neoplastic features, with sites, whereas their mislocalization leads to disruption Dlg and Scrib exhibiting the correct localization; animals of adherens and apical junctions (Woods et al., 1996, undergo a complete metamorphosis and hatch as viable 1997; Bilder and Perrimon, 2000). lgl, dlg and scrib are adults. These data demonstrate that Hugl-1 can act as a tumour suppressor genes in Drosophila, since their loss tumour suppressor in Drosophila and thus is the functional of function causes neoplastic overgrowth of larval brain homologue of lgl. Furthermore, our data suggest that the and imaginal epithelia. In lgl mutant imaginal tissues, genetic pathway including the tumour suppressors lgl, different cell populations show a gradual misreading of dlg and scrib may be conserved in mammals, since human positional cues, forming amorphous masses that closely scrib and mammalian dlg can also rescue their respective resemble mammalian in situ carcinomas (Agrawal et al., Drosophila mutations. Our results highlight the usefulness 1995; Wodarz, 2000). When these tumorous tissues of fruit fly as a model system for investigating in vivo the are transplanted into wild-type recipients, they are mechanisms linking loss of cell polarity and cell prolifera- able to grow and migrate to distant sites killing the tion control in human . host, thus behaving like mammalian metastatic tumours Oncogene (2004) 23, 8688–8694. doi:10.1038/sj.onc.1208023 (Woodhouse et al., 1998). Furthermore, lgl-induced Published online 27 September 2004 metastases overexpress type IV collagenase and NDP kinase, thus showing some of the biochemical features Keywords: Hugl-1; Lethal giant larvae; tumour suppres- observed in human secondary cancers (Timmons et al., sor; cell polarity; epithelial cancers; Drosophila 1993; Woodhouse et al., 1994). Two Lgl-related proteins have been found in mammalian genomes, which show the characteristic WD-40 repeats (Tomotsune et al., Cancer arises from cells that undergo multiple muta- 1993; Strand et al., 1994a, b). Human Lgl1 (Hugl-1/ tional events affecting cellular pathways involving cell Llgl1) maps to 17p11.2, within the interstitial deletion proliferation, apoptosis, differentiation and cell–cell associated to the Smith–Magenis syndrome (Strand interactions (Hanahan and Weinberg, 2000). Loss of et al., 1995), close to breakpoints of deletions found in apical–basal polarity in epithelial cells is one of the primitive neuroectodermal tumours (PNETs), pediatric hallmarks of aggressive and invasive cancers (Thiery, lesions of the CNS. The protein shows a global similarity of 62.5% with Lgl if conservative amino-acid changes are taken into account. spreads *Correspondence: D Grifoni; E-mail: [email protected] 4These authors contributed equally to this work all along the molecule and is significantly lower in the Received 21 January 2004; revised 23 June 2004; accepted 7 July 2004; C-terminal domain, which has previously been shown to published online 27 September 2004 be dispensable for Lgl tumour suppressor function Conservation of Lgl function D Grifoni et al 8689 (Jacob et al., 1987). Hugl-1 is mainly membrane- absent in oesophageal tumour samples, gastric carcino- associated, forms homo-oligomers and is a component mas and a number of tumour cell lines (Makino et al., of the multimeric cytoskeletal network that contains 1997; Ishidate et al., 2000; Liu et al., 2002); a low level nonmuscle type II heavy chain and a serine of Dlg1 expression has been associated to gastric kinase (Strand et al., 1995), similar to Lgl (Strand et al., cancers, with an additional decrease of Dlg4 expression 1994a, b; Kalmes et al., 1996). When expressed from correlating to the most aggressive types (Boussioutas baculovirus in insect cells, Hugl-1 is also transpho- et al., 2003). We have begun to collect data concerning sphorylated by an endogenous kinase (Strand et al., Hugl-1 expression in tumour samples. Here we per- 1995). Recently, a complex of PAR-6 and atypical formed a screen of 60 breast, , prostate, ovarian protein kinase C was shown to bind and phosphorylate cancers and melanomas to investigate the transcrip- mammalian Lgl (Plant et al., 2003; Yamanaka et al., tional profile of Hugl-1 in human solid malignancies. 2003). Both mammalian and Drosophila proteins are Interestingly, as shown in Figure 1, the transcript was able to rescue yeast strains double deficient for yeast reduced or absent in a high proportion of breast cancers Lgl homologues, Sop1 and Sop2 (Larsson et al., 1998; (13/17; 76%), lung cancers (12/19; 63%), prostate Kim et al., 2003). While the role of Lgl protein in cell cancers (8/15; 53%), ovarian cancers (2/4; 50%) and proliferation control has been assessed in Drosophila, melanomas (2/5; 40%), supporting a tumour suppressor the question must still be addressed for the human function for Hugl-1 in humans. counterpart. Evidence has emerged concerning a role In the light of this evidence, we were particularly for Lgl, Dlg and Scrib in tumour suppression also interested in studying the conservation of lgl tumour in mammals. Overexpression of different variants of suppressor function between fruit fly and humans. Hugl- human Dlg leads to a arrest in many types of 1, besides having a high structural conservation with Lgl cell lines (Hanada et al., 2000; Ishidate et al., 2000) protein (Figure 2a), shows similar biochemical proper- and preliminary studies indicate a similar behaviour for ties. We previously reported that the subcellular hScrib (Dow et al., 2003). Using an ecdysone-inducible localization of Hugl-1 is also conserved in human cells overexpression of Hugl-1 in human epithelial (Strand et al., 1995). Another important structural cells (HEK-293) we can show that Hugl-1 causes a cell feature of Lgl and Hugl-1 is that both proteins form cycle arrest and stabilizes the culture in a confluent homo-oligomers. Lgl homo-oligomerization domains monolayer, which can be maintained at least for 5 weeks have been identified and biochemically characterized without adverse effects (CC Schimanski and D Strand, (Strand et al., 1994a, b; Jakobs et al., 1996). In order to unpublished data). Taken together, all these analyses assess the ability of the two proteins to form hetero- support the concept that mammalian Lgl, Dlg and Scrib complexes, we transfected Sf9 insect cells with recombi- are involved in cell proliferation regulation. Moreover, nant baculoviruses containing the coding region of lgl the transcript of Dlg3 has been shown to be completely or Hugl-1. As shown in Figure 2c, the a-Hugl antibody

