Materials and Chemicals
The Fam cDNA clone was generated as described previously (Wood et al., 1997
). MDCKII cells, EL cells, and the mouse antibody against ZO-1 were kindly provided by Drs. A. Nagafuchi and S. Tsukita (University of Kyoto, Kyoto, Japan) (Itoh et al., 1991
; Nagafuchi et al., 1994
). pMT123 (HA-ubiquitin expression plasmid) was kindly provided by Dr. D. Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany) (Treier et al., 1994
). Rabbit polyclonal antibodies against AF-6 (914–1129 aa; #3), AF-6 (1130–1612 aa; #4), Fam (1–20 aa; N20), Fam (2441–2554 aa; C114), and Fam (1165–1967 aa; K2) were generated as described previously (Harlow and Lame, 1988
). FITC-conjugated anti-rabbit IgG antibody, Texas red–conjugated anti-mouse IgG antibody, and [35
S]methionine were purchased from Amersham Corp.
(Buckinghamshire, United Kingdom). Mouse monoclonal antibody against β-catenin was purchased from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies against α-catenin and β-catenin were purchased from Sigma Chemical Co.
(St. Louis, MO). Polyvinylidene difluoride membranes (Problott, 0.45-μm pore size) were purchased from PE Applied Biosystems (Foster, CA). Achromobacter
protease I was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Proteasome inhibitor ALLN (N
-acetyl-Leu-Leu-norleucinal) and calpain inhibitor II ALLM (N
-acetyl-Leu-Leu-methional) were purchased from Peptide Institute, Inc. (Osaka, Japan). All materials used in the nucleic acid study were purchased from Takara Shuzo Corp. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources.
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.
Preparation of Bovine Brain Cytosolic Fraction
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.
GST-AF-6 (1130–1612 aa) Affinity Column Chromatography
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.
Peptide Sequence Analysis of p220
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
Hydrolysis Assay of Ub-PEST
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 Na125
I 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 125
I-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
Immunofluorescence and Laser Scanning Confocal Microscopy
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 Binding Assay (In Vitro–translated Fam)
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).
In Vitro Binding Assay (Maltose-binding Protein [MBP]–Fam)
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).
Effect of Proteasome Inhibitor on Rat1 and EL Cells
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.
Ubiquitination Assay of AF-6 In Vivo
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.
Deubiquitination Assay of the Ubiquitinated AF-6 by Fam
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.