Negative regulation of condensin I by CK2-mediated phosphorylation
2006; Springer Nature; Volume: 25; Issue: 22 Linguagem: Inglês
10.1038/sj.emboj.7601394
ISSN1460-2075
AutoresAi Takemoto, Keiji Kimura, Junn Yanagisawa, Shigeyuki Yokoyama, Fumio Hanaoka,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle26 October 2006free access Negative regulation of condensin I by CK2-mediated phosphorylation Ai Takemoto Ai Takemoto Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Keiji Kimura Corresponding Author Keiji Kimura Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency, Wako, Saitama, Japan Search for more papers by this author Junn Yanagisawa Junn Yanagisawa Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan Search for more papers by this author Fumio Hanaoka Corresponding Author Fumio Hanaoka Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency, Wako, Saitama, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Ai Takemoto Ai Takemoto Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Keiji Kimura Corresponding Author Keiji Kimura Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency, Wako, Saitama, Japan Search for more papers by this author Junn Yanagisawa Junn Yanagisawa Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan Search for more papers by this author Shigeyuki Yokoyama Shigeyuki Yokoyama Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan Search for more papers by this author Fumio Hanaoka Corresponding Author Fumio Hanaoka Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency, Wako, Saitama, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan Search for more papers by this author Author Information Ai Takemoto1,2, Keiji Kimura 1,3,4, Junn Yanagisawa3, Shigeyuki Yokoyama2,5 and Fumio Hanaoka 1,4,6 1Cellular Physiology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan 2Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan 3Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki, Japan 4Solution Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency, Wako, Saitama, Japan 5RIKEN Genomic Sciences Center, Tsurumi, Yokohama, Japan 6Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan ‡These authors contributed equally to this work *Corresponding authors: Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tenno-dai, Tsukuba Science City, Ibaraki 305-8572, Japan. Tel.: +81 29 853 6632; Fax: +81 29 853 4605; E-mail: [email protected] School of Frontier Biosciences, Osaka University, 1-3 Yamada-Oka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 7975; Fax: +81 6 6877 9382. E-mail: [email protected] The EMBO Journal (2006)25:5339-5348https://doi.org/10.1038/sj.emboj.7601394 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis. Introduction The packaging of long DNA molecules into compact rod-like structures characteristic of mitotic chromosomes ensures faithful segregation of genomic material during mitosis and meiosis. Failure of this process may lead to aneuploidy, cancer, and cell death. Despite its importance, the molecular bases underlying dynamic changes in higher-order chromosome structure during mitosis have remained enigmatic. About a decade ago, studies using Xenopus egg extracts led to the identification of a five-subunit protein complex, 13S condensin, now referred to as condensin I, which plays a central role in the assembly and maintenance of mitotic chromosome structure (Hirano and Mitchison, 1994; Hirano et al, 1997). Condensin I is the main component of mitotic chromosomes (Saitoh et al, 1994; Hudson et al, 2003; Maeshima and Laemmli, 2003). The two core subunits of condensin I, CAP-C/SMC4 and CAP-E/SMC2, belong to the SMC (structural maintenance of chromosomes) family of adenosine triphosphatases (ATPases), which are involved in many aspects of chromosome dynamics (Jessberger, 2002; Losada and Hirano, 2005; Nasmyth and Haering, 2005; Uhlmann and Hopfner, 2006). The SMC2–SMC4 heterodimer adopts a V-shaped structure with an ATP-binding 'head' domain and Walker A and Walker B motifs at the distal ends of each arm (Melby et al, 1998; Anderson et al, 2002). It is thought that ATP binding and hydrolysis of each 'head' domain modulates engagement and disengagement of the 'head' domain, a cycle that is presumed to reconfigure DNA and chromatin structure. Purified SMC2–SMC4 heterodimers possess the ability to reanneal complementary single-stranded DNAs into double-stranded DNA in an ATP-independent manner (Sutani and Yanagida, 