Figure 1 Hugl-1 expression is lost in solid tumours. An RT–PCR assay was performed using total RNA isolated from breast, lung, prostate, ovarian and melanoma tumours or normal tissue. Normal and tumour tissues were obtained after informed consent during routine diagnostic or therapeutic procedures. The b-actin expression level is shown as a standard reaction control. Primers utilized for actin were as follows: b-Act-fwd 50-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-30, b-Act-rev 50-CTA GAA GCA TTT GCG GTG GAC GAC GGA GGG-30; Hugl-1-fwd: 50-CTG TGG GCC CGC ATT GTG AG-30, Hugl-1-rev: 50-GGG CCA GGC TGT CAG AGT GCT-30. Hugl-1 transcription was reduced or lost in 13/17 (76%) breast, 12/19 (63%) lung, 8/15 (53%) prostate, 2/4 (50%) ovarian cancers and 2/5 (40%) melanomas

Oncogene Conservation of Lgl function D Grifoni et al 8690

Figure 2 Hugl-1 and Lgl form heterocomplexes in vitro.(a) Functional domains of Lgl and Hugl-1 proteins with the most conserved regions highlighted in Hugl-1. (b) Eye and wing imaginal discs from wt and l(2)gl4 third instar larvae. (c) a-Hugl-1 co- immunoprecipitation assay from recombinant Sf9 cell lysates. Antibodies, immunoprecipitations, recombinant baculoviruses, buffers and Western blotting have previously been described (Strand et al., 1994a, 1995). Essentially, affinity-purified polyclonal antibodies raised against synthetic peptides derived from the C-terminal end of Lgl or Hugl-1 were used in combination with Protein A agarose to immunoprecipitate Hugl-1 or Lgl. Sf9 cells were infected with recombinant baculoviruses expressing Lgl or Hugl-1 alone or in combination. Components of the recovered complexes were resolved on two identical 8% SDS–PAGE gels and Western blotted. The filters were probed with either a-Hugl-1 or a-Lgl antibodies. Molecular weight markers were obtained from Sigma (SDS-7B prestained marker)

