Casein Kinase II Catalyzes a Mitotic Phosphorylation on Threonine 1342 of Human DNA Topoisomerase IIα, Which Is Recognized by the 3F3/2 Phosphoepitope Antibody
1998; Elsevier BV; Volume: 273; Issue: 46 Linguagem: Inglês
10.1074/jbc.273.46.30622
ISSN1083-351X
AutoresJohn R. Daum, Gary J. Gorbsky,
Tópico(s)Plant nutrient uptake and metabolism
ResumoThe 3F3/2 antibody recognizes a phosphoepitope that is implicated in the mitotic checkpoint regulating the metaphase-to-anaphase transition. Immunoprecipitation and Western blotting revealed that the 3F3/2 antibody binds to human DNA topoisomerase II α (HsTIIα) from mitotic but not interphase HeLa cells. Extracts from mitotic cells efficiently catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα expressed in bacteria. Expression and site-directed mutagenesis of various HsTIIα protein fragments mapped the 3F3/2 phosphoepitope to the region of HsTIIα containing phosphorylated threonine 1342. This threonine lies within a consensus sequence for phosphorylation by casein kinase II (CKII). CKII is present in cellular extracts and is associated with isolated mitotic chromosomes. The 3F3/2 phosphoepitope kinase present in mitotic cell extracts was able to create the epitope using GTP and was inhibited by heparin. A kinase associated with the isolated chromosomes also generated the 3F3/2 phosphoepitope on HsTIIα. Recombinant CKII catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα containing threonine 1342. These results indicate that the mitotic 3F3/2 phosphoepitope kinase activity is attributable to CKII. We suggest that the 3F3/2 phosphoepitope reflects a CKII-catalyzed phosphorylation of threonine 1342 that may regulate mitotic functions of HsTIIα. The 3F3/2 antibody recognizes a phosphoepitope that is implicated in the mitotic checkpoint regulating the metaphase-to-anaphase transition. Immunoprecipitation and Western blotting revealed that the 3F3/2 antibody binds to human DNA topoisomerase II α (HsTIIα) from mitotic but not interphase HeLa cells. Extracts from mitotic cells efficiently catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα expressed in bacteria. Expression and site-directed mutagenesis of various HsTIIα protein fragments mapped the 3F3/2 phosphoepitope to the region of HsTIIα containing phosphorylated threonine 1342. This threonine lies within a consensus sequence for phosphorylation by casein kinase II (CKII). CKII is present in cellular extracts and is associated with isolated mitotic chromosomes. The 3F3/2 phosphoepitope kinase present in mitotic cell extracts was able to create the epitope using GTP and was inhibited by heparin. A kinase associated with the isolated chromosomes also generated the 3F3/2 phosphoepitope on HsTIIα. Recombinant CKII catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα containing threonine 1342. These results indicate that the mitotic 3F3/2 phosphoepitope kinase activity is attributable to CKII. We suggest that the 3F3/2 phosphoepitope reflects a CKII-catalyzed phosphorylation of threonine 1342 that may regulate mitotic functions of HsTIIα. topoisomerase II casein kinase II human topoisomerase II adenosine 5′-O-(thiotriphosphate) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid dithiothreitol radioimmune precipitation. DNA topoisomerase II (TII)1 catalyzes the passage of one double-stranded DNA molecule through another double-stranded DNA molecule in an ATP-dependent manner by creating a transient DNA double strand break, passing the intact double strand through this break, and then ligating the break. This activity alters DNA topology during catenation/decatenation, enables knotting/unknotting, and relaxes DNA supercoils generated during DNA replication and RNA transcription (reviewed in Ref. 1Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2074) Google Scholar). 