The Escherichia coli expression plasmid pGEX-3X-AF-6 (1130–1612 aa) was constructed by subcloning the PvuII and EcoRI fragments of AF-6 into the SmaI and EcoRI sites of pGEX-3X. The plasmids for in vitro translation of Fam were constructed by amplifying the cDNA fragments encoding Fam (1–669 aa, 670–1213 aa, 1210–2100 aa, and 2097–2554 aa) by PCR from the full-length Fam cDNA in pBluescript SK(−). The fragment of Fam (1–669 aa) was amplified using the sense primer containing a BamHI site (5′-AATTGGATCCATGACAGCCACGACTCGTG-3′) and the antisense primer containing a BamHI site (5′-ATTAGGATCCTCACAGCTGGCCATCCTTCA-3′). The fragment of Fam (670–1213 aa) was amplified using the sense primer containing a BamHI site (5′-AATTGGATCCCTGTGGCTGTGTGCTCCAC-3′) and the antisense primer containing a BamHI site (5′-ATTAGGATCCTCAGCATGCATTCAGATGATGGG-3′). The fragment of Fam (1210–2100 aa) was amplified using the sense primer containing a BamHI site (5′-AATTGGATCCTGCATGCTTAGAAACGTGTC-3′) and the antisense primer containing a BamHI site (5′-ATTAGGATCCTTACTCAGAGAATCGATTTGAAAC-3′). The fragment of Fam (2097–2554 aa) was amplified using the sense primer containing a BamHI site (5′-AATTGGATCCCGATTCTCTGAGTACCTTTTG-3′) and the antisense primer containing a BamHI site (5′-ATTAGGATCCTTAATATGTGCATTTCACTGATC-3′). The fragments (1–669 aa, 670–1213 aa, and 2097–2554 aa) were cloned into the BamHI site of pRSET-A, and the fragment (1210–2100 aa) was cloned into the BamHI site of pBluescript SK(−). The Escherichia coli expression plasmid pMal-c2-Fam (1476–1918 aa) was constructed by inserting the blunt-ended PstI and BstXI fragment of Fam into pMal-c2. The mammalian expression plasmid pEF-BOS-AF-6-myc was constructed as follows: the cDNA fragments encoding AF-6 were subcloned into pBluescript SK(−) having a sequence encoding a myc epitope tag (MEQKLISEEDL), and AF-6-myc cDNA fragment was inserted into the XbaI site of pEF-BOS. For the preparation of pEF-BOS-HA-Fam-CAT (1210–2410 aa), the fragment (1210–2410 aa) was subcloned into pBluescript SK(−), and the fragment was inserted into the BamHI site of pEF-BOS-HA.

MDCKII and Rat1 cells were grown in DME containing 10% calf serum, penicillin, and streptomycin in an air-5% CO₂ atmosphere at constant humidity. EL cells were grown in DME containing 10% FBS and 100 μg/ml of G418 in an air-5% CO₂ atmosphere at constant humidity. COS7 cells were grown in DME containing 10% FBS, penicillin, and streptomycin in an air-5% CO₂ atmosphere at constant humidity.

100 g of bovine brain gray matter was cut into small pieces with scissors and suspended in 300 ml of homogenizing buffer A (25 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, 10 mM MgCl₂, 10 μM [p-amidino-phenyl]methanesulfonyl fluoride [p-APMSF], 1 μg/ml leupeptin, 10% sucrose). The suspension was homogenized with a Potter-Elvehjem Tefron-glass homogenizer and filtered through four layers of gauze. The homogenate was centrifuged at 20,000 g for 30 min at 4°C and then at 100,000 g for 60 min at 4°C as described (Yamamoto et al., 1995). The supernatant was stored at −80°C as the cytosolic fraction.

