CDK phosphorylation of Xenopus laevis M18 BP 1 promotes its metaphase centromere localization
2019; Springer Nature; Volume: 38; Issue: 4 Linguagem: Inglês
10.15252/embj.2018100093
ISSN1460-2075
AutoresBradley T. French, Aaron F. Straight,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle2 January 2019free access Transparent process CDK phosphorylation of Xenopus laevis M18BP1 promotes its metaphase centromere localization Bradley T French orcid.org/0000-0001-7397-7814 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Aaron F Straight Corresponding Author [email protected] orcid.org/0000-0001-5885-7881 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Bradley T French orcid.org/0000-0001-7397-7814 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Aaron F Straight Corresponding Author [email protected] orcid.org/0000-0001-5885-7881 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Author Information Bradley T French1 and Aaron F Straight *,1 1Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA *Corresponding author. Tel: +1-650-723-2941; E-mail: [email protected] EMBO J (2019)38:e100093https://doi.org/10.15252/embj.2018100093 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Chromosome segregation requires the centromere, the site on chromosomes where kinetochores assemble in mitosis to attach chromosomes to the mitotic spindle. Centromere identity is defined epigenetically by the presence of nucleosomes containing the histone H3 variant CENP-A. New CENP-A nucleosome assembly occurs at the centromere every cell cycle during G1, but how CENP-A nucleosome assembly is spatially and temporally restricted remains poorly understood. Centromere recruitment of factors required for CENP-A assembly is mediated in part by the three-protein Mis18 complex (Mis18α, Mis18β, M18BP1). Here, we show that Xenopus M18BP1 localizes to centromeres during metaphase—prior to CENP-A assembly—by binding to CENP-C using a highly conserved SANTA domain. We find that Cdk phosphorylation of M18BP1 is necessary for M18BP1 to bind CENP-C and localize to centromeres in metaphase. Surprisingly, mutations which disrupt the metaphase M18BP1/CENP-C interaction cause defective nuclear localization of M18BP1 in interphase, resulting in defective CENP-A nucleosome assembly. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase. Synopsis Epigenetic specification of vertebrate centromeres depends on assembly of CENP-A nucleosomes during interphase. In frogs, phosphorylation-dependent targeting of the CENP-A recruitment factor M18BP1 already during mitosis is crucial for its later nuclear localization and assembly role. CDK phosphorylation on Xenopus laevis M18BP1 is required for its interaction with CENP-C in metaphase. Mutations that disrupt metaphase M18BP1 localization result in defective interphase localization and CENP-A assembly. M18BP1 exchange across the nuclear envelope is limited. Metaphase M18BP1 localization may promote its retention on chromatin during nuclear envelope assembly. Introduction During cell division, eukaryotes segregate their genomes by attaching chromosomes to the microtubules of the mitotic spindle through the kinetochore (Westhorpe & Straight, 2015). Kinetochores assemble in mitosis on a specialized region of the chromosome termed the centromere (Musacchio & Desai, 2017). Mutations that disrupt the functions of centromere and kinetochore proteins cause chromosome missegregation, genome instability, and cell death (Stoler et al, 1995; Fachinetti et al, 2013). Centromeres are distinguished by the incorporation of a histone H3 variant termed CENP-A (CENtromere Protein A) into centromeric nucleosomes (Palmer et al, 1987; Yoda et al, 2000; Van Hooser et al, 2001; Guse et al, 2011; Mendiburo et al, 2011). CENP-A nucleosomes epigenetically determine the function of centromeres, and loss of CENP-A results in defective kinetochore assembly and improper chromosome segregation (Karpen & Allshire, 1997; McKinley & Cheeseman, 2016). Thus, a central question in chromosome