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. 2024 Jul 10;228(1):iyae108. doi: 10.1093/genetics/iyae108

Mck1-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in Saccharomyces cerevisiae

Tianyi Zhang 1,✉,#, Wei-Chun Au 2,#, Kentaro Ohkuni 3,#, Roshan L Shrestha 4, Peter Kaiser 5, Munira A Basrai 6,✉,4
Editor: J Surtees
PMCID: PMC11373516  PMID: 38984710

Abstract

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4–Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4–Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

Keywords: CENP-A, Cse4, Mck1, Cdc4, centromere

Introduction

The centromere (CEN) is a specialized region of the chromosome for the formation of kinetochore (CEN and associated proteins) which is essential for faithful chromosome segregation. Despite the divergence in DNA sequences in different species, centromeres contain an evolutionarily conserved histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, Cnp1 in Schizosaccharomyces pombe, and centromere identifier in Drosophila melanogaster) (Choy et al. 2012; Henikoff and Furuyama 2012; Biggins 2013). Mislocalization of overexpressed CENP-A or its homologs to noncentromeric regions contributes to chromosomal instability (CIN) in budding and fission yeasts, flies, and human cells (Heun et al. 2006; Au et al. 2008; Mishra et al. 2011; Lacoste et al. 2014; Athwal et al. 2015; Shrestha et al. 2017). Overexpression and mislocalization of CENP-A have been observed in many cancers and this correlates with poor prognosis and increased invasiveness (Tomonaga et al. 2003; Amato et al. 2009; Li et al. 2011; McGovern et al. 2012; Sun et al. 2016; Zhang et al. 2016). Hence, defining pathways that prevent mislocalization of CENP-A is an area of active research.

Ubiquitin-mediated protein degradation plays a critical role in regulating the cellular levels of CENP-A homologs in budding, fission yeasts, and flies, and this prevents mislocalization to noncentromeric regions (Collins, Furuyama and Biggins 2004; Moreno-Moreno et al. 2006; Moreno-Moreno et al. 2011; Au et al. 2013; Gonzalez et al. 2014). Studies with budding yeast have defined multiple E3 ubiquitin ligases such as Psh1, Sumo-targeted ubiquitin ligases (STUbLs) Slx5/Slx8, Skp-Cullin-F-box (SCF)-Rcy1, and SCF-Cdc4/Met30 for ubiquitin-mediated proteolysis of Cse4 (Hewawasam et al. 2010; Ranjitkar et al. 2010; Cheng et al. 2016, 2017; Ohkuni et al. 2016, 2018; Au et al. 2020). The latter describes an unusual heterodimeric SCF complex with the two different substrate acceptors, Cdc4 and Met30, a specific configuration required for Cse4 degradation (Au et al. 2020). Psh1-mediated proteolysis of overexpressed Cse4 has been well characterized and shown to be regulated by the facilitates chromatin transcription/transactions complex (Deyter and Biggins 2014), Casein kinase 2 (CK2; Hewawasam et al. 2014), Fpr3 (proline isomerase; Ohkuni et al. 2014), histone regulation histone chaperone complex (Ciftci-Yilmaz et al. 2018), and Dbf4-dependent kinase complex (Eisenstatt et al. 2020). Slx5-mediated Cse4 proteolysis is regulated by sumoylation of Cse4 K65 (Ohkuni et al. 2018). Removal of Psh1-mediated polyubiquitinated Cse4 from noncentromeric regions is facilitated by Cdc48Ufd1/Npl4 segregase (Ohkuni et al. 2022). We have also pursued studies to define pathways that promote mislocalization of Cse4, showing that sumoylation of Cse4 and an in vivo conformational change of Cse4 upon interaction with histone H4 contribute to mislocalization of Cse4 to noncentromeric regions (Ohkuni et al. 2020, 2024; Eisenstatt et al. 2021).

