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Human Chromatid Cohesin Component hRad21 Is Phosphorylated in M Phase and Associated with Metaphase Centromeres

2001; Elsevier BV; Volume: 276; Issue: 7 Linguagem: Inglês

10.1074/jbc.m007809200

1083-351X

Md. Tozammel Hoque, Fuyuki Ishikawa,

14-3-3 protein interactions

Sister chromatids duplicated in S phase are connected with each other during G2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells. Sister chromatids duplicated in S phase are connected with each other during G2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells. immunofluorescence human Rad21 polyacrylamide gel electrophoresis phosphate-buffered saline propidium iodide Accurate chromosome segregation during mitosis into two daughter cells is one of the requirements for stable genome maintenance. Until recently, the molecular mechanisms regulating chromatid cohesion had not been well understood. However, recent studies, mostly conducted in budding yeast, have outlined the process generally (reviewed in Ref.1Nasmyth K. Peters J.-M. Uhlmann F. Science. 2000; 288: 1379-1384Crossref PubMed Scopus (379) Google Scholar). Concurrent with DNA replication, the sister chromatids become connected along their entire length. Several distinct groups of proteins are involved in establishing and maintaining chromatid cohesion. The most intensively investigated protein complex, budding yeast cohesin, consists of four subunit proteins, Scc1p/Mcd1p, Scc3p, Smc1p, and Smc3p (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 4Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (500) Google Scholar), and serves as a physical glue between sister chromatids. Cohesin is phylogenetically conserved. Rad21p in fission yeast and human Rad21 (hRad21) are homologues of Scc1p/Mcd1p (5Birkenbihl R.P. Subramani S. Nucleic Acids Res. 1992; 20: 6605-6611Crossref PubMed Scopus (212) Google Scholar, 6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). Smc1p and Smc3p are found in budding yeast, Xenopus, and mammals (7Strunnikov A.V. Larionov V.L. Koshland D. J. Cell Biol. 1993; 123: 1635-1648Crossref PubMed Scopus (259) Google Scholar, 8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar, 9Schmiesing J.A. Ball J., A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (92) Google Scholar). They are members of the SMC (structuralmaintenance of chromosome) protein family that is characterized by the presence of coiled-coil domains and ATPase domains (reviewed in Ref. 10Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar). Very recently, two Scc3p homologues, SA1 and SA2, have been found in Xenopus and human cells (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). Interestingly, in Xenopus, two distinct classes of cohesin, termed x-cohesinSA1 and x-cohesinSA2, are present. These two complexes share Xenopus (X)SCC1, XSCC2, and XRAD21 and differ via containing either XSA1 or XSA2 (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). Immunodepletion of Xenopus cohesin from egg extracts led to a failure of chromatid cohesion (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). Therefore, the cohesin complex is likely to be conserved in all eukaryotes, including humans. However, the precise roles of cohesin may be different between budding yeast and other eukaryotes. In budding yeast, Scc1p/Mcd1p abruptly dissociates from chromatin at the onset of anaphase (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 4Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (500) Google Scholar). In contrast, ∼95% of Xenopus cohesion molecules (XSMC1, XSMC3, and XRAD21) dissociate from chromatin at the entry of mitosis, much earlier than the metaphase-anaphase transition (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). Similarly, in indirect immunofluorescence (IF)1 experiments, it has been found that human Smc1p and mouse Rad21 (called PW29) are mostly excluded from mitotic chromosomes (9Schmiesing J.A. Ball J., A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (92) Google Scholar, 12Darwiche N. Freeman L.A. Strunnikov A. Gene (Amst.). 