1997; Sakai et al, 2003). However, no ATP-dependent activities have been detected for the SMC heterodimer. The remaining three subunits (CAP-D2, -G and -H) are not related to SMC proteins and have possible roles in condensin regulation (Kimura and Hirano, 2000; Schleiffer et al, 2003). All five subunits of condensin I are highly conserved among eukaryotes ranging from yeast to the human (Hirano, 2000, 2005; Hagstrom and Meyer, 2003; Nasmyth and Haering, 2005). The purified condensin I holo-complex constrains positive superhelical tension into DNA in an ATP-hydrolysis-dependent manner, as has been shown using supercoiling and knotting assays (Kimura and Hirano, 1997; Kimura et al, 1999; Hagstrom et al, 2002). This activity is stimulated by the Cdc2-dependent phosphorylation of the non-SMC subunits during mitosis, suggesting that this process may contribute to compaction of chromatin fibers during mitosis (Kimura et al, 1998, 2001; Hagstrom et al, 2002). More recently, a second condensin complex, condensin II, which has the same two SMC subunits as condensin I but a different set of non-SMC subunits (CAP-D3, -G2 and -H2), was found in vertebrate cells (Ono et al, 2003; Yeong et al, 2003). Condensin II contributes to mitotic chromosome assembly in a manner distinct from that of condensin I (Ono et al, 2003; Hirota et al, 2004; Gerlich et al, 2006), but its biochemical activities are poorly characterized. Condensin II associates with chromatin throughout the cell cycle, whereas condensin I targets mitotic chromosomes after nuclear envelope breakdown and dissociates from chromosomes during anaphase (Hirano, 2005). However, it has been reported that a small amount of condensin I persists within the nucleus during interphase (Schmiesing et al, 2000; Cabello et al, 2001). In addition to their mitotic functions, condensins I and II have been implicated in chromatin regulation during interphase. For example, condensin is required for transcriptional repression in Saccharomyces cerevisiae and Drosophila (Bhalla et al, 2002; Dej et al, 2004; Machin et al, 2004). In S. pombe, the condensin I subunit, Cnd2, is involved in the DNA checkpoint response and the DNA excision repair pathway (Aono et al, 2002). Human condensin I interacts with the PARP–1–XRCC1 complex and plays a role in the repair of DNA single-strand breaks (Heale et al, 2006). We investigated the regulation of the human condensin I complex during interphase. We previously reported that human condensin I is phosphorylated throughout the cell cycle (Takemoto et al, 2004). However, the sites at which condensin I is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis (Takemoto et al, 2004). Now, we have found that CK2 is the main kinase that phosphorylates condensin I during interphase. CK2-mediated phosphorylation during interphase greatly reduced its supercoiling activity, in contrast to the slight stimulatory effect of mitosis-specific phosphorylation by Cdc2. In this report, we describe the effect of CK2-mediated phosphorylation of condensin I on cell-cycle regulation and discuss its biological significance. Results Supercoiling activity of condensin I is suppressed by phosphorylation during interphase We previously reported that condensin I is phosphorylated in logarithmically growing (log-phase) cells and in mitotic HeLa cells, but specific sites detected by the phospho-specific monoclonal antibody MPM-2 are phosphorylated only during mitosis (Takemoto et al, 2004). Very little is known about the effects of interphase phosphorylation on the functions of condensin I, whereas mitotic Cdc2-dependent phosphorylation stimulates the supercoiling activity of condensin I in Xenopus or human (Kimura et al, 1998, 2001) and is required for nuclear targeting of condensin I in S. pombe (Sutani et al, 1999). To characterize the roles of phosphorylation during interphase, we purified condensin I from log-phase cells (in which more than 95% cells were in interphase) and mitotic HeLa cells and assessed its phosphorylation status and the effects of phosphorylation on its biochemical activities. We first compared the phosphorylation levels of the interphase and mitotic forms of condensin I using Pro-Q Diamond phosphoprotein gel stain, which detects overall phosphorylation (Schulenberg et al, 2003). Four of the five subunits (hCAP-C, -D2, -G, and -H) were phosphorylated in both forms of condensin I, and their phosphorylation levels were indistinguishable (Figure 1A). However, two-dimensional tryptic phosphopeptide mapping (Russo et al, 1992) revealed that, for each subunit, the sites that were phosphorylated during interphase differed from those that were phosphorylated during mitosis (Figure 1B). Figure 1.Negative regulation of condensin I by interphase phosphorylation. (A) Total phosphorylation levels. Condensin I was purified from log-phase (log, lanes 1–4) and mitotic HeLa cells (M, lanes 5–8) and treated with λ-PPase (log-pp, lane 4; M-pp, lane 8). Samples were resolved by SDS–PAGE followed by staining with Pro-Q Diamond phosphoprotein gel stain, which detects overall phosphorylation (Schulenberg et al, 2003). The levels of the hCAP-G subunit were 125 ng, lanes 1 and 5; 250 ng, lanes 2 and 6; 500 ng, lanes 3, 4, 7, and 8. (B) Phosphopeptide analysis of mitotic and log-phase condensin I. Condensin I was purified from 32P-labeled log-phase cell extract (I, upper panel) or mitotic HeLa cell extract (M, lower panel). Phosphopeptide maps of the hCAP-C (C), hCAP-D2 (D2), hCAP-G (G), and hCAP-H (H) subunits are shown. (C) Supercoiling (upper panel) and DNA binding activities (lower panel) of log-phase (log, lanes 2–4) dephosphorylated log-phase (log-pp, lanes 5–7), M-phase (M, lanes 8–10), and dephosphorylated M-phase condensin I (M-pp, lanes 11–13). The supercoiling reactions, but not the DNA binding assay, were supplemented with topoisomerase I purified from HeLa cells. The molar ratios of condensin I to DNA in both reactions were 50:1 (lanes 2, 5, 8, and 11), 100:1 (lanes 3, 6, 9, and 12), or 200:1 (lanes 4, 7, 10, and 13). No protein was added to lane 1. The changes in the average linking number of the substrate DNA was measured to be +0.01 (lane 2), +0.05 (lane 3), +0.3 (lane 4), +0.6 (lane 5), +1.7 (lane 6), +4.1 (lane 7), +0.3 (lane 8), +2.3 (lane 9), +5.3 (lane 10), +0.2 (lane 11), +1.9 (lane 12), and +4.5 (lane 13). (D) Phosphorylation status and activity of condensin I following incubation in interphase extracts. Condensin I was purified from log-phase HeLa cells and treated with λ-PPase (lanes 5–10) or untreated (log, lanes 2–4). Condensin I treated with λ-PPase was incubated with buffer alone (log-pp, lanes 5–7) or with interphase Xenopus egg extract (log-pp-I ext., lanes 8–10) and repurified. The supercoiling activity of condensin I was assayed (upper panel). The molar ratios of condensin I to DNA were 100:1 (lanes 1, 4, and 7), 200:1 (lanes 2, 5, and 8), and 401:1 (lanes 3, 6, and 9). Protein was omitted from lane 1. The same condensin I samples (320 ng for hCAP-G) were subjected to SDS–PAGE and the gels were stained with Pro-Q Diamond (left lower panel) or Coomassie Brilliant Blue (right lower panel). Download figure Download PowerPoint To test the effect of interphase and mitotic phosphorylation on supercoiling activity, both forms of condensin I were dephosphorylated with lambda phosphatase (λ-PPase) (Figure 1A, lanes 4 and 8). Removal of phosphates from the interphase form of condensin I resulted in a great increase in its supercoiling activity (see Figure 1C, upper panel), whereas dephosphorylation of mitotic condensin I resulted in a slight decrease in supercoiling activity (Figure 1C, upper panel). In contrast, the DNA-binding activities of the interphase and mitotic forms of condensin I were almost unaffected by dephosphorylation (Figure 1C, lower panel). To further investigate the effects of interphase phosphorylation, the supercoiling activity and phosphorylation status of condensin I were determined after incubation of dephosphorylated condensin I with an interphase Xenopus egg extract and subsequent repurification. This treatment resulted in a decrease in supercoiling activity to a level similar to that of interphase condensin I before dephosphorylation (Figure 1D) accompanied by subunit rephosphorylation. On the other hand, supercoiling activity was increased by treatment with a mitotic egg extract (data not shown). These results suggest that supercoiling activity is greatly suppressed by interphase-specific phosphorylation. This is the first demonstration of negative regulation of condensin I. Condensin I is phosphorylated by CK2 during interphase In an attempt to identify the kinase or kinases that phosphorylate and inactivate condensin I during interphase, 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of CK2, was added to the log-phase HeLa cell culture because CK2 is abundant in eukaryotic