We previously defined a role for the essential E3 ligase SCF-Cdc4/Met30 in proteolysis of endogenous Cse4 to prevent mislocalization of Cse4 for chromosomal stability (Au et al. 2020). F-box subunits of SCF, such as Cdc4 or Met30, recruit substrates by interacting with degrons with specific amino acid sequence motifs. Phosphorylation of substrates for some F-box proteins such as Cdc4 is essential for the interaction between the F-box protein and the substrate. For example, phosphorylation of cell-cycle regulators, such as Sic1, Cdc6, Ash1, and Eco1, by Cdk1 is necessary for their recognition by Cdc4 (Perkins et al. 2001; Hao et al. 2007; Liu et al. 2011; Lyons et al. 2013). Subsequent studies indicated that Cdc4 prefers to bind peptides with two phosphorylated sites (Hao et al. 2007). Mck1 kinase or its metazoan homolog glycogen synthase kinase (GSK)-3β phosphorylates amino acid four residues upstream of a previously phosphorylated residue (consensus motif: S/T*-x-x-x-pS/pT; Fiol et al. 1987; Doble and Woodgett 2003; Mok et al. 2010). Phosphorylation by Mck1 increases affinity of the target substrates with Cdc4. Indeed, Mck1 phosphorylated sites have been demonstrated to be important in the degrons of several Cdc4 substrates in yeast, including Cdc6, Hsl1, Hst3, Rcn1, and Eco1 (Mizunuma et al. 2001; Kishi et al. 2007; Ikui et al. 2012; Lyons et al. 2013; Edenberg et al. 2014). Furthermore, the degrons of many substrates of vertebrate Fbw7, the Cdc4 homolog, are phosphorylated by GSK-3β (Welcker and Clurman 2008; Crusio et al. 2010; Yeh et al. 2018). Mck1 was reported to be a dosage suppressor of CDEIII CEN mutations and proposed to phosphorylate Ndc10 (Shero and Hieter 1991; Jiang et al. 1995). However, the molecular role for Mck1 in chromosome segregation has not been investigated.

In this study, we have defined a role for Mck1 in proteolysis of Cse4 to prevent its mislocalization for chromosomal stability. Our results showed that Mck1 interacts with Cse4 and the GAL-CSE4mck1Δ strain exhibits synthetic dosage lethality (SDL), defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced interaction of Cse4 with Cdc4. Studies with GAL-CSE4 strain with mutations corresponding to three potential Mck1 phosphorylation sites in Cse4 (cse4-3A: cse4 S10A S16A T166A) exhibits phenotypes similar to that for GAL-CSE4mck1Δ strain. Our results show that Mck1 and consensus Mck1 phosphorylation sties in Cse4 S10, S16, and T166 promote Cse4–Cdc4 interaction, and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and CIN.

Materials and methods

Strains, plasmids, and growth conditions

The yeast strains and plasmids used in this study are described in Supplementary Tables 1 and 2, respectively. Unless otherwise noted, the yeast strains used are derived from S. cerevisiae BY4741. Yeast strain of an indicated gene integration or deletion was created by homologous recombination (Longtine et al. 1998). Triple-hemagglutinin (HA)-Cse4 was tagged within the N-terminus at amino acids 80 (Baker and Rogers 2006; Ohkuni et al. 2016; Au et al. 2020). Triple-Myc-Cse4 was tagged to the N-terminus end.

Yeast strains were grown in rich media (YPD: 1% yeast extract, 2% bacto-peptone, and 2% glucose) or synthetic medium with glucose or raffinose and galactose (2% final concentration each) allowing selection of the indicated plasmids.

Protein stability assays

Protein stability assays were done as previously described (Au et al. 2020). For strains expressing GAL-induced genes, cultures were grown to logarithmic phase in glucose media, washed and resuspended in raffinose (2%)- and galactose (2%)-containing medium for 1.5–4 h so that initial steady-state levels of Cse4 in wild-type (WT) and mutants were similar. Cycloheximide (CHX) and glucose were then added to final concentrations of 10 µg/mL and 2%, respectively. Samples were taken at the indicated time points after CHX treatment. The protein samples were analyzed by SDS-PAGE and Western blot using anti-HA (12CA5) mouse (Roche, 11583816001) and anti-Tub2 rabbit (Basrai Laboratory) antibodies. Protein levels were quantified using Gene Tools software (version 3.8.8.0) from SynGene (Frederick, MD).

Chromosome spreads

Chromosome spreads were performed as described previously (Collins et al. 2004; Crotti and Basrai 2004). Three independent biological repeats were performed and scored in blind experiments. A more detailed description is provided in Supplementary material.

Co-immunoprecipitation experiments

For experiments with genes expressed from their native promoter, cultures were grown in selective medium with 2% glucose. For experiments with genes expressed from GAL promoter, cultures were overnight in selective medium containing 2% raffinose to logarithmic phase, diluted in the same selective medium containing 2% galactose and incubated at 25°C for 3 h. Cell lysis, beads incubation, and Western blot detection were done as described previously (Au et al. 2020). The protein samples were analyzed by SDS-PAGE and Western blot using anti-HA (12CA5) mouse (Roche, 11583816001), anti-Myc rabbit (Santa Cruz Inc, sc789), and anti-FLAG mouse (Sigma, F3165) antibodies.