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Anticancer Res. 1992; 12: 2093-2099PubMed Google Scholar). Yeast and Drosophila possess one form of TII. Earlier studies indicated that TII protein levels remained similar throughout the cell cycle in these organisms (25Whalen A.M. McConnell M. Fisher P.A. J. Cell Biol. 1991; 112: 203-213Crossref PubMed Scopus (29) Google Scholar, 26Cardenas M.E. Laroche T. Gasser S.M. J. Cell Sci. 1990; 96: 439-450PubMed Google Scholar), but subsequent studies have shown that yeast and Drosophila TII are expressed in a cell cycle-dependent manner similar to the expression of TIIα in other eukaryotes (27Cardenas M.E. Dang Q. Glover C.V. Gasser S.M. EMBO J. 1992; 11: 1785-1796Crossref PubMed Scopus (148) Google Scholar, 28Swedlow J.R. Sedat J.W. Agard D.A. Cell. 1993; 73: 97-108Abstract Full Text PDF PubMed Scopus (148) Google Scholar). TIIα is also phosphorylated in a cell cycle-dependent manner. Phosphorylation of TIIα is maximal in G2/M phase. Burden et al. (21Burden D.A. Goldsmith L.J. Sullivan D.M. Biochem. J. 1993; 293: 297-304Crossref PubMed Scopus (45) Google Scholar) detected phosphoserine residues on Chinese hamster TIIα in all phases of the cell cycle, whereas phosphothreonine residues were detected only in M phase cells. Several protein kinases including p34cdc2, protein kinase C, and casein kinase II (CKII) have been shown to phosphorylate TIIα. In G2/M phases, p34cdc2 is thought to phosphorylate serines 1212 and 1246 of human TIIα (HsTIIα) (29Wells N.J. Hickson I.D. Eur. J. Biochem. 1995; 231: 491-497Crossref PubMed Scopus (51) Google Scholar). Protein kinase C also phosphorylates HsTIIα in G2/M phases on serine 29 (30Wells N.J. Fry A.M. Guano F. Norbury C. Hickson I.D. J. Biol. Chem. 1995; 270: 28357-28363Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Taagepera et al. (31Taagepera S. Rao P.N. Drake F.H. Gorbsky G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8407-8411Crossref PubMed Scopus (162) Google Scholar) found that mouse TIIα is the major mitotic chromosomal protein recognized by MPM-2, an antibody that binds to certain mitosis-specific phosphorylations. TII is phosphorylated by CKII in yeast, Drosophila, and human cells (reviewed in Ref. 32Larsen A.K. Skladanowski A. Bojanowski K. Prog. Cell Cycle Res. 1996; 2: 229-239Crossref PubMed Scopus (52) Google Scholar). Wells et al. (33Wells N.J. Addison C.M. Fry A.M. Ganapathi R. Hickson I.D. J. Biol. Chem. 1994; 269: 29746-29751Abstract Full Text PDF PubMed Google Scholar) reported that the major phosphorylation target sites for CKII on HsTIIα were serine 1376 and serine 1524. CKII has also been reported to phosphorylate HsTIIα threonine 1342 (34Ishida R. Iwai M. Marsh K.L. Austin C.A. Yano T. Shibata M. Nozaki N. Hara A. J. Biol. Chem. 1996; 271: 30077-30082Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In yeast, CKII differentially phosphorylates TII in a cell cycle-dependent manner. Some CKII phosphoacceptor sites are preferentially phosphorylated in mitosis, while others are preferentially phosphorylated in G1 (27Cardenas M.E. Dang Q. Glover C.V. Gasser S.M. EMBO J. 1992; 11: 1785-1796Crossref PubMed Scopus (148) Google Scholar). CKII and TII form stable complexes in which both enzymes' respective catalytic activities are maintained even after the phosphorylation of TII by CKII (35Bojanowski K. Filhol O. Cochet C. Chambaz E.M. Larsen A.K. J. Biol. Chem. 1993; 268: 22920-22926Abstract Full Text PDF PubMed Google Scholar). In addition, it has been reported that binding of CKII to HsTIIα stabilizes enzymatic activity of HsTIIα without phosphorylation (36Redwood C. Davies S.L. Wells N.J. Fry A.M. Hickson I.D. J. Biol. Chem. 1998; 273: 3635-3642Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The consequences of TII phosphorylation on the activity of the enzyme have been examined in several studies. In yeast, dephosphorylation of TII eliminates decatenation activity, which can be restored by CKII phosphorylation (37Cardenas M.E. Walter R. Hanna D. Gasser S.M. J. Cell Sci. 1993; 104: 533-543Crossref PubMed Google Scholar). CKII phosphorylation of Drosophila TII also enhances enzymatic activity 2–3-fold (38Ackerman P. Glover C.V. Osheroff N. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3164-3168Crossref PubMed Scopus (178) Google Scholar). In mammals, the correlation between TII phosphorylation and its activity is less clear. Phosphorylation of HsTIIα by protein kinase C has been reported to increase its activity (28Swedlow J.R. Sedat J.W. Agard D.A. Cell. 1993; 73: 97-108Abstract Full Text PDF PubMed Scopus (148) Google Scholar). Saijo et al. (39Saijo M. Enomoto T. Hanaoka F. Ui M. Biochemistry. 