does not crossreact with Lgl (upper lane 3) and the a-Lgl homo-oligomerization domains or through common antibody does not recognize Hugl-1 (bottom lane 2), partners must still be determined, but the conserved but the complexes immunoprecipitated by a-Hugl-1 in nature of the homo-oligomerization domains strongly lysates from doubly transfected cells are composed of suggests that they could play a role in this interaction. molecules recognized by both a-Hugl-1 and a-Lgl In order to ascertain whether the human protein antibodies (upper and bottom lane 4), indicating that could substitute for the Lgl tumour suppressor function the two proteins are able to interact and form in vivo, we inserted the human Hugl-1 cDNA in the heterocomplexes in vitro. The three homo-oligomeriza- Drosophila genome. We generated several independent tion domains in Lgl (B50-amino-acid residues in length) lines of transgenic flies carrying the Hugl-1 cDNA under contain a relatively high number of hydrophobic amino the control of a Gal4-responsive UAS promoter (Brand acids interspersed with charged residues. The hydro- and Perrimon, 1993). The UAS-Hugl1-15A transgenic phobic nature of the domains is conserved between line showed the correct inducible transgene expression humans and flies, with 52, 56 and 56% homology and Hugl-1 molecular mass (data not shown) and was respectively, including stretches of 10–12 amino acids thus chosen for further analyses. Larval imaginal discs, with 470% identity (Jakobs et al., 1996). Whether with their monolayered columnar , are simple heterocomplex formation occurs directly through their systems in which it is feasible to alter expression

Oncogene Conservation of Lgl function D Grifoni et al 8691 and follow the consequent phenotypes throughout (data not shown), the incomplete differentiation of the development. We thus performed rescue experiments head structures is probably due to inherent limitations in using the EGUF system (Stowers and Schwarz, 1999), a the experimental system. genetic method suitable for the generation of Drosophila We next considered whether ubiquitous expression of eyes composed exclusively of mitotic clones of the Hugl-1 could rescue all the phenotypes of Drosophila lgl mutant genotype (Figure 3a). For all the rescue trials, mutants. To allow ubiquitous expression of Hugl-1 in we used the null allele lethal(2)gl4 (Gateff, 1978), whose lgl-null animals, the UAS-Hugl-1 transgenic line carry- imaginal phenotype is shown in Figure 2b. Flies ing the lgl4 allele was crossed to an actin5c-Gal4 homozygous for the mutant allele were always recovered activator line carrying the same mutant lgl allele. F1 as headless pharates, and third instar larvae showed flies homozygous for the lgl4 mutation, bearing both a complete loss of the eye imaginal disc structure the UAS-Hugl-1 transgene and the act5c-Gal4 driver, (Figure 3b, left). Flies carrying the human transgene, in hatched into viable adults (46%; n ¼ 1096) (Figure 4a) the same mutant background as above, were also or were recovered as completely developed pharates; recovered as pharates, but showed a partial eversion of the same viable phenotype was obtained utilizing the the head with the development of rudimental eyes and UAS-lgl transgene as a positive larval structures comparable to wild type (Figure 3b, control (52%; n ¼ 828) (Figure 4b). In the imaginal right). The penetrance of this phenotype was complete, tissues, the relative abundance of Lgl and Hugl-1 with a fluctuating expressivity observed in the head proteins expressed by the UAS-Gal4 system with respect everted portion; in the figure, the most represented to the wild-type Lgl, estimated through a Western blot phenotype is shown. Since the positive controls, which experiment, was approximately 5 : 1 (data not shown). consisted in individuals carrying the UAS-lgl endogen- Both genotypes showed complete sterility, with oogen- ous transgene (Ohshiro et al., 2000), showed the same esis arresting at stage 7; hence, this defect cannot be phenotypic defects and did not hatch as viable adults considered a failure in the rescue ability of the human

