PtK1, NIH3T3, HeLa, and HeLa S3 cells were obtained from the American Type Culture Collection (Rockville, MD). PtK1 and HeLa S3 cells were grown as previously described (Renzi et al., 1997
). In some experiments, the mitotic yield of PtK1 cells was increased by a 3–5-h treatment with 5 μg/ml of vinblastine (Sigma Chemical Co.
, St. Louis, MO). NIH3T3 cells and CCL39 hamster fibroblast were maintained at 37°C and 7.5% CO2
in DME containing 10% FCS. HeLa cells were grown at 37°C and 5% CO2
in Iscove's media containing 10% FCS.
To induce quiescence, NIH3T3 and CCL39 cells were grown in 10% FCS DME to 50% confluency, after which they were starved in DME containing 0.2% FCS for 25 h. For stimulation, 20% FCS was added for 15 or 5 min, respectively. PtK1 cells were grown to 80% confluency in 10% FCS MEM and then, without serum deprivation, were stimulated for 15 min with EGF (0.2 μg/ml).
For the CENP-E and MAP kinase coimmunoprecipitation experiments, HeLa cells were grown to 60% confluency. The cells were arrested at mitosis by a 15–17-h treatment with 0.03 μg/ml of nocodazole (Sigma Chemical Co.).
The rabbit phospho-MAP kinase antibody was raised against the phosphopeptide CHTGFLpTEpYVATR. The peptide was coupled to keyhole limpet hemocyanin (KLH) and injected into rabbits to raise polyclonal antisera (Quality Controlled Biochemicals, Inc., Hopkinton, MA). The polyclonal antibodies were negatively selected by repeated passage over a column of nonphosphorylated CHTGFLTEYVATR peptide coupled to KLH, and then positively selected by passage over a column of the phosphopeptide coupled to BSA. A concentration of 8.5 μg/ml was used for immunofluorescence and 1.7 μg/ml for Western blotting.
For ERK2-specific recognition by Western blotting, the monoclonal ERK2 antibody 05-157 (Upstate Biotechnology Inc., Lake Placid, NY) was used at a concentration of 0.1 μg/ml, at which it recognizes phosphorylated and unphosphorylated p42 ERK2. For hemagglutinin (HA)-tagged ERK2 immunoprecipitations, 18 μg of monoclonal anti-HA antibody 12CA5 (Berkeley Antibody Co., Richmond, CA) was used per immunoprecipitation. The human CENP-E protein was immunoprecipitated with the affinity-purified rabbit antibody 6A (9 μg/ml) that was generated against the central portion of CENP-E (amino acids 1,250–1,558), whereas Western blotting was performed with the same antibody at a concentration of 0.54 μg/ml. MAP kinase immunoprecipitations from HeLa cells were done with the polyclonal rabbit antibody TR10, made against recombinant ERK2 conjugated to KLH.
All the secondary fluorophore-conjugated antibodies used in indirect immunofluorescence were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The Texas red–conjugated goat anti–rabbit antibody and the fluorescein (FITC)-conjugated goat anti–human antibody were used at a concentration of 7.5 μg/ml. The human autoimmune CREST (calcinosis/Raynaud's phenomenon/esophageal dysmotility/sclerodactyly/telangiectasia variant of scleroderma) scleroderma serum was a gift of J.B. Rattner (University of Calgary, Alberta, Canada).
Preparation of Cell Lysates
For whole cell lysis experiments, PtK1 and NIH3T3 cells were grown to 80% confluency and then lysed in Laemmli sample buffer. The cell lysate was then sonicated, clarified by centrifugation, protein concentration determined, and then 100 μg of protein was used for SDS-PAGE.