1990; 29: 583-590Crossref PubMed Scopus (54) Google Scholar) have reported that dephosphorylated murine TII from Swiss 3T3 cells is almost completely inactive and that phosphorylation by a copurifying kinase increased its activity 8.6-fold over that of the dephosphorylated TII. In contrast, Redwood et al. (36Redwood C. Davies S.L. Wells N.J. Fry A.M. Hickson I.D. J. Biol. Chem. 1998; 273: 3635-3642Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) found that phosphorylation of HsTIIα by CKII did not increase decatenation activity. Kimuraet al. (40Kimura K. Saijo M. Tanaka M. Enomoto T. J. Biol. Chem. 1996; 271: 10990-10995Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) also indicated that murine TIIα from FM3A cells was unaffected either by phosphatase treatment or by phosphorylation with CKII. Phosphorylation of TII has been linked to chemotherapeutic drug resistance; phorbol 12-myristate 13-acetate induced hyperphosphorylation of HsTII by CKII in HL-60 cells treated with the anticancer drug etoposide, reduced the formation of cleavable complexes, and decreased cytotoxicity (41Chresta C.M. Hall B.F. Francis G.E. Leukemia. 1995; 9: 1373-1381PubMed Google Scholar). The precise roles of the phosphorylations of mammalian TII and their effects upon its postulated activities have yet to be fully characterized. The monoclonal phosphoepitope antibody, 3F3/2, was originally prepared by Cyert et al. (42Cyert M.S. Scherson T. Kirschner M.W. Dev. Biol. 1988; 129: 209-216Crossref PubMed Scopus (52) Google Scholar) against Xenopus egg extracts that had been supplemented with ATPγS. They found that the antibody recognized a large variety of thiophosphorylated proteins. Subsequently, we discovered that the antibody recognizes a small number of native phosphoproteins from mitotic cells that were never exposed to ATPγS. In addition, immunofluorescence and immunoelectron microscopy revealed that the antibody bound to proteins in kinetochores and centrosomes (43Gorbsky G.J. Ricketts W.A. J. Cell Biol. 1993; 122: 1311-1321Crossref PubMed Scopus (210) Google Scholar). Li et al. demonstrated that levels of expression of the 3F3/2 kinetochore phosphoepitope were linked to tension created by kinetochore and spindle microtubule interactions (44Li X. Nicklas R.B. J. Cell Sci. 1997; 110: 537-545PubMed Google Scholar). Moreover, when the 3F3/2 antibody was microinjected into living cells, the cells arrested at metaphase (45Campbell M.S. Gorbsky G.J. J. Cell Biol. 1995; 129: 1195-1204Crossref PubMed Scopus (118) Google Scholar). These results suggested that a 3F3/2 phosphoepitope may participate in a tension-sensitive signaling pathway and is a component of the cell cycle checkpoint regulating entry into anaphase. In this study, we have found that the 3F3/2 antibody binds to HsTIIα extracted from mitotically arrested cells. We have identified the specific phosphorylation site on HsTIIα required for the generation of the 3F3/2 phosphoepitope, investigated the cell cycle dependence of the phosphorylation, and characterized a mitotic kinase activity that can create this phosphoepitope. Tissue culture reagents including Dulbecco's modified Eagle's medium and nonessential amino acids were purchased from Life Technologies, Inc. (Gaithersburg, MD). Demecolcine, leupeptin, pepstatin A, Chaps, Triton X-100, EGTA, Trizma (Tris base), ATP, GTP, heparin, penicillin G, streptomycin, and β-mercaptoethanol were purchased from Sigma. DNase and Pefabloc SC were purchased from Boehringer Mannheim. Coomassie Protein Reagent Assay and protein A trisacryl beads were purchased from Pierce. Immobilon-P Western blotting polyvinylidene fluoride membrane was purchased from Millipore (Bedford, MA). The Renaissance Chemiluminescence Kit was purchased from NEN Life Science Products. Oligonucleotide primers were synthesized by NBI (Plymouth, MN). The Pet30 System and S-TagTM detection kit were purchased from Novagen (Madison, WI). The QuickChangeTM site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA). Purified human recombinant casein kinase II was purchased from New England Biolabs (Beverly, MA). The antiphosphoepitope antibody 3F3/2 was prepared as an ascites fluid in the Lymphocyte Culture Center at the University of Virginia. Anti-topoisomerase II α mouse monoclonal antibodies SWT3D1 and SWR1C2, herein designated T3D1 and R1C2, respectively, were originally produced by Robinson et al.