cells, is constitutively active during the cell cycle, and has a wide spectrum of substrates (Litchfield, 2003; Meggio and Pinna, 2003). Condensin I in log-phase HeLa cells was hardly phosphorylated in the presence of TBB (Figure 2A), suggesting that CK2 is a likely candidate for phosphorylation of condensin I during interphase. Figure 2.Phosphorylation of condensin I by CK2 during interphase. (A) Reduction of phosphorylation of condensin I in the presence of CK2 inhibitor (TBB). HeLa cells were cultured with 32P-orthophosphate in the absence (lanes 1and 3) or presence (lanes 2 and 4) of 200 μM TBB. Condensin I was immunoprecipitated from these cell extracts, and the precipitated proteins were separated by SDS–PAGE and analyzed using silver staining (lanes 1 and 2) or an image analyzer (BAS-2500 Fuji Photofilm) (lanes 3 and 4). (B) Phosphorylation of condensin I by CK2 in interphase Xenopus egg extracts. λ-PPase-treated condensin I (log-pp, lanes 4–10) was incubated with buffer alone (−, lane 4), interphase Xenopus egg extract (I, lane 5), CK2-depleted interphase extract (IΔCK2, lane 6), CK2-depleted interphase extract supplemented with CK2 (IΔCK2+CK2, lane 7), mitotic extract (M, lane 8), CK2-depleted mitotic extract (MΔCK2, lane 9), or CK2-depleted mitotic extract supplemented with CK2 (MΔCK2+CK2, lane 10) and repurified. Samples and control condensin I purified from log-phase cells (I, lanes 1–3) were resolved by SDS–PAGE and the gel was stained with Pro-Q Diamond (upper panel) or Coomassie Brilliant Blue (lower panel). Download figure Download PowerPoint To confirm that CK2 phosphorylates condensin I during interphase, we used a Xenopus egg extract because it faithfully reproduces cell cycle events and contains a large stockpile of structural proteins and regulatory factors. We first examined the effect of immunodepletion of CK2 from this extract. When dephosphorylated condensin I was incubated with an interphase egg extract, it was rephosphorylated to a level similar to that of the interphase complex, but it was almost unaffected by the CK2-depleted interphase extract (Figure 2B, lane 6). When purified CK2 was added to the CK2-depleted interphase extract, condensin I was rephosphorylated (Figure 2B, lane 7). In contrast, condensin I was phosphorylated in the mitotic extract depleted of CK2, even though the phosphorylation level was reduced (Figure 2B, lane 9). These results suggest that condensin I is phosphorylated by CK2 alone or by kinases activated by CK2 in the interphase extract, but is also phosphorylated by other kinases independent of CK2 in the mitotic extract, as has been reported for Cdc2 (Kimura et al, 1998, 2001), in addition to CK2. As condensin I subunits have several consensus phosphorylation sites that are recognized by CK2, we examined whether purified CK2 could phosphorylate condensin I. Purified CK2 phosphorylated the hCAP-C, -D2, -G and -H subunits of dephosphorylated condensin I, but not the sites that were recognized by MPM-2, indicating that CK2 phosphorylates these subunits at sites other than those phosphorylated by Cdc2 (Figure 3A). Two-dimensional tryptic phosphopeptide mapping indicated that the some spots of the phosphopeptides (Figure 3B, dashed circle) of the hCAP-C, -D2, -G and -H subunits phosphorylated by CK2 resembled those labeled in log-phase cells (compare Figure 3B with Figure 1B, upper panel). In contrast, the hCAP-D2, -G and -H subunits of condensin I were phosphorylated by Cdc2, and the phosphopeptide patterns of the subunits phosphorylated by Cdc2 were similar to those labeled in mitotic cells (data not shown). Figure 3.Inactivation of condensin I by CK2. (A) Phosphorylation of condensin I by CK2 and Cdc2. The interphase form of condensin I (log) or phosphatase-treated condensin (log-pp) was incubated with buffer alone (−, lanes 1 and 4), CK2 (CK2, lanes 2 and 5), or Cdc2 (Cdc2, lanes 3 and 6). Gels were stained with Pro-Q Diamond (upper panel), immunoblotted with the MPM-2 antibody (middle panel), and stained with Coomassie Brilliant Blue (lower panel). (B) Phosphopeptide analysis of CK2-labeled condensin I. Purified dephosphorylated condensin I was labeled by CK2 in vitro. Phosphopeptide maps of the hCAP-C (C), hCAP-D2 (D2), hCAP-G (G), and hCAP-H (H) subunits are shown. The phospho-spots that migrate similar to those of log phase condensin I are shown with dashed circle. (C) Inactivation of condensin I by CK2 and activation by Cdc2. A