segregation is how cells selectively maintain CENP-A chromatin at the centromere. CENP-A nucleosomes are equally distributed to each sister chromatid during DNA replication (Jansen et al, 2007). To prevent the replication-coupled dilution of CENP-A chromatin, new CENP-A nucleosomes are assembled once per cell cycle in vertebrates during G1. New CENP-A assembly is mediated by HJURP (Holliday junction-recognizing protein), the histone chaperone that binds soluble CENP-A/H4 dimers (Dunleavy et al, 2009; Foltz et al, 2009). HJURP localization is sufficient to promote local CENP-A deposition so its localization must be restricted to centromeres (Barnhart et al, 2011; Shono et al, 2015; French et al, 2017). Centromere-specific HJURP targeting requires a CENP-A nucleosome-binding protein, CENP-C (Carroll et al, 2010; Kato et al, 2013), and a three-protein complex termed the Mis18 complex (Barnhart et al, 2011; Moree et al, 2011; Tachiwana et al, 2015; Nardi et al, 2016; French et al, 2017; Pan et al, 2017; Spiller et al, 2017). The Mis18 complex recognizes centromeric chromatin either directly by binding to CENP-A nucleosomes (Kral, 2015; French et al, 2017; Hori et al, 2017; Sandmann et al, 2017) or indirectly by binding CCAN components (Moree et al, 2011; Dambacher et al, 2012; Shono et al, 2015; Stellfox Madison et al, 2016). Together, CENP-C and the Mis18 complex restrict HJURP localization to the pre-existing centromere. The process of CENP-A assembly is restricted to G1 in vertebrates in part by Cdk1 activity (Jansen et al, 2007; Bernad et al, 2011; Moree et al, 2011; Silva et al, 2012). When Cdk activity is inhibited, HJURP and the Mis18 complex localize to centromeres prior to G1 resulting in CENP-A assembly during G2 or even S phase (Silva et al, 2012; Muller et al, 2014). Cdk phosphorylation of HJURP inhibits HJURP binding to Mis18β, preventing its localization (Muller et al, 2014; Wang et al, 2014; Stankovic et al, 2017). Cdk phosphorylation of M18BP1 also inhibits M18BP1 binding to Mis18α/Mis18β and prevents M18BP1 centromere localization in human cells (Silva et al, 2012; McKinley & Cheeseman, 2014; Pan et al, 2017; Spiller et al, 2017; Stankovic et al, 2017). In addition, Cdk1 phosphorylation of CENP-A has been proposed to restrict CENP-A assembly by inhibiting CENP-A association with the HJURP chaperone, though conflicting data for this model exist (Hu et al, 2011; Yu et al, 2015; Fachinetti et al, 2017). While the Mis18 complex plays a clear role in localizing HJURP to centromeres in G1, whether it has additional roles in regulating CENP-A assembly remains unclear. Notably, M18BP1/KNL2 localizes throughout the cell cycle in Xenopus (Moree et al, 2011), Caenorhabditis elegans (Maddox et al, 2007), and chicken (Perpelescu et al, 2015; Hori et al, 2017), including all stages of mitosis. Metaphase M18BP1 localization has also been reported in human (McKinley & Cheeseman, 2014). The Mis18 complex has been proposed to “prime” centromeric chromatin for CENP-A assembly, perhaps by regulating centromeric histone acetylation (Hayashi et al, 2004; Fujita et al, 2007; Kim et al, 2012; Ohzeki et al, 2012, 2016; Shang et al, 2016). However, directly tethering HJURP to chromatin is sufficient to drive CENP-A assembly even in the absence of the Mis18 complex (Barnhart et al, 2011; French et al, 2017), suggesting that the primary role of the Mis18 complex in CENP-A assembly is HJURP localization in G1. To understand how M18BP1 controls CENP-A assembly, we identified the requirements for M18BP1 localization to metaphase and interphase centromeres using a cell-free CENP-A assembly system in Xenopus laevis egg extracts. We previously demonstrated that M18BP1 localization to metaphase centromeres in Xenopus requires CENP-C (Moree et al, 2011). CENP-C binding by the Mis18 complex is conserved in human, Xenopus, and mouse (Moree et al, 2011; Dambacher et al, 2012; Stellfox Madison et al, 2016). Here, we show that the interaction between M18BP1 and CENP-C is mediated by the highly