(46Robinson R.G. Rapp L. Bowdish K.J. Graham M.A. Huff A.C. Coughlin S.A. Hybridoma. 1993; 12: 407-415Crossref PubMed Scopus (9) Google Scholar) at Sterling Winthrop Pharmaceuticals (Collegeville, PA). T3D1 and R1C2 used in these experiments were generously provided by R. G. Robinson and Sterling Winthrop Pharmaceuticals as hybridoma cell supernatants. Rabbit anti-human casein kinase 2 α subunit (catalog no. 06-180) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Peroxidase conjugated goat anti-mouse (catalog no. 115-035-146) and goat anti-rabbit (catalog no. 111-035-144) IgG antibodies were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). The 16-amino acid peptide, FSDFDEKTDDEDFVPC, and the threonine-phosphorylated version were synthesized, purified, and characterized by mass spectroscopy by QCB (Hopkinton, MA). The first 15 residues of this peptide correspond to amino acids 1335–1349 of HsTIIα with the phosphorylated threonine corresponding to residue 1342. HeLa S3 cells (ATCC CCL 2.2) were grown in 1-liter spinner flasks in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) bovine calf serum, 20 mm Hepes, pH 7.2, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 0.05% (w/v) pluronic F68, 60 μg/ml penicillin G, and 100 μg/ml streptomycin sulfate. Cells defined as cycling cells were harvested from logarithmically growing cultures. Cells were arrested in S phase by the addition of 1 μg/ml aphidicolin and subsequent culture for 18 h. To arrest cells in M phase, cells were incubated for 18 h in the presence of 0.15 μg/ml demecolcine. The mitotic index, the percentage of cells containing condensed chromosomes, was determined for cycling, S phase, and M phase cells by propidium iodide fluorescent staining analysis. The mitotic index in cycling cells ranged from 4 to 10%, S phase cells from 0 to 1%, and M phase cells from 70 to 95%. Harvested cells were washed twice in 4 °C phosphate-buffered saline by centrifugation prior to extraction and use in various assays. Cell pellets not immediately used in assays were frozen in liquid nitrogen and stored at −70 °C. HeLa S3 chromosomes were isolated from mitotically arrested cultures as described previously (47Renzi L. Gersch M.S. Campbell M.S. Wu L. Osmani S.A. Gorbsky G.J. J. Cell Sci. 1997; 110: 2013-2025PubMed Google Scholar). Briefly, M phase-arrested cells were centrifuged, rinsed twice in swelling buffer (10 mmHepes, pH 7.4, 40 mm KCl, 5 mm EGTA, 4 mm MgSO4, protease inhibitor mixture), and lysed by vigorous pipetting in 4 °C extraction buffer (60 mm Pipes, 25 mm Hepes, pH 6.9, 10 mm EGTA, 4 mm MgSO4, 1 mm DTT, protease inhibitor mixture, and either 1% CHAPS or 0.5% Triton X-100). When indicated, the extraction buffer also contained 200 nm microcystin-LR, a serine-threonine phosphatase inhibitor. Protease inhibitor mixture additions result in final concentrations of 5 μg/ml pepstatin A, 5 μg/ml leupeptin, and 5 μg/ml Pefabloc SC. Centrifugation at 200 × g for 5 min at 4 °C removed nonmitotic nuclei and other cellular debris from the suspended chromosomes. The exclusion of nuclei was confirmed by propidium iodide fluorescent staining analysis. Chromosomes were pelleted by centrifugation at 1600 × g for 10 min at 4 °C and washed three times in extraction buffer by repeated centrifugation. Nuclei were isolated from cycling cell cultures (described above) using identical buffers and techniques as used during the isolation of chromosomes with the exception that the nuclei were pelleted from cellular debris and extract by centrifugation at 64 × g for 5 min at 4 °C. The nuclei preparations contained greater than 99% nonmitotic nuclei as determined by propidium iodide fluorescent staining analysis. Chromosomes or nuclei not immediately used in assays were frozen in liquid nitrogen and stored at −70 °C. Protein A trisacryl beads were washed three times by centrifugation in 50 mm Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS (RIPA). 