Figure 3 Hugl-1 sustains Lgl function in the developing eye. (a) Schematic representation of the EGUF system. In the territories where eyeless promoter is active, the Gal4 transactivator is expressed and binds to the UAS cassettes upstream of the Flippase gene, which is thus transcribed. FLPproduct cuts its pericentromeric targets (FRT in cytological position 40A) promoting chromosomal arm exchange. In the rescue cross, Hugl-1 is also transcribed under the control of the eyeless promoter in the mutant cells. Otherwise, the clones stemmed by twin wild-type cells (and heterozygous cells in which recombination did not occur) bear the proapoptotic gene hid under the control of a promoter active at metamorphosis and will not be present in the adult eye. (b) (Left) lgl4, FRT40A/lgl4, FRT40A;ey-Gal4, UAS-FLP/ey-Gal4, UAS-FLP larval neoplastic eye disc and headless pharate, dorsal view. (Right) lgl4, FRT40A/lgl4, FRT40A;ey-Gal4, UAS-FLP/UAS-Hugl-1 rescued larval eye disc and pharate, dorsal and side view

Oncogene Conservation of Lgl function D Grifoni et al 8692

Figure 4 Hugl-1 sustains Lgl tumour suppressor function throughout development. (a) lgl4/lgl4;UAS-Hugl-1/act5c-Gal4 rescued fly. (b) lgl4/lgl4;UAS-lgl/act5c-Gal4 rescued fly. Scanning (c) and transmission (e) microscopy of lgl4/lgl4;UAS-lgl/act5c-Gal4 adult eye; the same analyses performed in lgl4/lgl4;UAS-Hugl-1/act5c-Gal4 flies (d, f). The red arrows mark the photoreceptor R7 in (e, f); the red asterisks mark ommatidia that lack R7 photoreceptor in (f). (g, h) Wing blade: Â 100 optical magnification of lgl4/lgl4;UAS-lgl/act5c- Gal4 (g) and lgl4/lgl4;UAS-Hugl-1/act5c-Gal4 (h) rescued flies. Note the misorganized bristle pattern in the Hugl-1 rescued wing margin (h) with respect to that shown in (g). For scanning microscopy, samples were dehydrated in hexamethyldisilazane, gold-coated in a Biorad SC502 SEM Coating System and analysed in a JEOL JSM-5200 scanning microscope. For transmission microscopy, tissues were treated according to standard protocols and ultrathin sections were prepared with a Reichert OmU3 ultramicrotome, coloured with uranyl acetate, contrasted with lead citrate according to Reynolds and observed in a 75 kV Hitachi H-800 transmission electron microscope

transgene. Both genotypes lived as long as a wild-type swept’ phenotype, which could result from underlying strain, did not develop any tumorous lesions through- defects in cell polarity (Figure 4h). To address this out their lifespan, and showed imaginal structures question, we analysed by confocal microscopy the indistinguishable from wild type. The complete rescue subcellular localization of Hugl-1, Dlg and Scrib in the of larval lethality by human Hugl-1 with animals imaginal epithelia of the rescued animals. progressing through all developmental stages is As shown in Figure 5b and d, in the imaginal eye disc conclusive evidence for the conservation of the Lgl columnar epithelium, Hugl-1 is cortically recruited as tumour suppressor function between Drosophila and the endogenous Lgl (Figure 5a and c) and Dlg and Scrib humans. in the rescued tissues show the same pattern as in the We observed some phenotypic alterations associated wild-type controls. Moreover, projections of the same to flies rescued by the human cDNA. These phenotypes discs along the Z-axis show that Dlg and Scrib are showed a complete penetrance and a comparable highly polarized and concentrate to the subapical region expressivity. At a stereoscopic level, the eye appeared of the cell membrane in rescued tissues (Figure 5b0 and rough, scutellar bristles were shortened and thickened d0) as in wild-type controls (Figure 5a0 and c0). These and the wing blade was bent downward, with margin data strongly suggest that the subtle impairments bristles irregularly spaced. At a cellular level, we observed in the rescued animals are not due to defects observed fused ommatidia with severe complanarity in apical–basal polarity; they are hence probably defects and accessory cells duplication (Figure 4d). imputable to some differences existing between human These eyes also exhibited a variable frequency in the and Drosophila proteins affecting a still unascertained percentage of ommatidia lacking photoreceptor R7 biological mechanism, or to an embryonic interference (Figure 4f). Concerning the wing phenotypes, the triple occurring between the maternal Lgl function and the row of the margin bristles was highly misorganized and, human protein; further analyses are required for this throughout the wing blade, bristles showed a ‘wind- issue to be addressed.