Glutathione-S-transferase (GST)-AF-6 (1130–1612 aa) was expressed in Escherichia coli BL21 (DE3) and purified according to the manufacturer's instructions. Glutathione-Sepharose 4B (1 ml) was coated with GST-AF-6 (1130–1612 aa; 30 nmol). The brain cytosolic fraction was then preabsorbed to remove the native GST with glutathione-Sepharose 4B and loaded onto the GST-AF-6 (1130–1612 aa) affinity column. The column was washed with 10 ml of buffer B (20 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, 5 mM MgCl₂, 10 μM p-APMSF, 1 μg/ml leupeptin) followed by washing with 10 ml of buffer B containing 75 mM NaCl. The protein bound to the affinity column was eluted 10 times with 1 ml of buffer B containing 10 mM reduced glutathione.

The 10-mM reduced glutathione eluates from the first to seventh fractions were dialyzed three times against distilled water and concentrated by freeze-drying. The concentrated samples were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Iwamatsu, 1992). The immobilized p220 was reduced and S-carboxymethylated, followed by in situ digestion with Achromobacter protease I and Asp-N. The digested peptides were fractionated by C18 column chromatography and subjected to amino acid sequencing (Iwamatsu, 1992).

For purification of Fam, the brain cytosolic fraction was loaded onto the GST-AF-6 (1130–1612 aa) affinity column. Fam was eluted by adding buffer B containing 200 mM NaCl from the GST-AF-6 affinity column. Ubiquitin-αNH-MHISPPEPESEEEEEHYC (Ub-PEST) was radiolabeled with Na¹²⁵I using IODO-BEADS (Markwell, 1982). Fam (1.6 μg) was incubated for various periods at 37°C in a final volume (50 μl) containing 1 μg of ¹²⁵I-labeled Ub-PEST, 100 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1 mM DTT, and 5% (vol/vol) glycerol. The reaction was terminated by adding 50 μl of acetone, and the mixture was frozen overnight at −80°C. The pellet fractions were boiled in sample buffer for SDS-PAGE, and were subjected to SDS-PAGE detected by Coomassie Brilliant Blue staining and by autoradiography (Woo et al., 1995).

MDCKII cells plated on 13-mm round glass coverslips were fixed with methanol for 10 min. The fixed cells were incubated for 2 h with mouse monoclonal antibodies against ZO-1 or β-catenin, and were washed three times for 10 min with PBS. The cells were then incubated for 2 h with anti-Fam antibody (K2) and washed three times for 10 min with PBS. The cells were incubated for 1 h with FITC-conjugated anti-rabbit IgG antibody and Texas red–conjugated anti-mouse IgG antibody, and washed three times for 10 min with PBS. The distributions of Fam, ZO-1, and β-catenin were examined using a laser scanning confocal microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with an argon laser and a helium-neon laser for double fluorescence at 488 and 543 nm (emission filter; BP510–525 and LP590).

MDCKII cells were grown in 100-mm tissue culture dishes. After being washed with PBS, the cells were lysed with 1 ml of buffer C (50 mM Tris/ HCl at pH 8.0, 1 mM EDTA, 75 mM NaCl, 1 mM MgCl₂, 0.2% Triton X-100, 10 μM p-APMSF, 10 μg/ml leupeptin). The lysate was removed from the dishes with a rubber policeman. The lysate was sonicated, incubated in a 1.5-ml tube for 15 min on ice, and then clarified by centrifugation at 12,000 g for 30 min at 4°C. The soluble supernatant was incubated with 10 μg of anti-Fam antibody (K2), anti-AF-6 antibody (#3), or preimmuneserum. The immunocomplexes were then precipitated with protein A-Sepharose 4B (Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The immunocomplexes were washed six times with buffer C, eluted by boiling in sample buffer for SDS-PAGE and subjected to immunoblot analysis using anti-Fam antibody (N20) or anti-AF-6 antibody (#4).

In vitro translation of pRSET-Fam (1–669 aa), pRSET-Fam (670–1213 aa), pBluescript SK(−)-Fam (1210–2100 aa), and pRSET-Fam (2097– 2554 aa) were performed using a TNT T7-coupled reticulocyte lysate system (Promega Corp., Madison, WI) under the conditions described in the manufacturer's instruction manual. Glutathione-Sepharose 4B beads (31 μl) were coated with GST fusion proteins and washed with 310 μl of buffer B. The coated beads were added to 40 μl of the in vitro–translated products labeled with [³⁵S]methionine containing 1 mg/ml BSA, and were incubated for 1 h at 4°C with gentle mixing. The beads were washed six times with 102 μl of buffer B, and the bound proteins were coeluted with GST fusion proteins three times by adding 102 μl of buffer B containing 10 mM reduced glutathione. The eluates were subjected to SDS-PAGE and vacuum-dried. The [³⁵S]-labeled bands corresponding to in vitro–translated Fam were visualized with an image analyzer (BAS-2000; Fuji Photo Film Co., Tokyo, Japan).