conserved SANTA domain in M18BP1. Their interaction requires M18BP1 phosphorylation at threonine 166, a Cdk site, and is therefore restricted to mitosis. Surprisingly, mutations that disrupt the interaction between M18BP1 and CENP-C not only prevent proper metaphase localization, but also interphase localization. This results in part from defective nuclear localization. We propose that M18BP1 may identify centromeric sites in metaphase for subsequent CENP-A nucleosome assembly in interphase. Results Metaphase localization of M18BP1 requires the conserved SANTA domain Xenopus laevis expresses two M18BP1 isoforms that are both capable of supporting interphase CENP-A assembly but differ in the timing of their localization to centromeres (Moree et al, 2011; Session et al, 2016). We confirmed that Myc-tagged versions of M18BP1-1 and M18BP1-2, made by in vitro translation and added to Xenopus egg extract, recapitulated this localization to sperm chromatin. Consistent with previous results, we found that M18BP1-1 localized to metaphase centromeres while M18BP1-2 was only weakly detectable (Moree et al, 2011; Fig EV1A). Click here to expand this figure. Figure EV1. Comparison of M18BP1-1 and M18BP1-2 metaphase localization Full-length M18BP1-1 and M18BP1-2 differ in their metaphase localization. Immunofluorescence images of M18BP1 isoform localization to metaphase sperm centromeres. The M18BP1 isoform is indicated to the left; immunolocalized protein is indicated above. Myc-tagged, in vitro translated M18BP1-1 or M18BP1-2 was incubated with sperm chromatin in metaphase-arrested Xenopus egg extract depleted of endogenous M18BP1. M18BP1-1 localizes robustly to metaphase centromeres whereas M18BP1-2 was only weakly detectable. This image represents the maximum amount of metaphase M18BP1-2 localization observed. Scale bar, 10 μm. Insets are magnified 3×. CENP-A nucleosome binding-deficient mutant of M18BP1-1 remains at metaphase centromeres. Quantification of immunofluorescence intensity of full-length Flag-M18BP1-1 (mutant species indicated at bottom) at metaphase centromeres normalized to WT. R774A shows 85 ± 2% of WT localization. Error bars represent SEM from two independent experiments. Significance determined by Welch's unpaired two-tailed t-test. M18BP1-2161–570 exhibits CENP-C-dependent localization to metaphase centromeres similar to M18BP1-1161–580. Representative immunofluorescence images showing M18BP1 truncation localization (indicated at left) in metaphase extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or depleted of CENP-C as indicated at left. Immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3×. M18BP1-1 images are the same as those in Fig 3E. Schematic showing M18BP1 chimeras in which the indicated domains from M18BP1-1 (dark green) were substituted into M18BP1-2 (light green) and vice versa for assessing their role in metaphase centromere localization. Quantification of metaphase centromere localization of M18BP1 chimeras in (D). M18BP1 species indicated at bottom. Graph shows mean centromere intensity normalized to M18BP1-1WT. Error bars represent SEM from three independent experiments. Significance determined by Welch's unpaired two-tailed t-test, *P < 0.05, **P < 0.005. Download figure Download PowerPoint To determine which regions of the M18BP1-1 protein were important for metaphase localization, we measured the extent of localization of Myc-tagged M18BP1-1 truncations at sperm centromeres. In metaphase extracts depleted of both M18BP1 isoforms, a truncated version of M18BP1-1 containing only the first 580 amino acids (M18BP1-11–580) was sufficient for centromeric localization. This result demonstrates that residues 581–1356 of M18BP1-1—which contain both the CENP-A nucleosome-binding domain (French et al, 2017) and the DNA-binding SANT/Myb domain (Maddox et al, 2007)—are dispensable for metaphase localization (Fig 1A–C). Consistent with this, mutation of the CENP-A nucleosome-binding domain in M18BP1-1 had little effect on metaphase centromere