standard supercoiling assay was performed using the same condensin fractions as in (A). The approximate molar ratios of condensin I to DNA were 250:1 (lanes 2, 4, 6, 8, 10, and 12) and 500:1 (lanes 3, 5, 7, 9, 11, and 13). No protein was added to lane 1. (D) CK2-mediated phosphorylation did not affect Cdc2-mediated phosphorylation level, but inactivated condensin I. Dephosphorylated condensin I was incubated with buffer alone (lane 1), CK2 (lane 2), CK2 and then Cdc2 (lane 3), Cdc2 (lane 4), or Cdc2 and then CK2 (lane 5). An aliquot of each sample was immunoblotted with MPM-2 (upper panel). A supercoiling assay was performed using the same samples (lower panel). The approximate molar ratios of condensin I to DNA for the supercoiling assay 546:1. Download figure Download PowerPoint Next, we tested whether the supercoiling activity of condensin I was reduced by CK2 phosphorylation. When dephosphorylated condensin I was treated with CK2, its supercoiling activity was decreased to a level similar to that of interphase condensin I before dephosphorylation (Figure 3C). In contrast, the supercoiling activity of interphase condensin I was stimulated by Cdc2 treatment, but this activity was still weaker than that of the dephosphorylated form of condensin I (Figure 3C, lane 7). When dephosphorylated condensin I was phosphorylated by Cdc2, its supercoiling activity increased further (Figure 3C, lane 8) to a level similar to that of mitotic condensin I (data not shown), which suggests that both phosphorylation by Cdc2 and dephosphorylation of CK2-specific sites are required for full activation of condensin I. Taken together, we consider that some sites are labeled by CK2 both in vivo and in vitro, and these sites are implicated in the inactivation of condensin I during interphase. To test further the role of CK2-mediated phosphorylation on the regulation of condensin I, dephosphorylated condensin I was treated by any combinations of CK2 and Cdc2 (Figure 3D). The Cdc2-mediated phosphorylation level was not reduced by CK2-mediated phosphorylation, as detected by immunoblotting using MPM-2 that recognizes phosphorylation of Cdk-consensus sequence (Figure 3D, upper). However, the stimulation of its supercoiling activity was weak when it was phosphorylated by CK2 and followed by Cdc2 (Figure 3D, lower, lanes 2 and 3). Alternatively, condensin I, which had been activated by Cdc2, was greatly inactivated by subsequent phosphorylation by CK2 (Figure 3D, lower, lanes 4 and 5). Thus, CK2-mediated phosphorylation reduces the supercoiling activity of condensin I in opposition to its stimulation by Cdc2. The similar results were obtained as to another Cdk kinase, Cdk2/CyclinA, that are active during interphase (Supplementary Figure 1). Dephosphorylation of CK2 consensus sites is insufficient for mitotic chromosome condensation We then determined whether the dephosphorylated form of condensin I could support chromosome condensation in interphase Xenopus egg extracts lacking Cdc2 activity because the supercoiling activity of dephosphorylated condensin I was almost as strong as that of the mitotic form (Figure 1C). To manipulate the phosphorylation level of condensin I in extracts, we tested several protocols for differentially immunodepleting and restoring purified condensin I. The efficiency of immunodepletion was monitored using immunoblotting (Figure 4A). We did not expect the CK2-specific sites of dephosphorylated condensin I to be phosphorylated after exposure to condensin- and CK2-depleted extracts. Figure 4.Effects of CK2 depletion on chromosome condensation and the chromosomal targeting of condensin. (A) Efficiency of immunodepletion. Mitotic extracts (M, lanes 1–3) or interphase extracts (I, lanes 4–6) were immunodepleted with control IgG (mock, lanes 1 and 4), with anti-XCAP-E and anti-XCAP-G antibodies (Δcond., lanes 2 and 5), or with a mixture of anti-XCAP-E, anti-XCAP-G, and anti-CK2β antibodies (Δcond. and ΔCK2, lanes 3 and 6). Equal volumes of each extract were analyzed by immunoblotting with the indicated antibodies. (B) Chromosomal morphology. Mitotic extracts were mock-depleted (panel 1), depleted of condensin I (panels 2–4), or depleted of condensin I and CK2 (panels 5–7). Interphase extracts were mock-depleted (panel 8), depleted of condensin I (panels 9–11), or condensin I and CK2 (panels 12–14). Log-phase condensin I (panels 3, 6, 10, and 13) or dephosphorylated condensin I (panels 4, 7, 11, and 