100 μl of protein A bead slurry was then combined with 800 μl of RIPA and either 1 μl of 3F3/2 ascites or 150 μl each of T3D1 and R1C2 anti-human topoisomerase II α antibodies. The mixtures were agitated for 2 h at 4 °C. Antibody-conjugated protein A beads were washed three times in RIPA containing 200 nmmicrocystin-LR and protease inhibitor mixture. Chromosomes were treated with 0.1 units/μl DNase for 5 min in extraction buffer (described above) at 37 °C. SDS was added to achieve a concentration of 1% (w/v), and the sample was vortexed. RIPA, without any SDS but containing 200 nm microcystin-LR and protease inhibitor mixture, was then added to adjust the final SDS concentration to 0.1%. The extract was centrifuged at 15,000 × g for 15 min, and the supernatant was retained for immunoprecipitation. The chromosomal extracts were combined with the antibody-conjugated beads and gently agitated at 4 °C for 2–3 h. The immunoprecipitates were then washed three times by centrifugation with RIPA containing 200 nm microcystin-LR and protease inhibitor mixture. Supernatants were also retained for analysis. For SDS-PAGE and immunoblotting analysis, the immunoprecipitates and supernatants were treated with SDS-PAGE loading buffer and heated at 95 °C for 5 min. Chromosomes were isolated as described above in the absence of phosphatase inhibitors. The chromosomes were then suspended in extraction buffer with and without 200 nm microcystin-LR. ATP was added to 1 mm, and the chromosomes were incubated at 37 °C for 20 min. Chromosomes were then treated with 0.1 units/μl DNase for 5 min at 37 °C, mixed 1:1 (v/v) with SDS-PAGE loading buffer, and heated at 95 °C for 5 min for gel electrophoresis and immunoblotting. For SDS-PAGE analysis, whole cell extracts were prepared by dissolving cell pellets in SDS-PAGE loading buffer. Soluble cell extracts for SDS-PAGE were prepared by lysing cell pellets in 50 mm Tris-HCl, pH 7.5, 10 mm EGTA, 4 mm MgSO4 (TEM) containing 0.5% Triton X-100, 200 nm microcystin-LR, and protease inhibitor mixture. The lysate was centrifuged at 15,000 × g for 15 min at 4 °C, and the resulting supernatant was mixed 1:1 (v/v) with 2× SDS-PAGE loading buffer. Isolated chromosomes or nuclei were treated with 0.1 units/μl DNase for 5 min in extraction buffer (described above) and mixed 1:1 (v/v) with 2× SDS-PAGE loading buffer. All SDS-PAGE samples were heated at 95 °C for 5 min prior to electrophoresis. Cellular extracts used in phosphorylation assays were prepared by lysing freshly isolated cells in TEM containing 200 nmmicrocystin-LR, 1% Chaps, 1 mm DTT, and protease inhibitor mixture or by thawing frozen cells in TEM containing 200 nmmicrocystin-LR, 1 mm DTT, and protease inhibitor mixture. These mixtures were intermittently vortexed and kept on ice for 5 min. The extracts were cleared by 15,000 × g centrifugation at 4 °C for 15 min. Protein concentrations for the extracts were determined using a Coomassie protein assay. Proteins were separated by electrophoresis using 5–20% gradient or 6% SDS-polyacrylamide gels and transferred to Immobilon-P membranes. For Western analysis, the membranes were blocked for 30 min with 10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.05% Tween 20 (TBST) containing 5% bovine serum albumin and 0.02% sodium azide. For detection of proteins recognized by the 3F3/2 antibody, 3F3/2 ascites was diluted 1:5000 in TBST and incubated with the membrane for 1–2 h. For detection of human topoisomerase II α, T3D1 and R1C2 mouse monoclonal antibodies from hybridoma culture supernatants were diluted 1:1000 in TBST and incubated with the membrane for 1–2 h. For detection of CKII, rabbit anti-CKII α subunit antibody at 1 μg/ml in TBST was incubated with the membrane for 1–2 h. After washing in TBST, membranes were incubated with peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibodies diluted 1:20,000 to 1:30,000 in TBST for 1 h. Immunoblots were washed again in TBST and then developed using the Renaissance chemiluminescence system. Immunoblotting inhibition assays for the 3F3/2 antibody were