localization (Fig EV1B). A fragment of M18BP1-1 containing amino acids 161–580 retained 67 ± 4% of the protein at centromeres compared to the wild-type protein (Fig 1A–C); however, further truncation of this region abolished metaphase localization (Fig 1A–C). Thus, M18BP1-1161–580 is capable of localizing to metaphase centromeres. Figure 1. M18BP1-1161–580 is sufficient for centromere localization in metaphase egg extract Representative immunofluorescence images showing localization of Myc-tagged M18BP1-1 truncations schematized in (B) to metaphase sperm centromeres in M18BP1-depleted extract. Truncations analyzed are indicated on the left; immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3×. Schematic of M18BP1-1 truncations used to define the metaphase targeting domain in (A) and (C). Quantification of the average Myc intensity at centromeres from (A). Values are normalized to centromeric signal of full-length M18BP1-1. Graph shows the mean ± SEM of at least three experiments. Significance determined by Welch's unpaired two-tailed t-test, *P < 0.05, **P < 0.005. Download figure Download PowerPoint We hypothesized that amino acid differences between M18BP1-1 and M18BP1-2 within this region (161–580 in M18BP1-1; 161–570 in M18BP1-2) would account for their differential localization to metaphase centromeres and that mutagenesis of these sites might specifically prevent metaphase M18BP1-1 localization. To test this, we generated chimeric M18BP1 proteins in which regions from one isoform encompassing the CENP-C binding domain, the CENP-A nucleosome-binding domain, or the C terminus were exchanged for the corresponding regions of the other isoform (Fig EV1D). We then assayed localization of these chimeras to metaphase centromeres in M18BP1-depleted extract. While substitution of M18BP1-21–570 into M18BP1-1 partially reduced its localization to metaphase centromeres, substitution of M18BP1-11–580 into M18BP1-2 did not confer appreciable metaphase centromeres localization, suggesting that amino acid differences in this region do not strictly control metaphase localization (Fig EV1E). A truncation of M18BP1-2 comprising amino acids 161–570 was able to localize to metaphase centromeres at levels comparable to M18BP1-1161–580 and in a CENP-C-dependent manner (Fig EV1C). Thus, metaphase localization of M18BP1-2 may be inhibited by interactions with other regions of the protein outside of 161–570. We next turned our attention toward conserved features of M18BP1-1 that might mediate metaphase localization. M18BP1-1161–580 contains a highly conserved SANTA (SANT-Associated) domain (Fig 1B), named for its co-occurrence with DNA-binding SANT/Myb domains. The SANTA domain contains five highly conserved hydrophobic residues (Fig 2A; Zhang et al, 2006). We previously showed that mutation of these residues to alanine in the SANTA domain of M18BP1-2 reduced its localization to interphase centromeres by > 90% (French et al, 2017). We tested whether mutation of the SANTA domain in full-length M18BP1-1 also affected metaphase localization and found that M18BP1-1L416A, W419A, F491A, F495A, W499A (M18BP1-1SANTA) showed a 45 ± 5% reduction in metaphase localization (Fig 2C and D) indicating that the SANTA domain is required for normal levels of metaphase M18BP1 localization. Figure 2. Mutation of the conserved SANTA domain disrupts M18BP1-1 binding to CENP-C and metaphase centromere localization Alignment of SANTA domain among select eukaryotes. Red circles indicate conserved hydrophobic residues predicted to play a role in protein–protein interactions that were mutated in this study (Zhang et al, 2006). Darker shades of blue represent increased conservation of amino acids in alignment of ˜300 M18BP1 homologues. Secondary structure prediction was reported by Zhang et al (2006). Representative Western blot showing disruption of the interaction between M18BP1-1 and CENP-C by mutation of the SANTA domain by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Flag-M18BP1-1WT or