Differential Nuclear Translocation And
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Published June 15, 1998 Differential Nuclear Translocation and Transactivation Potential of b-Catenin and Plakoglobin Inbal Simcha, Michael Shtutman, Daniela Salomon, Jacob Zhurinsky, Einat Sadot, Benjamin Geiger, and Avri Ben-Ze'ev Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Abstract. b-Catenin and plakoglobin are homologous pro- or b-catenin were equally potent in transactivating a Gal4- teins that function in cell adhesion by linking cadherins to responsive reporter, whereas activation of LEF-1– the cytoskeleton and in signaling by transactivation together responsive transcription was significantly higher with with lymphoid-enhancing binding/T cell (LEF/TCF) tran- b-catenin. Overexpression of wild-type plakoglobin or mu- scription factors. Here we compared the nuclear transloca- tant b-catenin lacking the transactivation domain induced tion and transactivation abilities of b-catenin and plakoglo- accumulation of the endogenous b-catenin in the nucleus bin in mammalian cells. Overexpression of each of the two and LEF-1–responsive transactivation. It is further shown proteins in MDCK cells resulted in nuclear translocation that the constitutive b-catenin–dependent transactivation in Downloaded from and formation of nuclear aggregates. The b-catenin-contain- SW480 colon carcinoma cells and its nuclear localization ing nuclear structures also contained LEF-1 and vinculin, can be inhibited by overexpressing N-cadherin or a-catenin. while plakoglobin was inefficient in recruiting these mole- The results indicate that (a) plakoglobin and b-catenin dif- cules, suggesting that its interaction with LEF-1 and vincu- fer in their nuclear translocation and complexing with LEF-1 lin is significantly weaker. Moreover, transfection of LEF-1 and vinculin; (b) LEF-1–dependent transactivation is prefer- jcb.rupress.org translocated endogenous b-catenin, but not plakoglobin to entially driven by b-catenin; and (c) the cytoplasmic part- the nucleus. Chimeras consisting of Gal4 DNA-binding do- ners of b-catenin, cadherin and a-catenin, can sequester it main and the transactivation domains of either plakoglobin to the cytoplasm and inhibit its transcriptional activity. on May 19, 2009 ell–cell adhesion plays an important role in tissue involved in the wingless (wg) signaling pathway that regu- morphogenesis and homeostasis (Gumbiner, 1996; lates cell fate during development (Peifer, 1995; Orsulic C Huber et al., 1996a), and is commonly mediated and Peifer, 1996). In Xenopus, b-catenin participates in by cadherins, a family of Ca21-dependent transmembrane the wnt-signaling pathway that determines body axis for- adhesion receptors (for review see Geiger and Ayalon, mation (for review see Miller and Moon, 1996; Gumbiner, 1992; Kemler, 1992; Takeichi, 1991, 1995). Cadherins were 1995, 1996), and overexpression of b-catenin and plako- shown to form homophilic interactions with similar recep- globin in Xenopus embryos was shown to induce double tors on neighboring cells, whereas their cytoplasmic do- axis formation (Funayama et al., 1995; Karnovsky and mains interact with the cytoskeleton. The latter interac- Klymkowsky, 1995; Merriam et al., 1997; Miller and Moon tions are essential for stable adhesion and are mediated 1997; Rubenstein et al., 1997). In cultured cells, wnt over- via b-catenin, or its closely related homologue g-catenin expression elicits adhesion-related responses and increased (plakoglobin), which interacts with microfilaments through levels of b-catenin and plakoglobin (Bradley et al., 1993; a-catenin (Kemler, 1993; Geiger et al., 1995; Jou et al., Hinck et al., 1994; Papkoff et al., 1996). b-Catenin levels 1995; Rimm et al., 1995) and a-actinin (Knudsen et al., are regulated by glycogen synthase kinase-3b and ade- 1995). nomatous polyposis coli (APC)1 tumor suppressor protein In addition to the direct role of b-catenin/plakoglobin in (Papkoff et al., 1996; Rubinfeld et al., 1996; Yost et al., promoting adhesion, their homologue in Drosophila arma- 1996) which are thought to target b-catenin for degrada- dillo (Peifer and Weischaus, 1990), was also shown to be tion by the ubiquitin–proteasome system (Aberle et al., Address all correspondence to: Avri Ben-Ze'ev, Department of Molecu- 1. Abbreviations used in this paper: aa, amino acid(s); APC, adenomatous lar Cell Biology, Weizman Institute of Science, Rehovot 76100, Israel. polyposis coli; DBD, DNA-binding domain; HA, hemagglutinin; LEF, Tel.: (972) 8-934-2422. Fax: (972) 8-934-4125. E-mail: lgbenzev@weizmann. lymphoid-enhancing binding factor; TCF, T cell factor; VSV, vesicular weizmann.ac.il stomatitis virus; wt, wild-type. The Rockefeller University Press, 0021-9525/98/06/1433/16 $2.00 The