Chromosome transmission fidelity (ctf) assays

Assays for the loss of the reporter chromosome (RC) were done as previously reported (Spencer et al. 1990; Basrai et al. 1996). Chromosome loss was quantified by measuring % of colonies with ≥ 50% red sectors and visible white sectors. Colonies that were completely red were not counted.

Ub pull-down assays

The levels of ubiquitinated protein were determined by Ub pull-down assay as described previously (Au et al. 2020; Ohkuni et al. 2022). A more detailed description is provided in Supplementary material.

Results

Mck1 interacts with Cse4 and regulates ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization

Mutants of SCF-Cdc4/Met30 components exhibit SDL when Cse4 is overexpressed from a galactose-inducible promoter (GAL-CSE4; Au et al. 2020). Since Mck1 kinase phosphorylates substrates for SCF-Cdc4, we examined whether mck1Δ strain also exhibits SDL phenotype with GAL-CSE4. Growth assays showed that mck1Δ strain exhibits SDL with GAL-CSE4 on galactose plates at the permissive temperature of 25 °C (Fig. 1a). The observed SDL is linked to deletion of MCK1 as the lethality was suppressed by expressing the cognate wild-type gene in the mck1Δ strain (Fig. 1a).

Fig. 1.

Fig. 1.

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Mck1 interacts with Cse4 and regulates ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization. a) SDL phenotype in mck1Δ GAL-CSE4 strain. Growth assays were performed with WT (BY4741) and mck1Δ (YMB10154) strains transformed with vector (pMB433), or GAL-HA-CSE4 (pMB1458) plated on either 2% glucose or 2% galactose medium. Complementation of mck1Δ GAL-CSE4 SDL was performed with mck1Δ strain with or without GAL-CSE4 transformed with vector (pRS425) or plasmid expressing MCK1 (pMB1872). The plates were incubated at 25 °C for 6 days and photographed. b) Mck1 regulates Cse4 proteolysis. Protein stability assays were done with extracts from WT (YMB4741) and mck1Δ (YMB10154) strains transformed with GAL-HA-CSE4 (pMB1807) grown in galactose (2%) medium for 1.5 and 4 h at 25 °C and later shift to glucose (2%) containing medium, followed by addition of CHX (10 µg/mL). Protein extracts were prepared from cells collected at the indicated time points. Western blots were probed with anti-HA (HA-Cse4) or anti-Tub2 (loading control) antibody. Cse4 protein half-lives (t1/2) are reported as mean ± SD of three independent experiments. The difference in t1/2 is statistically significant (P = 0.0022). c) Cse4 ubiquitination is reduced in mck1Δ strain. Protein extracts were prepared from WT and mck1Δ strains used in (b). WT strain expressing either vector (pMB430) or cse4 16KR (pMB1892) were used as negative controls. These strains were grown in galactose medium for 2–3.5 h to express comparable levels of Cse4. Agarose-TUBE1 (tandem ubiquitin binding entities) was used to pull-down and ubiquitination levels of Cse4 were detected by Western blot analysis with anti-HA antibody. Input samples were analyzed using anti-HA (Cse4) and anti-Tub2 antibodies. Asterisk shows nonmodified Cse4. d) Levels of polyubiquitinated Cse4 from (c) were quantified after normalization to input Cse4. Statistical significance from three biological repeats was assessed by unpaired t-test. Error bars indicate standard deviation from the mean. e) Cse4 is mislocalized to noncentromeric regions in mck1Δ strain. Chromosome spreads were prepared from WT (BY4741) and mck1Δ (YMB10154) strains transformed with GAL-HA-CSE4 (pMB1597) after transient induction in galactose (2%) medium at 25 °C for 1.5 h. Cse4 localization was determined using Cy3-conjugated secondary antibody and DNA was stained with DAPI. Centromeric Cse4 localization is restricted to 1–2 foci. Mislocalization of Cse4 results in more than three foci or increased area of Cse4 localization within the nucleus. Images were acquired with 63 × objective with the same exposure time. Error bars represent the standard deviation of three independent experiments. The total number of cells used is indicated (n) and the numbers (mislocalized/total) of three replicates are 10/83, 10/75, and 9/96 in WT and 32/104, 30/95, and 24/112 in mck1Δ. f) Mck1 interacts with Cse4 in vivo. Protein extracts from WT strain (BY4741) expressing GAL-Myc-CSE4 (pMB1878) with either vector (pMB433) or GAL-MCK1-HA-3C-ZZ (MCK1 MORF, GE Healthcare) were prepared after transient induction in 2% galactose medium for 3 h at 25 °C. Input or IP (IgG beads, 3C protease treatment, and anti-HA beads) samples were analyzed by Western blot and probed with anti-Myc and anti-HA antibodies.