The deubiquitinating catalytic domain of Fam (1476–1918 aa) was expressed as a MBP fusion protein, and was purified with amylose resin (New England Biolabs, Inc., Beverly, MA). MBP-Fam (1476–1918 aa; 0.05, 0.125, 0.25, 1.25, 2.5, and 7.5 nmol) was mixed with glutathione-Sepharose 4B beads (30 μl) coated with 0.3 nmol of either GST or GST-AF-6 (1130–1612 aa) in 250 μl of buffer B. The beads were washed six times with 100 μl of buffer B, and were then washed three times with 100 μl of buffer B containing 50 mM NaCl. The bound MBP-Fam (1476–1918 aa) was coeluted with GST or GST-AF-6 (1130–1612 aa) by adding 100 μl of buffer B containing 10 mM glutathione three times. Portions (30 μl each) of the three fractions of the glutathione-eluates were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. MBP-Fam (1476– 1918 aa) was visualized and estimated with a densitograph (ATTO, Tokyo, Japan).

Rat1 and EL cells were seeded in 100-mm tissue culture dishes at a cell density of 1 × 10⁶ cells/dish, and were cultured for 24 h. The cells were then treated with 25 μM ALLN or ALLM for 3 h. ALLN and ALLM were dissolved in DMSO to a final concentration of 10 mM. The cells were washed twice with PBS and lysed in 360 μl of buffer D (50 mM Tris/ HCl at pH 7.5, 100 mM KCl, 4 mM EGTA, 1 mM NaF, 1 mM sodium vanadate, 0.25% Triton X-100, 10 μM p-APMSF, 1 μg/ml leupeptin, 25 μM ALLN, and 300 mM sucrose). The cell lysates of Rat1 and EL cells were subjected to SDS-PAGE and immunoblotted with anti-AF-6 (#4) antibody, polyclonal antibody against β-catenin, or anti-α-catenin antibody.

COS7 cells were seeded on 60-mm tissue culture dishes at a cell density of 6 × 10⁵ cells/dish, and were cultured for 16 h. COS7 cells were transfected with pEF-BOS-AF-6-myc (12 μg) and pMT123 (HA-ubiquitin; 3 μg) by the standard DEAE-dextran method (Lopata et al., 1984). To examine the effect of the proteasome inhibitor ALLN, COS7 cells were grown for 30 h after transfection, and were then treated with 25 μM ALLN for 18 h. The cells were washed twice with PBS and lysed in 360 μl of buffer D. An immunoprecipitation analysis with anti-AF-6 antibody (#3) was performed as described above. The samples were subjected to SDS-PAGE followed by immunoblot analysis using anti-HA antibody.

The plasmids pEF-BOS-AF-6-myc (12 μg) and pMT123 (3 μg) were transfected with or without pEF-BOS-HA-Fam-CAT (5 μg) in COS7 cells. Immunoprecipitation and an immunoblot analysis were carried out as described above.

First we examined whether Fam has deubiquitinating activity. We used the 200-mM NaCl eluate from the GST-AF-6 (1130–1612 aa) affinity column as a source of Fam. Fam was incubated with the ¹²⁵I-labeled ubiquitin-conjugated PEST sequence (Ub-PEST) for various periods of time. As shown in Fig. ​Fig.2,2, Fam could release ubiquitin from Ub-PEST (a), and could produce the hydrolyzed ¹²⁵I-PEST peptides from Ub-PEST (b). In the Fam minus control of Fig. ​Fig.2,2, the 200-mM NaCl buffer that did not contain any enzyme was used. These results indicate that Fam is able to generate free ubiquitin from Ub-PEST. It is well-known that ubiquitin-aldehyde (Ub-CHO) inhibits Ubps. Release of ubiquitin from Ub-PEST or production of the PEST peptides was almost abolished in the presence of Ub-CHO. The deubiquitinating activity was not detected in the 200-mM NaCl eluate from the GST affinity column (data not shown). These results demonstrated that Fam has deubiquitinating activity.