Journal of Cell Biology, Volume 141, Number 6, June 15, 1998 1433–1448 http://www.jcb.org 1433 Published June 15, 1998 1997; Orford et al., 1997; Salomon et al., 1997). When 1990). HA–b-catenin was obtained by PCR amplification of the 59 end of b-catenin levels are high, it can associate with architectural mouse b-catenin (Butz et al., 1994) using oligonucleotides 59-ACCT- TCTAGAATGGCTACTCAAGCTGACCTG-39 and 59-ATGAGCAG- transcription factors of the lymphoid-enhancing binding CGTCAAACTGCG-39. The XbaI/SphI fragment of the PCR product factor/T cell factor (LEF/TCF) family and translocate into and the SphI/BamHI fragment of mouse b-catenin were joined and cloned the nucleus (Behrens et al., 1996; Huber et al., 1996b; Mo- into the pCGN vector. A b-catenin mutant containing armadillo repeats lenaar et al., 1996). In the nucleus, the b-catenin–LEF/ 1–10 (see Fig. 1, HA b-catenin 1-ins) was obtained using oligonucleotides TCF complex activates transcription of LEF/TCF-respon- 59-ACCTTCTAGATTGAAACATGCAGTTGTCAATTTG-39 and 59- ACCTGGATCCAGCTCCAGTACACCCTTCG-39. The amplified frag- sive genes that are not yet known in mammalian cells ment was cloned into pCGN as an XbaI/BamHI fragment. Vesicular sto- (Morin et al., 1997), but have partially been characterized matitis virus (VSV)-tagged b-catenin at the COOH terminus was obtained in Xenopus (Brannon et al., 1997) and Drosophila (Riese by PCR amplification of the 39 end of Xenopus b-catenin (McCrea et al., et al., 1997; van de Wetering et al., 1997). 1991) using 59-AACTGCTCCTCTTACTGA-39 and 59-TATCCCGGGT- CAAGTCAGTGTCAAACCA-39. An XbaI/SmaI fragment of the ampli- Elevation of b-catenin in colon carcinoma cells that ex- fied product was joined to an XbaI/EcoRI digest of Xenopus b-catenin press a mutant APC molecule (Powell et al., 1992; Polakis, from Bluescript and then cloned into the pSY-1 plasmid containing an 11-aa 1997), or in melanoma where mutations in the NH2-termi- VSV glycoprotein tag (Kreis, 1986) at the COOH terminus. The product nal domain of b-catenin were detected (both inhibiting was subcloned into pCI-neo (Promega Corp., Madison, WI). b-catenin degradation) is oncogenic, most probably due to HA-tagged human plakoglobin (see Fig. 1, HA plakoglobin) was ob- tained by PCR amplification of the 59 end of human plakoglobin cDNA constitutive activation of target genes which contributes to (pHPG 5.1) (Franke et al., 1989) using 59-ACCTTCTAGAATGGAG- tumor progression (Korinek et al., 1997; Morin et al., 1997; GTGATGAACCTGATGG-39 and 59-AGCTGAGCATGCGGACCAG- Peifer, 1997; Rubinfeld et al., 1997). Interestingly, plako- AGC-39 oligonucleotide primers. The XbaI/SphI fragment of the PCR globin was shown to suppress tumorigenicity when overex- product and the SphI/BamHI fragment of human plakoglobin cDNA were cloned into the pCGN vector. A plakoglobin mutant containing pressed in various cells (Simcha et al., 1996; Ben-Ze'ev, armadillo repeats 1–10 (see Fig. 1, HA plakoglobin 1-ins) was amplified 1997), and displays loss of heterozigosity in sporadic ovar- using 59-ACCTTCTAGACTCAAGTCGGCCATTGTGC-39 and 59-ACC- ian and breast carcinoma (Aberle et al., 1995). Moreover, TGGATCCTGCTCCGGTGCAGCCCTCC-39 oligonucleotides. The am- upon induction of plakoglobin expression in human fibro- plified fragment was cloned into the XbaI/BamHI site of pCGN. A Downloaded from sarcoma and SV-40–transformed 3T3 cells b-catenin is dis- COOH terminus VSV-tagged plakoglobin (see Fig. 1, plakoglobin-VSV) was obtained by amplifying the 39 terminus of plakoglobin cDNA using placed from its complex with cadherin and directed to deg- 59-AGGCCGCCCGGGCAGCATG-39 and 59-CGCATGGAGATCTTC- radation (Salomon et al., 1997). CGGCTC-39 oligonucleotide primers. The EcoRI/BglII fragment of the In the present study we characterized the mechanisms plakoglobin cDNA and the BglII/SmaI fragment of the amplified product underlying nuclear accumulation of b-catenin and/or pla- were cloned in frame with the COOH terminus VSV tag into the pSY-1 plasmid. VSV-tagged plakoglobin was recloned into the pCI neo- koglobin and identified some of the partners associated expression vector (Promega Corp.) as an EcoRI/NotI fragment. jcb.rupress.org with both proteins in the nucleus. Furthermore, we com- HA-tagged a-catenin (see Fig. 1, HA a-catenin) was constructed by pared the nuclear translocation and transactivation abili- amplification of the 59 end of chicken a-catenin (Johnson et al., 1993) us- ties of wild-type (wt) and mutant b-catenin and plakoglo- ing 59-ACCTTCTAGAATGACGGCTGTTACTGCAGG-39 and 59-GCC- bin constructs and found that these two proteins differ TTCTTAGAGCGCCCTCG-39 oligonucleotide primers. The XbaI/ApaI fragment of the PCR product and the ApaI/KpnI fragment of chicken considerably in these properties, and demonstrated that a-catenin from pLK–a-catenin