CCL39 fibroblasts, stably transfected with HA-tagged wild type ERK2 or ERK2 phosphorylation site mutants (Her et al., 1993
), were washed twice in ice-cold PBS and then lysed in cold lysis buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% NP-40, 1 μM pepstatin, 1 μg/ml leupeptin, 1 mM PMSF, 0.2 mM Na vanadate, 2 μg/ml aprotinin, 40 mM 4-nitrophenyl phosphate-disodium salt [PNPP], 50 mM β-glycero-phosphate, 2 mM DTT). Samples were centrifuged at 14,000 rpm for 15 min in a microfuge and protein concentration was estimated by Coomassie dye labeling (Bio-Rad Laboratories, Hercules, CA) before immunoprecipitation.
For CENP-E immunoprecipitations, cells were washed twice in ice-cold PBS and lysed in cold hypotonic buffer (20 mM Hepes, pH 7.4, 2 mM EGTA, 2 mM MgCl2
plus above mentioned inhibitors; used in Fukuda et al., 1997
) and centrifuged for 20 min at 14,000 rpm.
Monoclonal anti-HA antibody 12CA5, affinity-purified rabbit anti-CENP-E 6A antibody or rabbit TR10 antibody were preabsorbed for 1 h at 4°C to protein A–agarose (Boehringer Mannheim Corp., Indianapolis, IN) before incubation with 500 μg of CCL39 cell extract for 4 h, 300 or 800 μg of HeLa cell extract for 3 h at 4°C, respectively. CENP-E immunoprecipitates were transferred to Wizard minicolumns and washed on a vacuum manifold (Promega Corp., Madison, WI). Immunoprecipitates were washed four times with lysis buffer before Laemmli sample buffer was added. The samples were resolved by SDS-PAGE and transferred to nitrocellulose for blotting.
All blotting was performed using nitrocellulose (Protran; Schleicher & Schuell, Dassel, Germany). The blocking and primary antibody steps were done at 37°C for 1 h in 5% dry milk in PBS/0.1% Tween 20. The secondary HRP-conjugated anti-mouse and anti-rabbit antibodies or protein A–HRP were incubated for 1 h at room temperature in PBS/0.1% Tween 20. Subsequently, enhanced chemiluminescence was performed using the Amersham Life Science kit (Arlington Heights, IL). Membranes were stripped with stripping buffer (62.5 mM Tris, pH 6.8, 2% SDS, 1% β-mercaptoethanol) for 10 min at 65°C before reprobing.
Cell Fixation and Immunofluorescence
NIH3T3 and PtK1 cells (see Fig. , C and D) were stimulated with FCS or EGF, rinsed once with PBS, and then fixed in 0.2% acrolein (Polyscience, Inc., Warrington, PA), 4% formaldehyde, 0.2% Triton X-100, and 2 mM Na vanadate in PBS for 20 min at room temperature. Cells were then quenched with 0.025% NaBH4, 2% glycine in PBS twice for 5 min, and then once for 10 min. Cells were then washed twice with PBS/0.05% Tween 20.
Figure 1 Specificity of phospho-MAP kinase antibody. (A) Phospho-MAP kinase immunoblot of whole cell lysate from NIH3T3 fibroblasts maintained in 10% FCS containing media (asynch.); serum deprived (0.2% FCS) for 25 h (− FCS) or serum deprived and (more ...)
PtK1 cells in Figs. , , and were first rinsed with PHEM (60 mM Pipes, 25 mM Hepes, pH 6.9, 10 mM EGTA, 4 mM MgSO4) and extracted for 5 min at room temperature with PHEM plus 1% CHAPS and 1 μM pepstatin, 1 μg/ml leupeptin, 2 μg/ml aprotinin, 50 mM β-glycero-phosphate and 0.2 mM Na vanadate. Cells were then fixed in 1% formaldehyde in PHEM for 15 min. Cells were rinsed twice with MBST (10 mM MOPS, 150 mM NaCl, 0.05% Tween 20, pH 7.4).
Figure 3 Phospho-MAP kinase localization at kinetochores of a vinblastine-treated cell. Indirect immunofluorescence was applied on this vinblastine treated, extracted, and fixed PtK1 cell. It was double labeled with phospho-MAP kinase antibody (red) and (more ...)
