Yeast Two-Hybrid Screening and Sequence Analysis
The yeast two-hybrid screening (Chien et al., 1991
) of a rat brain cDNA library (Clontech
) was conducted with a yeast strain CG-1945 [MATa ura3-52 his3-200 lys2-801 trp1-901 ade2-101 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1-HIS3 cyhr2 URA3::(GAL4 17-mers)3-CYC1-lacZ
], a derivative of HF7c (Feilotter et al., 1994
), by using the regulatory domain of rat PKCζ (residues 1–250) (Ono et al., 1988b
) fused with the yeast GAL4 DNA-binding domain as a bait. The cDNA fragment of a positive clone obtained was sequenced with a DNA sequencer (model 373S; Perkin Elmer
/Applied Biosystems). The full-length cDNA was obtained from a rat brain Marathon-Ready cDNA library (Clontech
) by the method of rapid amplification of cDNA ends (RACE) (Frohman et al., 1988
). Prediction for the coiled–coil structure (Lupas, 1996
) was performed by software available at the web site of the Swiss Institute for Experimental Cancer Research (http://ulrec3.unil.ch/software/COILS_form.html
Plate Assay for β-Galactosidase (β-Gal) Activity in Yeast Cells
β-Gal activity in yeast cells was measured by the plate assay method. Yeast transformants (Leu+, Trp+, His+) were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3MM papers that had been soaked in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM MgCl2, 50 mM 2-mercaptoethanol, pH 7.0) containing 1 mg/ml 5-bromo-4-chloro-3-indoryl-β-d-galactoside (X-Gal). After developing at 37°C for 30 min, the yeast cells forming dark blue colonies were classified into a strongly positive group (+++). After developing at 37°C for 10 h, the yeast cells forming either dark blue or blue colonies were classified into a moderately positive (++) and a weakly positive group (+), respectively. Those forming white colonies were classified into a negative group (−). All measurements were repeated at least four times.
Northern Blot Analysis
Northern blots containing poly(A)+ RNA (~2 μg/lane) from eight tissues of adult rats and mouse embryos in four different developmental stages were obtained from Clontech. The amount of poly(A)+ RNA in each lane was calibrated using the rat β-actin gene. The full-length FEZ1 cDNA fragment labeled with [α-32P]dCTP (~110 TBq/mmol) by a Ready-To-Go DNA labeling kit (Pharmacia Biotech) was used as a probe. Hybridization was carried out under highly stringent conditions. The blots were autoradiographed by using a BAS-2000 bioimage analyzing system (Fuji).
Expression of Epitope-tagged Proteins in COS-7 Cells
For expression of the NH2
-terminally FLAG-tagged FEZ1 protein (FEZ1-FLAG), a pTB701-FLAG-FEZ1 plasmid was constructed by placing in frame FEZ1
cDNA 3′ downstream of the FLAG epitope sequence of pTB701-FLAG (Kuroda et al., 1996
). Similarly, for expression of the NH2
-terminally HA-tagged PKCζ (PKCζ-HA), a pTB701-HA-PKCζ plasmid was constructed from pTB701-HA (Kuroda et al., 1996
). An expression plasmid for a kinase-negative mutant protein of PKCζ-HA (K281M PKCζ-HA), pTB701-HA-K281M PKCζ, was prepared by replacing the ATP-binding Lys-281 residue by Met with a Quick-Change site-directed mutagenesis kit (Stratagene Cloning Systems). An expression plasmid for a constitutively active mutant of PKCζ-HA (caPKCζ-HA), pTB701-HA-caPKCζ, was prepared by deleting the pseudosubstrate region from Arg-116 to Trp-122 (Schonwasser et al., 1998
). These plasmids were transferred into COS-7 cells by electroporation using a Gene Pulser II (Bio-Rad Laboratories).
Subcellular Fractionation of COS-7 Cells
COS-7 cells (~5.0 × 107 cells) expressing FEZ1-FLAG were suspended in 1 ml of PBS and sonicated on ice for 15 s. After centrifugation at 10,000 g at 4°C for 10 min, the supernatant was collected as a cytoplasmic fraction. The pellet was resuspended in 1 ml of PBS containing 1% (vol/vol) Triton X-100 and sonicated on ice for 15 s. After centrifugation at 10,000 g at 4°C for 10 min, the supernatant was collected as a membrane fraction. Samples (10 μl) derived from ~5.0 × 105 cells were subjected to SDS-PAGE (12.5%) and analyzed by Western blotting using an anti-FLAG mAb M2 (Eastman Kodak).
In Vitro Transcription and Translation
In vitro synthesis of FEZ1-FLAG was performed with a Single Tube Protein System 2 (Novagen). In brief, the cDNA for FEZ1-FLAG was placed 3′ downstream of the T7 promoter and then was transcribed with T7 RNA polymerase in the presence of dNTPs at 30°C for 15 min. The synthesized mRNA was translated in the rabbit reticulocyte lysate containing 1.5 MBq of [35S]Met (~37 TBq/mmol) at 30°C for 60 min. Samples were analyzed by SDS-PAGE (12.5%) and subsequent autoradiography.