Flag-M18BP1-1SANTA. The M18BP1-1 species added to each reaction, and the cell cycle state of the extract is indicated above. The immunoblotted species is indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control. Representative images showing that mutation of the SANTA domain in M18BP1-1 causes decreased localization to sperm centromeres. Metaphase extracts were depleted of endogenous M18BP1 and either mock-depleted or depleted of CENP-C. The extracts were then complemented with WT or SANTA mutant M18BP1-1. M18BP1-1 species and depletion conditions are indicated at left; immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3×. Quantification of the average Flag-M18BP1-1 intensity at centromeres from (C). Values are normalized to centromeric signal of M18BP1-1WT in mock-depleted extract. Graph shows the mean ± SEM of three experiments. Significance determined by Welch's unpaired two-tailed t-test, **P < 0.005. Download figure Download PowerPoint Xenopus M18BP1-1 directly interacts with CENP-C, and immunodepletion of CENP-C from egg extract prevents metaphase M18BP1-1 localization (Moree et al, 2011). We used co-immunoprecipitation to test whether the reduction in centromere localization of the M18BP1-1SANTA mutant was due to a disruption of the interaction between M18BP1-1 and CENP-C. We added in vitro translated M18BP1-1WT or M18BP1-1SANTA to metaphase Xenopus extracts depleted of endogenous M18BP1. While M18BP1-1WT co-precipitated CENP-C from metaphase extract, M18BP1-1SANTA precipitated little CENP-C (Fig 2B) suggesting that the SANTA mutant disrupts CENP-C binding. We predicted that if metaphase M18BP1 localization were solely mediated by interaction of the SANTA domain with CENP-C, then M18BP1-1SANTA localization would not be further reduced following CENP-C depletion. To test the role of CENP-C in localizing M18BP1, we depleted CENP-C from Xenopus egg extracts and measured the localization of M18BP1-1WT and M18BP1-1SANTA at centromeres. CENP-C depletion reduced both M18BP1-1WT and M18BP1-1SANTA localization to 8 ± 2 and 4 ± 2% of mock-depleted levels, respectively (Fig 2C and D). Given that the M18BP1-1SANTA mutation reduced but did not prevent co-immunoprecipitation of CENP-C (Fig 2B), there likely exist additional points of interaction between M18BP1 and CENP-C or unidentified factors that are depleted with CENP-C and required for M18BP1 localization. Amino acids 161–580 comprise the CENP-C binding domain of M18BP1-1 To better characterize the regions of M18BP1 required for CENP-C binding in metaphase, we purified GST-CENP-C1191–1400, the previously identified M18BP1-binding domain (Moree et al, 2011), from Escherichia coli and performed GST pulldowns with a series of M18BP1-1 truncations expressed by in vitro translation (Figs 3A–C and EV2B). M18BP1-11–413 and M18BP1-1414–749, two adjacent but non-overlapping truncations, bound to CENP-C, suggesting that portions of each contribute to the interaction between M18BP1 and CENP-C (Fig 3A–C). M18BP1-1161–580, which spans regions of M18BP1-11–413 and M18BP1-1414–749, bound CENP-C in vitro (Fig 3A–C). We found that the homologous regions in Xenopus M18BP1-2 (M18BP1-2161–570) and in human M18BP1 (HsM18BP1325–480) also bound CENP-C in vitro (Fig EV2A–D). In contrast with a previous report in mouse (Dambacher et al, 2012), we found that the SANT/Myb domain was not required for CENP-C binding (Figs 3A–C, and EV2C and D). Figure 3. M18BP1-1161–580 recapitulates metaphase-specific binding to CENP-C A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191–1400. Material bound to glutathione agarose was resolved by SDS–PAGE and visualized by autoradiography (see also Fig EV2). Schematic showing the cognate binding domains on M18BP1-1 (top) and CENP-C (bottom), indicated by the black boxes, relative to other functional domains. Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161–580, the CENP-C binding domain. Representative Western blot showing metaphase-specific binding of M18BP1-1161–580 to CENP-C by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Myc-M18BP1-1 or Myc-M18BP1-1161–580. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated above. Immunoblotted species indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control. Representative immunofluorescence images showing Myc-M18BP1-1161–580 localization at sperm centromeres in extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or immunodepleted of CENP-C (indicated at left). Cell cycle state indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets magnified 3×. Quantification of (E). Graph shows mean Myc-M18BP1-1161–580 intensity at centromeres ± SEM of two independent experiments normalized to the mock-depleted, metaphase condition. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. In vitro identification of the CENP-C binding domain in M18BP1-1, M18BP1-2, and human M18BP1 Coomassie-stained gel showing the purity of the GST-xlCENP-C1191–1400 and GST-hsCENP-C723–943 proteins used for in vitro binding assays. Reproduction of the representative GST-pulldown data from Fig 3 which includes the Coomassie stain showing equivalent recovery of GST-CENP-C1191–1400 for all truncations analyzed (left). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161–580, the CENP-C binding domain of M18BP1-1. Gel and quantification of GST-pulldown to map the CENP-C binding domain of M18BP1-2. Myc-M18BP1-2 truncations (amino acids indicated at the bottom) were translated in reticulocyte lysate in the presence of [35S]-methionine and mixed with recombinant GST-CENP-C1191–1400. Material bound to glutathione agarose was resolved by SDS–PAGE and visualized by autoradiography to assess binding of M18BP1-2 truncations. Bound material was quantified as in Fig 3B and normalized to M18BP1-2161–570, the CENP-C binding domain of M18BP1-2. Notably, M18BP1-2161–415 was not sufficient to bind CENP-C (data not shown). Error bars represent SD of two independent experiments. Gel and quantification of GST-pulldown to map the CENP-C binding domain of human M18BP1. Pulldowns were performed as in (C), except radiolabeled truncations were mixed with recombinant GST-hCENP-C723–943, the M18BP1-binding domain on human CENP-C. Data are normalized to M18BP1325–480, the CENP-C binding domain on human M18BP1. Error bars represent SD of three independent experiments. Download figure Download PowerPoint M18BP1-1 binds directly to CENP-C in metaphase, but not in interphase (Moree et al, 2011). To test whether M18BP1-1161–580 retained the cell cycle-dependent interaction with CENP-C in Xenopus egg extract, we added M18BP1-1161–580 to metaphase and interphase extract, immunoprecipitated the protein, and assayed CENP-C co-precipitation by Western blotting. Similar to full-length M18BP1-1, we found that M18BP1-1161–580 co-precipitates CENP-C only from metaphase egg extract (Fig 3D). We next assayed the localization of M18BP1-1161–580 to sperm centromeres in egg extract and found that M18BP1-1161–580 associates only with metaphase centromeres. Immunodepletion of CENP-C from these extracts prevented the localization of M18BP1-1161–580 to the centromere (Fig 3E and F). Thus, M18BP1-1161–580 binds directly to CENP-C, co-precipitates CENP-C in metaphase, and localizes to metaphase centromeres dependent on the presence of CENP-C. CDK phosphorylation of threonine 166 is required for metaphase M18BP1-1 localization and CENP-C binding During the transition from metaphase to interphase, M18BP1 switches from binding directly to CENP-C for metaphase centromere localization to binding directly to CENP-A nucleosomes for interphase centromere localization (Moree et al, 2011; French et al, 2017). We tested whether the interaction between M18BP1 and CENP-C might be regulated by a shift in mitotic kinase activity as cells exit metaphase. We immunoprecipitated either full-length M18BP1-1 or M18BP1-1161–580 from metaphase extract and treated the immunoprecipitate with λ-phosphatase. Dephosphorylation by λ-phosphatase caused dissociation of CENP-C from both full-length M18BP1-1 and