1999; 233: 39-47Crossref PubMed Scopus (104) Google Scholar). Therefore, most cohesins are apparently absent on metaphase chromatids in higher eukaryotes. It is not known what molecules or conditions are responsible for the chromatid cohesion immediately before anaphase. Two models have been proposed to explain the apparent inconsistency between the timings of the cohesin-chromatin dissociation and the mitotic chromatid separation (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). The first model proposes that cohesin molecules are responsible for interphase-specific chromatid cohesion and that some yet unidentified mitosis-specific cohesion machinery is responsible for the chromatid cohesion from prophase until the onset of anaphase. The second model hypothesizes that the same cohesin complex is required for both interphase- and mitosis-specific chromatid cohesions. However, in this model, the complex dissociates from chromatin in two steps, whereby most cohesin is released from chromatin at the entry into mitosis. The remaining cohesin connects chromatids in metaphase and dissociates from chromatin at the onset of anaphase. The recent discovery of Xenopus (X)SA proteins revealed that a small population of XSA1 is associated with the metaphase chromosomes formed in Xenopus cell-free extracts, thus supporting the second model (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). In this paper, we describe the biochemical and cytological behaviors of hRad21. We aimed to better understand the roles of hRad21 particularly in metaphase. We observed a small but significant population of hRad21 associated with colcemid-induced mitotic chromosomes. These results suggest that the mitotic cohesion is mediated by cohesin, further underscoring the conserved mechanisms regulating chromatid cohesion and separation in eukaryotes. Full-length hRad21 cDNA was isolated from a HeLa cell cDNA library by reference to the published hRad21 cDNA sequence (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). The cloned cDNA was completely sequenced. The resulting sequence was identical to the published clone. The full-length hRad21 cDNA was then subcloned into pGEX5X-1 (Amersham Pharmacia Biotech). glutathione S-transferase-fused hRad21 was expressed in Escherichia coli. The recombinant protein contained in the inclusion body was denatured and purified using Prep Cell Model 491 (Bio-Rad). The purified protein was mixed with Freund's complete adjuvant and injected into rabbits to obtain the anti-hRad21 antisera. To generate the anti-C-hRad21 antibodies, an oligopeptide possessing a C-terminal amino acid sequence of hRad21 (QQAIELTQEEPYSD, amino acids 606–619) was conjugated with keyhole limpet hemocyanine and was used to immunize the rabbits. The resulting antibodies were purified by affinity chromatography. Mouse anti-α-tubulin monoclonal antibody was purchased from Sigma. Mouse anti-PCNA, anti-lamin B, and anti-cyclin B1 monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti-CENP-B antibody was a kind gift from Dr. H. Masumoto (Nagoya University). Western blotting was performed according to Ref. 13Harlow E. Lane D. Antibodies. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 471-510Google Scholar. Briefly, membranes were first pretreated for 1 h in Block-Ace solution (Dai Nippon Pharmaceuticals). Then all subsequent incubations and washes were carried out in 1× TNT buffer (20× TNT: 0.4 m Tris-HCl, 2.8 m NaCl, 1.0% Tween 20). Membranes were incubated for 1 h with primary antibodies at room temperature, followed by three washes. Then membranes were incubated for 30 min with horseradish peroxidase-conjugated anti-rabbit antibodies (Amersham Pharmacia Biotech), followed by three washes. Signals were detected using an ECL kit (Amersham Pharmacia Biotech). In vitro transcription and translation experiments were performed using a TnT Quick Coupled Transcription/Translation System (Promega). 