To understand the functions of Fam, we examined its intracellular distribution in MDCKII epithelial cells that show characteristics of polarized epithelial cells and form the junctional complex, including the tight junction and adherens junction at cell–cell contact sites (Gonzalez-Mariscal et al., 1985). The immunoblot analysis of cell lysates from MDCKII cells showed that anti-Fam antibody recognized a single band corresponding to a molecular weight of ~220 kD (Fig. ​(Fig.33 A) as in the case of bovine brain extract (Fig. ​(Fig.11 b). Antibody preincubation with the recombinant Fam abolished the immunoreactivity (data not shown). The immunoreactivity of Fam was specifically localized at sites where a cell contacted a neighboring cell, and not at free ends of plasma membranes (Fig. ​(Fig.33 B). The cytoplasm exhibited a relatively low level of immunoreactivity. To further examine whether Fam exists in the apical or basal site of the lateral membrane, cellular distribution of Fam was compared with that of ZO-1 and β-catenin (Fig. ​(Fig.3,3, C, b and e). ZO-1 was concentrated at the apical sections, whereas β-catenin was found at more basal sections as described previously (Nagafuchi and Takeichi, 1989; Itoh et al., 1993). In contrast, Fam immunoreactivity was colocalized at the immunofluorescence microscopic level with ZO-1 at the apical sites, and with β-catenin at the basal sites (Fig. ​(Fig.3,3, C, c and f). Since we have previously shown that AF-6 interacts with ZO-1 and is colocalized with ZO-1 at cell–cell contact sites including tight junctions (Yamamoto et al., 1997), part of the Fam that is localized at the same sites with ZO-1 may be colocalized with AF-6. We also found that accumulation of Fam is induced by the formation of cell–cell adhesions by using a Ca²⁺ switch assay in MDCKII cells (data not shown). These results suggest that Fam is partly colocalized with AF-6 at apical sites of the lateral membrane in confluent MDCKII cells.

Because Fam was partly colocalized with AF-6 at apical sites of the lateral membrane, we examined whether Fam interacts with AF-6 in vivo. When Fam was immunoprecipitated with anti-Fam antibody from confluent MDCKII cells, AF-6 was coimmunoprecipitated with Fam (Fig. ​(Fig.44 a). AF-6 was not coimmunoprecipitated with control preimmuneserum. AF-6 appeared to associate with Fam with a stoichiometry of about one AF-6 per ten Fam under these conditions. Similarly, Fam was also coimmunoprecipitated with AF-6 (Fig. ​(Fig.44 b) when AF-6 was immunoprecipitated with anti-AF-6 antibody from confluent MDCKII cells. These results indicate that Fam interacts with AF-6 in vivo.

Since the AF-6–interacting domain of Fam involves the deubiquitinating catalytic domain of Fam, we speculate that AF-6 is the substrate of Fam, and is subjected to the ubiquitin–proteasome pathway. As described above, when Rat1 and EL cells were treated with the proteasome inhibitor ALLN, the higher molecular weight forms of AF-6 were detected. We also performed an assay for detecting the ubiquitinated proteins (Treier et al., 1994; Aberle et al., 1997) and found that AF-6 was ubiquitinated in vivo. Many proteins involved in cell cycle control, transcription activation, cell growth, antigen presentation, and so on are known to be regulated by the ubiquitin–proteasome pathway. These proteins include c-fos, NFκB-IκB complex, p53 and growth factor receptors such as PDGFR and FGFR-1, and it has been gradually clarified how these proteins are subjected to the ubiquitin–proteasome pathway (Hochstrasser, 1995; Hicke, 1997). It remains to be clarified how AF-6 is ubiquitinated and subsequently degraded by the proteasome pathway.