14) was restored to the depleted extracts. Sperm DNA was incubated with these extracts for 2 h, and chromosomal morphology was observed after fixation followed by staining with Hoechst dye (bar: 10 μm). (C) Quantification of condensin I in chromatin-bound fractions. Log-phase condensin I was added to condensin I-depleted interphase extracts (lanes 1–2) or to CK2- and condensin I-depleted interphase extracts (lanes 3–4). Dephosphorylated condensin I was added to condensin I-depleted interphase extracts (lanes 5–6), CK2- and condensin I-depleted interphase extracts (lanes 7–8), condensin I-depleted mitotic extracts (lanes 9–13), or CK2- and condensin I-depleted mitotic extracts (lanes 14–18). Sperm chromatin was incubated with each extract at 22°C for 2 h, chromatin was isolated, and 50% (lanes 1, 3, 5, and 7), 100% (lanes 2, 4, 6, and 8), 1.2% (lanes 9 and 14), 2.4% (lanes 10 and 15), 4.8% (lanes 11 and 16), 9.5% (lanes 12 and 17), and 19% (lanes 13 and 18) of the bound proteins were analyzed by quantitative immunoblotting (upper panel). The amounts of the hCAP-E subunit bound to sperm chromatin were determined using the Las 1000 imaging system (Fujifilm). The amount of chromosome-bound hCAP-E in each extract relative to that in the mock-depleted mitotic extract is indicated as a percentage (lower graph). Download figure Download PowerPoint When sperm chromatin was incubated in a control mitotic extract as a positive control, it was converted into a cluster of mitotic chromosomes (Figure 4B, panel 1). Chromosome condensation was not observed in condensin-depleted and condensin- and CK2-depleted mitotic extracts (Figure 4B, panels 2 and 5). When purified condensin I from HeLa cell extracts in either the interphase or dephosphorylated forms was included with condensin-depleted mitotic extracts, chromosome condensation was restored (Figure 4B, panels 3, 4, 6, and 7). Thus, CK2 depletion does not affect chromosome condensation in mitotic extracts. On the other hand, chromosome condensation did not occur in interphase extracts under any conditions (Figure 4B, panels 8–14), even when dephosphorylated condensin I was added to a condensin- and CK2-depleted extract in which condensin I was not phosphorylated (Figure 4B, panel 14). We also examined time-course experiments using 'interphase extract that was depleted of condensin I, and added back log phase condensin I', and 'interphase extract that was depleted of CK2 and condensin, and added back dephosphorylated condensin I'; however, chromosome condensation did not occur at any time points (Supplementary Figure 2). Therefore, we examined the association of condensin I with chromatin using quantitative immunoblotting. The amount of condensin I bound to sperm chromatin in interphase extracts was <10% of that of mitotic extracts under all conditions (Figure 4C). Depletion of Cdc2 from mitotic extract also resulted in the great reduction of chromosomal binding of condensin I (Supplementary Figure 3). These results suggest that Cdc2 activity is required for most chromosomal binding activity of condensin I in Xenopus egg extract. Therefore, sperm chromatin is not converted into mitotic chromosome-like structures in interphase extracts that lack Cdc2 activity, even when the supercoiling activity is stimulated by dephosphorylation. However, when dephosphorylated condensin I was included in a CK2-depleted interphase extract, the amount of condensin I targeted to sperm chromatin was more than twice that observed when its phosphorylated interphase form was included (Figure 4C). Moreover, the supercoiling activity of dephosphorylated condensin I was more than four times stronger than that of interphase condensin I. Thus, it is tempting to speculate that CK2-dependent phosphorylation of condensin I may control chromatin structure and function during interphase by regulating the chromatin-binding and supercoiling activities of condensin I. The CK2-specific consensus sites in condensin I are dephosphorylated on mitotic chromosomes To determine if the CK2-specific consensus sites in condensin I subunits are phosphorylated in HeLa cells, we synthesized phosphopeptides containing single phosphoserine or phosphothreonine residues and prepared phospho-specific antibodies. Among them, an antibody against the hCAP-H sequence (PGLQAADphosphoS570DDEDLDD) was available for detection of phospho-epitopes. The affinity
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