M18BP1-1161–580 precipitates (Fig 4A and B). This suggests that phosphorylation is required to maintain the interaction between M18BP1 and CENP-C and that regulation of this interaction is preserved in M18BP1-1161–580. Figure 4. Identification of metaphase phosphorylation sites in M18BP1-1 by mass spectrometry A, B. Metaphase binding of M18BP1-1 to CENP-C requires phosphorylation. Either full-length Myc-M18BP1-1 (A) or Myc-M18BP1-1161–580 (B) was immunoprecipitated from metaphase extract. Half of the immunoprecipitate was treated with λ-protein phosphatase (+ LPPase) to assess whether binding to CENP-C required phosphorylation. Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control. Immunoblotted species is indicated at left. C. Cdk inhibition with flavopiridol prevents M18BP1-1161–580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161–580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, and immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3×. See Fig EV3 for demonstration of inhibitor efficacy. D. Schematic of phosphorylation sites in M18BP1-1161–580 mutated for this study. M18BP1-1 residue numbers indicated above. Positions of phosphorylation sites are indicated by orange lines and labels below. Bold indicates consensus Cdk phosphorylation sites. E. Quantification of immunofluorescence experiments examining localization of M18BP1-1161–580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1. Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition. Significance determined by Welch's unpaired two-tailed t-test, *P < 0.05, **P < 0.005. See also Fig EV4B. Download figure Download PowerPoint To identify kinases that might regulate M18BP1 localization, we analyzed M18BP1-1161–580 localization in M18BP1-depleted metaphase extract treated with inhibitors of several mitotic kinases: cyclin-dependent kinase (Cdk; flavopiridol), Polo-like kinase (Plx; BI2536), Aurora kinase (ZM-447439), and Mps1 (reversine; Fig EV3C–F). Of these, only Cdk inhibition caused loss of M18BP1-1161–580 localization (Fig 4C). Although Cdk inhibition also drives the extract into interphase, inhibitors of other mitotic kinases had no effect on M18BP1 localization suggesting that Cdk phosphorylation may directly regulate M18BP1. Click here to expand this figure. Figure EV3. Identification of phosphosites in M18BP1-1161–580 by mass spectrometry Pulldowns from metaphase or interphase extract depleted of M18BP1 with (+) or without (−) MBP-M18BP1-1161–580. (Top) Immunoblot showing that MBP-M18BP1-1161–580 specifically co-immunoprecipitates CENP-C from metaphase extract. (Bottom) Coomassie colloidal blue-stained gel showing material precipitated with α-MBP antibody-coated beads. Red boxes indicate bands that were excised and submitted for mass spectrometry. Immunoblot showing levels of MBP-M18BP1-1161–580 in M18BP1-depleted extract relative to endogenous M18BP1 levels (undepleted, left lane). Non-specific band recognized by α-M18BP1 antibody indicated by asterisk. MBP-M18BP1-1161–580 was ˜56-fold in excess of endogenous M18BP1. Representative images showing spindle morphology in cycled metaphase egg extracts treated with DMSO or with the Polo kinase (Plx) inhibitor BI2536. Spindles in BI2536-treated extract were largely monopolar or asymmetric whereas control extracts showed largely bipolar spindles, similar to the effect of Plx immunodepletion from metaphase egg extract (Budde et al, 2001). Scale bar, 10 μm. Autoradiograph showing histone H1 phosphorylation by Cdk in control DMSO-treated metaphase extracts or extracts treated with the Cdk inhibitor flavopiridol. Western blot showing reduction in histone H3 serine 10 phosphorylation by Aurora kinase in extracts treated with ZM447439. See (Gadea & Ruderman, 2005) Autoradiographs showing the change in Cdk activity (as determined by histone H1 phosphorylation) in control extracts with and without spindle assembly checkpoint activation brought about by treatment with nocodazole (top,
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