Oncogene Conservation of Lgl function D Grifoni et al 8693

Figure 5 Dlg and Scrib show a wild-type subcellular localization in the rescued imaginal tissues. (a) Lgl (red) and Scrib (green) localization in wild-type imaginal eye disc. (a0) Section of the same tissue along the Z-axis; the apical–basal orientation of the epithelium is shown. (b) Hugl-1 (red) and Scrib (green) localization in lgl4/lgl4;UAS-Hugl-1/act5c-Gal4 imaginal eye disc. (b0) Section of the same tissue along the Z-axis. (c) Lgl (red) and Dlg (green) localization in wild-type imaginal eye disc. (c0) Section of the same tissue along the Z-axis. (d) Hugl-1 (red) and Dlg (green) localization in lgl4/lgl4;UAS-Hugl-1/act5c-Gal4 imaginal eye disc. (d0) Section of the same tissue along the Z-axis. Images were captured with a LEICA TSC SP2 AOBS confocal microscope at  630 magnification and  4 optical zoom. Immunofluorescent labelling of third instar imaginal discs was performed according to standard protocols. The antibodies utilized were a-Lgl and a-Hugl-1 (1 : 200 of a 1 mg/ml solution; Strand et al., 1994a, 1995), a-Dlg (1 : 500, mouse monoclonal 4F3, Developmental Studies Hybridoma Bank, University of Iowa), a-Scrib (1 : 500; Albertson and Doe, 2003), and Texas red goat a- rabbit (1 : 50) and FITC goat a-mouse (1 : 25) from Jackson Laboratories

Approximately 50 Drosophila tumour suppressors Drosophila and humans is convincing evidence, with the have been so far described (Watson et al., 1994), but results obtained with dlg and scrib (Thomas et al., 1997; only lgl, dlg and scrib mutations show loss of apical– Dow et al., 2003), that the entire genetic network may be basal polarity and lead to neoplastic growth of nervous conserved between Drosophila and mammals. These and epithelial tissues (Agrawal et al., 1995; Goode and results set the basis for using the powerful fruit fly Perrimon, 1997). A wealth of literature exists describing genetic system to gather relevant information regarding mechanisms in which cell polarity and proliferation are molecular pathological mechanisms involved in the inversely correlated (Bissell and Radisky, 2001; Thiery, onset and progression of human cancers. 2002); such a link prevents undesired cell proliferation in monolayered epithelia. Our observation that Hugl-1 transcription is lost in human cancers fits into the Acknowledgements We thank all members of our laboratories for their support, emerging concept that lgl, dlg and scrib are involved V Politi for helpful discussions, F Matsuzaki for UAS-lgl in proliferation control in humans (Boussioutas et al., stocks, CQ Doe and V Robinson for a-Scrib antibodies and 2003; Makino et al., 1997; Hanada et al., 2000; Ishidate M Ziosi and E Tosti for their kind collaboration. These studies et al., 2000; Liu et al., 2002; Dow et al., 2003). Our were funded by a grant to DS through the MAIFOR program demonstration that Lgl function is conserved between and a grant to SC from the Italian MIUR.

References

Agrawal N, Kango M, Mishra A and Sinha P. (1995). Dev. Boussioutas A, Li H, Liu J, Waring P, Lade S, Holloway AJ, Biol., 172, 218–229. Taupin D, Gorringe K, Haviv I, Desmond PV and Bowtell Albertson R and Doe CQ. (2003). Nat. Cell Biol., 5, DD. (2003). Cancer Res., 63, 2569–2577. 166–170. Brand AH and Perrimon N. (1993). Development, 118, Bilder D, Li M and Perrimon N. (2000). Science, 289, 113–116. 401–415. Bilder D and Perrimon N. (2000). Nature, 403, 676–680. Dow LE, Brumby AM, Muratore R, Coombe ML, Sedelies Bissell MJ and Radisky D. (2001). Nat. Rev. Cancer, 1, KA, Trapani JA, Russell SM, Richardson HE and Humbert 46–54. PO. (2003). Oncogene, 22, 9225–9230.