Immunoprecipitation and Phosphorylation Assay
COS-7 cells (~5.0 × 107 cells) coexpressing FEZ1-FLAG and either PKCζ-HA or K281M PKCζ-HA were suspended in 500 μl of the lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM 2-mercaptoethanol, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, and 1% [vol/vol] Triton X-100, pH 7.5) containing 1 tablet of the complete protease inhibitor cocktail (Boehringer Mannheim) per 50 ml of the buffer. After centrifugation at 10,000 g at 4°C for 10 min, the lysates (500 μl) were incubated on ice for 1 h with 2 μg of either an anti-FLAG or anti-HA 12CA5 (Boehringer Mannheim) mAb and then mixed with 20 μl of protein G–Sepharose 4 fast flow beads (50% slurry; Pharmacia Biotech). After shaking at 4°C for 1 h, the beads were washed four times with the lysis buffer. For Western blotting, the beads were subjected to SDS-PAGE (12.5%). Tagged proteins were detected with either an anti-FLAG or anti-HA mAb as a primary antibody and an alkaline phosphatase-conjugated anti–mouse IgG (Promega) as a secondary antibody. For phosphorylation assay, the beads were mixed with 25 μl of the reaction mixture containing 20 mM Tris, 10 mM MgCl2, 20 μM ATP, pH 7.5 (without PKC activators). After addition of 3.7 KBq of [γ-32P]ATP (~220 TBq/ mmol), the beads were incubated at 30°C for 30 min. Samples were analyzed by SDS-PAGE (12.5%) and subsequent autoradiography.
In Vitro Phosphorylation Assay
-transferase (GST)-fused FEZ1 protein was synthesized in Escherichia coli
BL21 cells by using a pGEX6P-1 vector (Pharmacia Biotech
) and purified by a glutathione-Sepharose 4B column (Pharmacia Biotech
) according to the supplier's protocol. The phosphorylation reaction mixture (see above) (25 μl) and 50 ng of the conventional PKC isoforms (mixture of α, βI, βII, and γ) purified from the rat brain (Kikkawa et al., 1986
) were mixed with 5 μg of the purified GST-fused FEZ1 protein and the reaction was started by addition of 3.7 KBq of [γ-32
P]ATP (~220 TBq/mmol). The mixture was incubated at 30°C until incorporation of phosphate was saturated (~30 min). Samples were analyzed by SDS-PAGE (12.5%) and autoradiography, followed by measurement of radioactivities with a BAS-2000 image analyzer. Under the same conditions, ~1.8 mol of phosphate was incorporated into each mole of H1 histone, a commonly used substrate of PKC (Kikkawa et al., 1986
; Kuroda et al., 1996
Essential regions in the PKC isoforms for interaction with FEZ1 were investigated by a yeast two-hybrid assay using the following 12 PKC-deletion mutants: α-R, residues 1–336 of rat PKCα (Ono et al., 1988a
); β-R, residues 1–340 of rat PKCβI (Ono et al., 1986
); γ-R, residues 1–349 of rat PKCγ (Ono et al., 1988a
); δ-R, residues 1–345 of rat PKCδ (Ono et al., 1988b
); ε-R, residues 1–406 of rat PKCε (similarly, ε-V1, residues 1–133; ε-V1/C1, residues 1–297; and ε-C1/V3, residues 134–406) (Ono et al., 1988b
); and ζ-R, residues 1–250 of rat PKCζ (similarly, ζ-V1, residues 1–113; ζ-V1/C1, residues 1–180; and ζ-C1/V3, residues 114–250).
COS-7 cells coexpressing FEZ1-FLAG and either PKCζ-HA, K281M PKCζ-HA, or caPKCζ-HA were seeded in a 3.5-cm glass-bottom plate (MetTek Co.) at a concentration of ~5.0 × 104
cells/plate. For experiments involving the treatment with a PKC inhibitor, cells were treated with 0.1 μM staurosporin (Wako Pure Chemical Ind., Ltd.) (Tamaoki et al., 1986
) at 37°C for 2 h. Cells were fixed in 4% (wt/vol) paraformaldehyde at room temperature for 30 min, and then permeabilized and blocked in the mixture of 0.25% (vol/vol) Triton X-100, 5% (vol/vol) normal goat serum, and 5% (wt/vol) skim milk at room temperature for 30 min. The cells were incubated at room temperature for 2 h with 1 μg/ml of either an anti-FLAG or anti-HA mAb in PBS containing 0.03% (vol/vol) Triton X-100. Subsequently, the cells were incubated at room temperature for 30 min with 1 μg/ml of an FITC-conjugated anti–mouse IgG (Amersham International plc
.). The fluorescence was visualized under a Zeiss
LSM410 confocal laser scanning microscope (Carl Zeiss, Inc.
). Parental COS-7 cells showed no fluorescence with either an anti-FLAG or anti-HA mAb.
Transient Expression Assay for Neuronal Differentiation of PC12 Cells
PC12 cells (~5.0 × 105
cells) were seeded in a 10-cm plate and cultured for 24 h in DME supplemented with 10% (vol/vol) horse serum (GIBCO BRL
) and 5% (vol/vol) FCS. Cells were transfected with 9 μg of pTB701-FLAG-FEZ1, 3 μg of a pTB701-HA-PKCζ derivative, and 1 μg of a reporter plasmid, pRc-CMV-β-Gal (Higuchi et al., 1997
) by the liposome method (SuperFect; QIAGEN GmbH). After 72 h, cells were washed with PBS and fixed with 1% (vol/vol) glutaraldehyde at 4°C for 5 min, followed by washing twice with PBS containing 5 mM MgCl2
. Cells were stained by incubation at 37°C for 3 h in PBS containing 20 mM K3
, 20 mM K4
, 1 mM MgCl2
, and 1 mg/ml X-Gal. The β-Gal–positive blue cells were scored by phase-contrast microscopy. Morphologically altered cells were judged from neurite outgrowth with a flattened shape and increased body mass, as typified previously (Higuchi et al., 1997