Figure 4 Localization of phospho-MAP kinase during different stages of mitosis. Indirect immunofluorescence was performed on 1% Chaps extracted and 1% formaldehyde fixed PtK1 epithelial cells. The cells were labeled with the phospho-MAP kinase antibody (red (more ...)
Figure 5 Inhibition of phospho-MAP kinase antibody by blocking with the phosphopeptide. Vinblastine-treated PtK1 cells were extracted and fixed, after which indirect immunofluorescence was performed. The cell in A–C was blocked with the phosphopeptide (more ...)
Cells for immunofluorescence were blocked with 20% boiled normal goat serum (NGS; Sigma Chemical Co.) in MBST whereas the primary and secondary antibodies were diluted in MBST containing 5% boiled NGS. Cells were washed with PBS/0.05% Tween 20 for acrolein-fixed cells and MBST for extracted and formaldehyde-fixed cells. All incubations were done by rocking at room temperature for 1 h. After the last wash, cells were counterstained with the DNA dye 4′,6-diamidino-2-phenylindole (DAPI; Sigma Chemical Co.) at 0.5 μg/ml and were mounted on slides with Vectashield mounting media (Vector Laboratories, Burlingame, CA) containing 10 mM MgSO4. Slides were viewed with a Leitz DMRBE microscope and pictures were taken under the same conditions with the Leitz Vario Orthomat camera (Leica Ltd., Wetzlar, Germany). Subsequently, scanned images were processed using Adobe Photoshop and Illustrator software (Adobe Systems, San Jose, CA).
For the peptide-blocking experiments, the peptides (in an excess of 10-fold mol/mol over antibodies) were preincubated with phospho-MAP kinase antibody and CREST serum for 1 h at room temperature in PBS. After the incubation, the antibodies were applied to extracted and fixed cells that were previously blocked in 20% NGS in MBST.
Chromosome Isolation and Immunofluorescence
Chromosomes were prepared from a 500-ml HeLa S3 culture containing ~5 × 105 cells/ml as previously described (Renzi et al., 1997
), except that the glycerol gradient step was omitted and the final chromosome pellet was resuspended in 30 ml of the extraction/lysis buffer.
The isolated chromosomes were pelleted onto precoated (1 mg/ml poly-l-lysine in water for 30 min) 18-mm-round coverslips (Fisher Scientific Co., Pittsburgh, PA) by centrifugation at 1,200 g for 5 min and then fixed with 1% formaldehyde in PHEM. Immunofluorescence was then performed as described above.
In Vitro Phosphorylation Assay
p42 ERK2 was expressed as a soluble nonfusion protein in Escherichia coli
and purified as described (Wu et al., 1991
). It was then in vitro phosphorylated and activated by constitutively active recombinant MEK1 enzyme. The specific activity of MAP kinase was estimated to be 1.2 μmol/ min per milligram, using myelin basic protein as a substrate. The COOH-terminal portion (amino acids 2,295–2,663) of human CENP-E proteins (wild-type and mutant) were made from bacteria as a glutathione-S-transferase (GST) fusion proteins. The CENP-E purification and removal of GST was done as previously described (Liao et al., 1994
). The CENP-E mutant (CENP-E4A), created by site-directed mutagenesis, has alanines in the place of serines 2,567, 2,570, 2,601, and 2,616. The in vitro phosphorylation of 1.5 μg of the COOH-terminal CENP-E protein (~45 kD) by 160 ng of active MAP kinase was performed at 30°C for 15 min in a final volume of 40 μl containing 25 mM Hepes, pH 7.5, 10 mM Mg (CH3
, 1 mM dithiothreitol, and 0.02 mM [γ-32
P]ATP (~22,500 cpm/ pmol). Reactions were terminated with sample buffer, products resolved by SDS-PAGE, transferred to nitrocellulose, and then exposed to film.