Oncogene Conservation of Lgl function D Grifoni et al 8694 Gateff E. (1978). Science, 200, 1448–1459. Stowers RS and Schwarz TL. (1999). Genetics, 152, 1631–1639. Goode S and Perrimon N. (1997). Genes Dev., 11, 2532–2544. Strand D, Jakobs R, Merdes G, Neumann B, Kalmes A, Heid Hanada N, Makino K, Koga H, Morisaki T, Kuwahara H, HW, Husmann I and Mechler BM. (1994a). J. Cell Biol., Masuko N, Tabira Y, Hiraoka T, Kitamura N, Kikuchi A 127, 1361–1373. and Saya H. (2000). Int. J. Cancer, 86, 480–488. Strand D, Raska I and Mechler BM. (1994b). J. Cell Biol., 127, Hanahan D and Weinberg RA. (2000). Cell, 100, 57–70. 1345–1360. Ishidate T, Matsumine A, Toyoshima K and Akiyama T. Strand D, Unger S, Corvi R, Hartenstein K, Schenkel H, (2000). Oncogene, 19, 365–372. Kalmes A, Merdes G, Neumann B, Krieg-Schneider F and Jacob L, Opper M, Metzroth B, Phannavong B and Mechler Coy JF. (1995). Oncogene, 11, 291–301. BM. (1987). Cell, 50, 215–225. Thiery JP. (2002). Nat. Rev. Cancer, 2, 442–454. Jakobs R, De Lorenzo C, Spiess E, Strand D and Mechler Thomas U, Phannavong B, Muller B, Garner CC and BM. (1996). J. Mol. Biol., 264, 484–496. Gundelfinger ED. (1997). Mech. Dev., 62, 161–174. Kalmes A, Merdes G, Neumann B, Strand D and Mechler Timmons L, Hersperger E, Woodhouse E, Xu J, Liu LZ and BM. (1996). J. Cell Sci., 109, 1359–1368. Shearn A. (1993). Dev. Biol., 158, 364–379. Kim YS, Song J, Kim Y, Kim IO, Kang I and Baek KH. Tomotsune D, Shoji H, Wakamatsu Y, Kondoh H and (2003). Int. J. Oncol., 23, 1515–1519. Takahashi N. (1993). Nature, 365, 69–72. Larsson K, Bohl F, Sjostrom I, Akhtar N, Strand D, Mechler Watson KL, Justice RW and Bryant PJ. (1994). J. Cell Sci. BM, Grabowski R and Adler L. (1998). J. Biol. Chem., 273, Suppl., 18, 19–33. 33610–33618. Wodarz A. (2000). Curr. Biol., 10, R624–R626. Liu LX, Liu ZH, Jiang HC, Qu X, Zhang WH, Wu LF, Zhu Woodhouse E, Hersperger E, Setler-Stevenson WG, Liotta LA AL, Wang XQ and Wu M. (2002). World J. Gastroenterol., and Shearn A. (1994). Cell Growth Differ., 5, 151–159. 8, 580–585. Woodhouse EE, Hersperger E and Shearn A. (1998). Dev. Makino K, Kuwahara H, Masuko N, Nishiyama Y, Morisaki Genes Evol., 207, 542–550. T, Sasaki J, Nakao M, Kuwano A, Nakata M, Ushio Y and Woods DF, Hough C, Peel D, Callaini G and Bryant PJ. Saya H. (1997). Oncogene, 14, 2425–2433. (1996). J. Cell Biol., 134, 1469–1482. Ohshiro T, Yagami T, Zhang C and Matsuzaki F. (2000). Woods DF, Wu JW and Bryant PJ. (1997). Dev. Genet., 20, Nature, 408, 593–596. 111–118. Plant PJ, Fawcett JP, Lin DC, Holdorf AD, Binns K, Yamanaka T, Horikoshi Y, Sugiyama Y, Ishiyama C, Suzuki Kulkarni S and Pawson T. (2003). Nat. Cell Biol., 5, A, Hirose T, Iwamatsu A, Shinohara A and Ohno S. (2003). 301–308. Curr. Biol., 13, 734–743.

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