1 μg of template plasmid DNA was used in each reaction. For autoradiography, proteins were labeled by addition of [35S]methionine (1000 Ci/mmol at 10 mCi/ml; Amersham Pharmacia Biotech) to the reaction mixture. For the Western analyses, cold methionine (0.02 mm) was used during synthesis of the protein. Cells were grown on coverslips. Two fixation protocols were used, producing essentially similar results. In the first protocol, cells were fixed with 100% methanol for 20 min at −20 °C. In the second protocol, cells were fixed with 4% paraformaldehyde at room temperature for 10 min, and then permeabilized with 0.1% Triton X-100 for 10 min. Fixed cells were pretreated with PBS containing 0.1% bovine serum albumin and 0.1% skim milk and then incubated with primary antibodies for 1.0 h at 37 °C. Cells were washed by PBS three times and incubated with secondary antibodies for 1 h at 37 °C. Coverslips were washed as above, mounted in mounting solution containing 0.25 μg/ml propidium iodide (PI) or TOTO3, and examined by laser confocal microscopy (Zeiss). HeLa cells were synchronized at early S phase by a thymidine and aphidicolin double-block protocol as described in (14Bischoff J.R. Anderson L. Zhu Y. Mossie K. Ng L. Souza B. Schryver B. Flanagan P. Clairvoyant F. Ginther C. Chan C.S. Novotny M. Slamon D.J. Plowman G.D. EMBO J. 1998; 17: 3052-3065Crossref PubMed Scopus (1111) Google Scholar). In the32P-metabolic labeling experiments, cells were incubated in a phosphate-free Dulbecco's modified Eagle's medium supplemented with [32P]orthophosphate to a final concentration of 0.5 mCi/ml for 1 h prior to harvesting. Cells were lysed by the modified RIPA buffer. Cell lysates were pretreated with normal rabbit IgG. Then the precleared cell lysates were incubated with primary antibodies, and the antigen-antibody complex was immunoprecipitated by protein A-Sepharose (Amersham Pharmacia Biotech). Obtained proteins were fractionated on 6% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The membrane was first subjected to autoradiography. Then the same membrane was analyzed for hRad21 by Western blotting using anti-yhRad21 antibodies. The signals were detected by a peroxidase immunostaining kit (Wako). 1 × 107 HeLa cells were washed with cold PBS, and collected. Cell pellets were suspended in five volumes of hypotonic buffer (10 mm HEPES, pH 7.5, 5 mm KCl, 1.5 mm MgCl2, and 1 mm dithiothreitol). Low speed cell pellets were resuspended in original cell volumes of hypotonic buffer and homogenized with a Dounce homogenizer using the tight pestle. Cells were examined with a light microscope to confirm that most cells had been disrupted. Cytoplasmic and nuclear fractions were separated by centrifugation at 1,000 × g. Cytosolic fractions were obtained from the cytoplasmic fractions following high speed centrifugation at 15,000 × g. Nuclear pellet fractions were washed twice with the hypotonic buffer and extracted by suspending the nuclei in hypotonic buffer containing either salt or detergents for 30 min on ice. Nuclear extract was separated from insoluble materials by high speed centrifugation. DNase I digestion of the crude nuclei was done according to Ref. 15Mittnacht S. Weinberg R.A. Cell. 1991; 65: 381-393Abstract Full Text PDF PubMed Scopus (271) Google Scholar. HeLa cells were synchronized at early S phase following a thymidine and aphidicolin double-block protocol. To obtain metaphase-like chromosomes, cells were released from the block and cultured for 8 h, followed by an additional culture for 3 h in the presence of 0.5 μg/ml colcemid. To obtain prometaphase-like chromosomes, cells were released from the block and cultured for 8.5 or 9 h, followed by an additional culture for 10 min in the presence of 0.5 μg/ml colcemid. The cells were harvested, treated with a hypotonic buffer (10 mm Tris, 10 mm NaCl, 5 mmMgCl2) for 15 min, and attached to a glass slides by Cytospin. The cells were fixed using cold methanol for 20 min, permeabilized with 0.1% Triton X-100 for 10 min, pretreated by blocking solution (0.1% bovine serum albumin and 0.1% skimmilk in PBS), and finally incubated with rabbit anti-hRad21 antibodies and mouse anti-CENP-B monoclonal antibodies. These antibodies were detected by Alexia Fluor 488-conjugated anti-rabbit Ig antibodies (Molecular Probes) and Cy3-conjugated anti-mouse Ig antibodies (Amersham Pharmacia Biotech), respectively. DNA was stained with TOTO3 (Molecular Probes). Images were captured by a lazer confocal microscope (Zeiss). Budding yeast Scc1p/Mcd1p is a nuclear protein that associates with chromatin from late G1 through the metaphase-anaphase transition (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). Scc1p/Mcd1p abundance is strictly regulated in a cell cycle-dependent manner. The protein is absent in early G1, accumulates in S, G2, and metaphase, and declines in anaphase (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). In addition, SCC1/MCD1 mRNA is absent in early G1 and most abundant in late G1/S (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). hRad21 mRNA is most abundant in late S through G2 (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). Therefore, we first investigated hRad21 protein levels throughout the cell cycle. Full-length human Rad21 (hRad21) cDNA was isolated from a HeLa cell cDNA library. Nucleotide sequencing of the obtained cDNA yielded a sequence of complete identity to the published one (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar), thus having an ORF that potentially encodes a protein of 631 amino acid residues with a calculated molecular mass of 72 kDa. Two different rabbit antisera were raised, one (anti-hRad21 antibody) against the glutathione S-transferase-fused recombinant full-length hRad21 protein expressed in E. coli and the other (anti-C-hRad21 antibody) against a C-terminal synthetic oligopeptide (see "Experimental Procedures"). These antibodies were purified by affinity chromatography using the respective cognate antigens. Following hRad21 cDNA transfection both antibodies recognized the recombinant hRad21 protein that was expressed in the 293T cells (human kidney cells transfected with SV40 T antigen) (Fig.1 A). Both antibodies also recognized an endogenous protein in untransfected 293T cells that showed the same mobility in SDS-PAGE with that of the overexpressed hRad21 (Fig. 1 B). These reactions were specific because preincubating the antibodies with the antigens prior to Western blotting prevented detection of hRad21 (Fig. 1, A andB). The apparent molecular mass of the hRad21 protein (about 120 kDa), as estimated using SDS-PAGE electrophoresis, was greater than that predicted from its supposed amino acid sequence. Similar observations were made of budding yeast Scc1p (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar), fission yeast rad21 protein (16Birkenbihl R. Subramani S. J. Biol. Chem. 1995; 270: 7703-7711Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and Xenopus Rad21 homologue, XRAD21 (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). To confirm that the 120-kDa band indeed represents hRad21 protein, we transcribed and translated hRad21 cDNA in vitro using a rabbit reticulocyte lysate system. Recombinant proteins were synthesized in the presence of either [35S]methionine or cold methionine and then analyzed by SDS-PAGE. Labeled proteins were detected by autoradiography, whereas unlabeled proteins were subjected to Western blotting with anti-hRad21 and anti-C-hRad21 antibodies. As shown in Fig. 1 C, the [35S]methionine-labeled hRad21 protein synthesized in vitro was detected as a 120-kDa protein band after SDS-PAGE electrophoretic migration. Because this 120-kDa protein was specifically recognized by both anti-hRad21 and anti-C-hRad21 antibodies (Fig. 1 C), we concluded that it is indeed hRad21 protein. HeLa cells were synchronized using a thymidine and aphidicolin double-block protocol (Fig. 2). The cells were harvested at intervals after release from the block and first extracted by 1% Triton X (Fractions T), followed by 0.5 mNaCl (Fractions N). The insoluble pellet fractions (Fractions P) were also examined. These fractions were analyzed using Western blotting. α-Tubulin served as the control cytoplasmic protein and was efficiently extracted to the T fractions, indicating that soluble proteins were successfully extracted with the Triton X-100 treatment (Fig. 2 B). It is known that PCNA is insoluble during S phase (17Li R. Hannon G.J. Beach D. Stillman B. Curr. Biol. 1996; 6: 189-199Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). PCNA was detected in Fractions P most strongly at 0–4 h and to a lesser level at 8 h (Fig. 2 C), indicating that most of the cells were in S phase at 0–4 h. In contrast, Cyclin B1 was most abundant at 8 and 10 h and suddenly disappeared at 12 and 14 h (Fig. 2 B). These results indicate that the cells were in G2/M phase at 8 to 10 h and exited from M phase at 12–14 h. FACscan analysis strongly supported the interpretation of the cell cycle progression inferred from the marker proteins (Fig.2 A). In these cells, the abundance of hRad21, as detected by anti-C-hRad21 antibody, remained essentially unchanged during the cell cycle. It is known that Scc1p/Mcd1p is proteolytically cleaved at the onset of anaphase (18Uhlmann F. Lottspeich F. Nasmyth K. Nature. 