GAL-CSE4-mediated SDL correlates with defects in Cse4 proteolysis which contributes to its mislocalization (Ohkuni et al. 2016; Ciftci-Yilmaz et al. 2018; Au et al. 2020; Eisenstatt et al. 2020). Hence, we examined the stability, ubiquitination, and chromosomal localization of Cse4 in a mck1Δ strain. Protein stability assays showed that Cse4, which was rapidly degraded in wild-type strain (t1/2 = 24.3 ± 2.5 min), was stabilized in the mck1Δ strain (t1/2 = 42.0 ± 3.6 min; Fig. 1b). Our results showed that consistent with defects in proteolysis of Cse4, the levels of ubiquitinated Cse4 were reduced in the mck1Δ strain (Fig. 1, c and d). We performed chromosome spreads to examine if defects in Cse4 proteolysis contribute to mislocalization of Cse4 to noncentromeric regions in mck1Δ strain. In wild-type cells, Cse4 localization is usually restricted to one or two foci coincident with DAPI signal, which correspond to kinetochore clusters (Supplementary Fig. 1a). In contrast, a higher incidence of multiple or diffused Cse4 foci within DAPI stained region were observed in mck1Δ cells (Fig. 1e and Supplementary Fig. 1a). To rule out the possibility that the mislocalization of Cse4 is due to declustering of centromeres, we examined the localization of kinetochore protein Mtw1-GFP. We previously showed that Mtw1-GFP is not declustered in mutants that show mislocalization of Cse4 (Ciftci-Yilmaz et al. 2018; Au et al. 2020; Eisenstatt et al. 2020). We determined that the localization pattern of Mtw1-GFP was restricted to one or two foci in both wild-type and mck1Δ strains (Supplementary Fig. 2). These results support a role for Mck1 in Cse4 proteolysis to prevent Cse4 mislocalization.

We next examined whether Mck1 interacts with Cse4 in vivo. Co-immunoprecipitation (Co-IP) experiments were done with strains co-expressing Myc-CSE4 with MCK1-HA. Immunoprecipitation with anti-HA antibody revealed an interaction between Mck1 and Cse4, which was not observed in control strain (Fig. 1f). Taken together, we conclude that Mck1 interacts with Cse4 in vivo and regulates ubiquitin-mediated proteolysis of Cse4 to prevent Cse4 mislocalization.

Consensus Mck1 phosphorylation sites in Cse4 (Cse4 S10, S16, and T166) regulate ubiquitin-mediated proteolysis of Cse4

Our results for Mck1-mediated Cse4 proteolysis led us to investigate the possibility that Cse4 is a potential substrate for Mck1 kinase. The consensus motif (S/T*-x-x-x-pS/pT) is frequently found in Mck1 substrates (Ikui et al. 2012; Lyons et al. 2013; Edenberg et al. 2014). We identified S10, S16, and T166 as potential phosphorylation sites in Cse4 (Fig. 2a). To test the role of these sites for Mck1-mediated proteolysis of Cse4, we generated a strain expressing GAL-cse4-3A (S10A, S16A, and T166A) and examined this strain for growth, protein stability, ubiquitination, and mislocalization phenotypes that we have shown in the mck1Δ strain (Fig. 1). Growth assays showed that GAL-cse4-3A mutant exhibits increased lethality when compared to that observed for GAL-CSE4 in a wild-type strain (Fig. 2b and Supplementary Fig. 3). Furthermore, the growth defect of GAL-cse4-3A mck1Δ strain was similar to that of GAL-CSE4mck1Δ strain, suggesting that mutation of S10, S16, and T166 phenocopies loss of Mck1 in the context of Cse4 biology (Fig. 2b and Supplementary Fig. 3). These results suggest that Cse4 S10, S16, and T166 contribute to the growth defects of the GAL-CSE4mck1Δ strain.

Fig. 2.

Fig. 2.