carried out by incubating 3F3/2 ascites at a 1:25,000 dilution in TBST with either the phosphopeptide or the corresponding nonphosphorylated peptide (described above). The peptide concentrations ranged from 0.1 to 100 μg/ml. The 3F3/2 antibody was incubated with the peptides for 1 h at 4 °C. This solution was then used for immunoblotting as described above. A Bluescript II KS +/− plasmid containing the complete coding sequence for HsTIIα was obtained from the ATCC. This sequence was digested using appropriate restriction endonucleases, and the resulting DNA fragments were inserted into the pET30 vector (pET System, Novagen). pET30 vectors are bacterial expression vectors that encode an S-TagTM and a 6× histidine sequence on the N terminus of expressed proteins. When necessary, oligonucleotide primers containing restriction endonuclease sites were designed and used in conjunction with polymerase chain reaction methods to create sequences encoding portions of HsTIIα suitable for insertion into pET30 vectors. Mutagenesis of HsTIIα coding sequences was achieved using the QuickChangeTM site-directed mutagenesis kit. Sequence analysis of mutagenized constructs was performed by the University of Virginia Biomolecular Research Facility using dye terminator chemistry and an Applied Biosystems 377 Prism DNA sequencer. BL21(DE3)Escherichia coli were transformed with pET30 vectors containing HsTIIα constructs and induced to express HsTIIα protein fragments. Synthesized proteins were separated by SDS-PAGE (described above), transferred to Immobilon-P membranes, and detected using the S-TagTM alkaline phosphatase-conjugated detection system. In addition, these HsTIIα fragments were used as substrates for the 3F3/2 kinase assay described below. Fragments of HsTIIα protein were separated by gel electrophoresis and transferred to Immobilon-P membranes as described above. The membranes were then cut into strips. Nonspecific protein binding sites were blocked by incubating the strips with TBST containing 5% bovine serum albumin and 0.02% sodium azide for 15 min. Strips were rinsed twice in TEM and then equilibrated in TEM containing 1 mm DTT, 200 nm microcystin-LR, and protease inhibitor mixture. After equilibration, the strips were placed in either HeLa S3 cellular extracts (prepared above) or casein kinase II diluted in TEM containing 1 mm DTT, 200 nmmicrocystin-LR, and protease inhibitor mixture. When indicated, 1 mm ATP or GTP was added. Final protein concentrations of cellular extracts used in this assay ranged from 20 to 150 μg/ml. To compare kinase activity between different cellular extracts, equivalent extract protein concentrations were used. Heparin at 50 μg/ml was included in the assay to test its inhibitory effects. The reactions were incubated at room temperature or 37 °C for 3–30 min. Multiple washes in TBST terminated the reactions. The strips were then immunoblotted with the 3F3/2 antibody as described above. Whole mitotic cells, extracts from mitotic cells, and mitotic chromosomal extracts isolated in the presence of the serine/threonine phosphatase inhibitor, microcystin-LR, were immunoblotted with the 3F3/2 anti-phosphoepitope antibody (Fig. 1). In whole cell and soluble cell extract preparations the major immunoreactive proteins were found at approximately 220- and 170-kDa. Whole cell and chromosome samples shared 220-, 170-, and 18-kDa immunoreactive proteins. The 170-kDa protein was concentrated in the isolated chromosome fraction that, along with the 220- and 18-kDa proteins, also contained immunoreactive proteins of approximately 100 and 55 kDa. Because HsTIIα is a 170-kDa protein known to be concentrated in mitotic chromosomes, we sought to determine whether the 170-kDa band identified by the 3F3/2 anti-phosphoepitope antibody might be HsTIIα. Immunoprecipitations from chromosomal extracts with 3F3/2 and anti-HsTIIα antibodies were performed. Both immunoprecipitates and their supernatants were then immunoblotted with 3F3/2 and anti-HsTIIα antibodies (Fig. 2). Immunoblotting with anti-HsTIIα antibody shows that some but not all of the HsTIIα was immunoprecipitated by the 3F3/2 a
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