1999; 400: 37-42Crossref PubMed Scopus (754) Google Scholar). We reproducibly observed some more quickly migrating proteins in hRad21 Western blots. At this moment, we do not know whether these fragments were derived from hRad21 or whether they had any physiological relevance. However, we did not see an increase of these small fragments in M phase, suggesting that they were not related to the chromatid separation event. We found that hRad21 was adequately extracted in 0.5 m NaCl but not in 1% Triton X-100 at all examined stages of the cell cycle (Fig. 2 B). We also found that hRad21 was not present in Fractions P (data not shown). Furthermore, hRad21 was not extracted by DNase I (data not shown). These results indicate that hRad21 is not a soluble protein or a protein loosely binding to chromatin. hRad21 may be associated with nuclear structures at all cell cycle stages including M phase. It has been shown that fission yeast Rad21p and budding yeast Scc1p are phosphorylated from S phase to anaphase (16Birkenbihl R. Subramani S. J. Biol. Chem. 1995; 270: 7703-7711Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We next examined whether hRad21 is phosphorylated in a cell cycle-dependent manner. For this purpose, HeLa cells were synchronized following a thymidine and aphidicolin double-block protocol. The cells were harvested at intervals after being released from the block. Prior to each harvest, the cells were cultured for 1 h in the presence of [32P]orthophosphate. A parallel culture was used for determining the cell cycle by FACscan analysis. hRad21 was immunoprecipitated using anti-hRad21 antibodies from cell extracts containing approximately the same number of cells. The immunoprecipitates were separated in an SDS-PAGE gel, and the proteins were blotted onto a membrane. First, the membrane was subjected to autoradiography (Fig. 3 B). Then the same membrane was analyzed for total hRad21 levels using a Western analysis with anti-C-hRad21 antibodies (Fig. 3 C). From these experiments, it was found that hRad21 is phosphorylated most intensely at 10, 12, and 14 h after release from the block. Because the FACscan analysis revealed that these samples were derived mostly from M phase cells, these results imply that hRad21 becomes hyperphosphorylated in M phase. To test the hypothesis that hRad21 is hyperphosphorylated in M phase more vigorously, we repeated the experiment in a quantitative manner. HeLa cells were arrested at G1/S following a thymidine and aphidicolin double-block protocol. A portion of the cells were released from the block and cultured for 8 h and then arrested at the following metaphase by incubating the cells with colcemid for 4 h. These G1/S-arrested and metaphase-arrested cells, along with the control exponentially growing cells, were labeled with [32P]orthophosphate for 2 h prior to each harvest. FACscan analysis of parallel cultures indicated that the two populations of cells were indeed arrested at G1/S and metaphase, respectively (Fig.4 A). hRad21 was immunoprecipitated from the exponentially growing, G1/S-arrested and metaphase-arrested cell lysates using anti-hRad21 antibodies. Three samples containing different protein amounts (1×, 3×, and 9×; where 3× and 9× samples contained 3- and 9-fold more protein than 1× samples, respectively) were analyzed for each type of immunoprecipitate using SDS-PAGE and blotted onto a membrane. The membrane was subsequently subjected to autoradiography (Fig. 4 B), followed by Western blotting with anti-hRad21 antibodies (Fig. 4 C). When the anti-hRad21-positive signals in the Western analysis were quantified, the 3× and 9× samples showed an almost 3-fold difference in their signal intensities, whereas the 1× samples were undetectable. This apparently linear dose-response relationship observed in the Western blotting data for 3× and 9× samples indicates that the intensities reliably reflect protein abundance in this range of protein amounts. In contrast, autoradiography intensities of the 9× samples displayed more than a 3-fold intensity difference relative to the 3× samples and therefore seemed to lack a linear dose-response relationship. Nevertheless, when we calculated relative phosphorylation levels by dividing the autoradiography intensities by the Western blot intensities, we saw consistent cell cycle-specific changes in hRad21 phosphorylation levels both among the 3× samples and among the 9× samples (Fig.4 D). In both comparisons, the colcemid-arrested cells showed 3-fold increases in the specific phosphorylation levels of hRad21 compared with the G1/S-arrested cells. Therefore, we concluded that hRad21 is hyperphosphorylated in M phase. In the autoradiographs of interphase immunoprecipitates, we observed three major and several minor 32P-labeled bands additional to hRad21 (Figs. 3 B and 4 B). The apparent molecular masses of the major bands as determined from SDS-PAGE were about 150, 140, and 80 kDa (Fig. 3 B). Because these three bands were not reactive with anti-hRad21 antibodies in the Western blotting analysis, we concluded that these three bands are not hRad21. We interpret them as being hRad21-associated phosphorylated proteins and have designated them p150, p140, and p80. Interestingly, the phosphorylation levels of hRad21 and its putative associated proteins (p150, p140, and p80) in the metaphase-arrested cells were significantly higher than those found with the interphase cells (Figs.3 B and 4 B). Furthermore, additional phosphorylated proteins, with apparent molecular masses of 180, 95, and 85 kDa, were specifically found in the hRad21 immunoprecipitate derived from the metaphase-arrested cells. Again, these three proteins were interpreted as putative hRad21-associated proteins and designated p180, p95, and p85. Although we do not know the identity of these bands, the results suggest that cohesin components may be coordinately modified and/or regulated by phosphorylation during M phase. We next examined the subcellular localization of hRad21 in the asynchronous HeLa cells by IF experiments using anti-hRad21 antibodies. HeLa cells were fixed with methanol, with or without being permeabilized in 0.1% Triton X-100. The samples were stained with TOTO3 to stain the DNA and examined by IF using anti-hRad21 and anti-α-tubulin antibodies. hRad21 was detected in interphase nuclei and was not present in nucleoli (Fig. 5). When the cells were pretreated with Triton X-100 prior to fixation, most of the α-tubulin was extracted to disappearance, whereas hRad21 remained. These results confirm the biochemical results described above and indicate that hRad21 is a nuclear protein associated with nuclear structures. If hRad21 is involved in sister chromatid cohesion, as has been proposed, one would expect the subcellular localization of hRad21 to change in M phase. However, it has been reported that mostXenopus Rad21 is dissociated from prophase-like chromosomes (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). We therefore were interested in the intracellular distribution of hRad21 in metaphase cells. Two fixation methods, one employing cold methanol and the other involving paraformaldehyde (see "Experimental Procedures"), were used. We found these two protocols essentially gave rise to the same results, and the results obtained from methanol-fixed cells are shown in Fig. 6and described below. Asynchronous HeLa cells stained with DNA dye TOTO3 were examined by IF using anti-α-tubulin and anti-hRad21 antibodies (Fig. 6). Although some hRad21 seemed to be associated with chromatin during early prophase when the two centrosomes are not yet separated, a significant amount was already dissociated from chromatin (Fig. 6,row a). hRad21 was quite heterogeneously distributed in late prophase cells, with some fraction of it still apparently bound to chromatin (Fig. 6, row b). However, in metaphase and anaphase, it seemed that most hRad21 had completely dissociated from the chromatin and was associated with the spindles (