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cse4-3A mutant for Mck1 consensus phosphorylation sites exhibits defects in ubiquitin-mediated proteolysis. a) Cartoon showing the three potential Mck1 phosphorylation sites (S/T*-X-X-X-pS/pT): Cse4 S10, S16, and T166. cse4-3A mutant was generated by mutating S10, S16, and T166 sites to alanine. HFD, histone-fold domain. b) SDL phenotype of GAL-cse4-3A mck1Δ is similar to that of GAL-CSE4mck1Δ. Growth assays were performed with WT (BY4741) and mck1Δ (YMB10154) strains transformed with vector (pMB433), GAL-HA-CSE4 (pMB1597), or GAL-HA-cse4-3A (pMB1909) plated on either 2% glucose or 2% galactose medium. The plates were incubated at 25 °C for 4 days and photographed. c) Cse4 S10, S16, and T166 regulate its proteolysis. Protein stability assays were done using extracts from WT (BY4741) strain transformed with GAL-HA-CSE4 (pMB1807) or GAL-HA-cse4-3A (pMB1833) grown in galactose (2%) medium for 1.5 h at 25 °C and later shift to glucose (2%) containing medium, followed by addition of CHX (10 µg/mL). Protein extracts were prepared from cells collected at the indicated time points. Western blots were probed with anti-HA (HA-Cse4) or anti-Tub2 (loading control) antibody. Cse4 protein half-lives (t1/2) are reported as mean ± SD of four independent experiments. The difference in t1/2 is statistically significant (P = 0.0095). d) Cse4-3A ubiquitination is reduced. Protein extracts were prepared from same strains used in (c). WT strain expressing cse4 16KR was used as a negative control. These strains were grown in galactose medium for 2 h. Agarose-TUBE1 was used to pull-down and ubiquitination levels of Cse4 were detected by Western blot analysis with anti-HA antibody. Input samples were analyzed using anti-HA (Cse4) and anti-Tub2 antibodies. Asterisk shows nonmodified Cse4. e) Levels of polyubiquitinated Cse4 from (d) were quantified after normalization to input Cse4. Statistical significance from three biological repeats was assessed by unpaired t-test. Error bars indicate standard deviation from the mean.

We next examined the protein stability and levels of ubiquitinated Cse4-3A. Cse4 was rapidly degraded in a wild-type strain (t1/2 = 28.0 ± 5.4 min), whereas Cse4-3A protein was stabilized (t1/2 = 41.0 ± 4.4 min; Fig. 2c). Consistent with these results, Cse4-3A showed reduced ubiquitination (Fig. 2, d and e), similar to what was observed in mck1Δ strain (Fig. 1c) and described in previous reports (Ciftci-Yilmaz et al. 2018; Eisenstatt et al. 2020). Based on these results, we conclude that consensus Mck1 phosphorylation sites in Cse4 (Cse4 S10, S16, and T166) regulate ubiquitin-mediated proteolysis of Cse4.

Strain expressing GAL-cse4-3a (Cse4 S10A, S16A, and T166A) exhibits mislocalization of Cse4-3a and elevated CIN

To determine whether increased stability of Cse4-3A contributes to its mislocalization, we performed chromosome spreads. Similar to results observed in mck1Δ strain (Fig. 1e and Supplementary Fig. 1a), higher incidence of multiple or diffused Cse4 foci within DAPI stained region were observed in cse4-3A expressing cells (Fig. 3a and Supplementary Fig. 1b). To determine the physiological consequence of these mutations on chromosomal stability, we performed a colony color assay to measure the rate of loss of a nonessential RC in wild-type strain transformed with vector, GAL-CSE4, and GAL-cse4-3A. Consistent with previous studies (Au et al. 2008, 2020), GAL-CSE4 strain exhibits higher chromosome loss compared to strain with vector alone (Fig. 3b). In contrast, GAL-cse4-3A showed more than 10-fold increase in loss of RC when compared to that observed in wild-type GAL-CSE4 strain.

Fig. 3.

Fig. 3.

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Cse4-3A is mislocalized and this contributes to CIN. a) Cse4-3A is mislocalized to noncentromeric regions. Localization of Cse4 was examined by chromosome spreads as described in Fig. 1e in WT (BY4741) strain transformed with GAL-HA-CSE4 (pMB1807) or GAL-HA-cse4-3A (pMB1833). Error bars represent the standard deviation of three independent experiments. The total number of cells used is indicated (n) and the numbers (mislocalized/total) of three replicates are 13/95, 11/91, and 9/85 in CSE4 and 88/107, 67/92, and 70/106 in cse4-3A. b) GAL-cse4-3A strain exhibits CIN. Loss of RC was measured in wild-type strain (YPH1015) transformed with vector (pMB430), GAL-CSE4 (pMB1807), or GAL-cse4-3A (pMB1833). Strains with reporter RC were spread on 2% galactose plates and incubated at 25 °C. Loss of the RC was determined by counting the number of colonies that show loss of the RC in the first division with greater than or equal to≥ half sector phenotype. At least 4,000 colonies from three independent transformants were assayed and the total number of colonies counted is indicated as (n). c) CIN phenotype of cse4-3A strain is suppressed by constitutive expression of histone H3 (Δ16H3). Loss of RC was measured as described in (b) using wild-type (YPH1015) or Δ16H3 (YMB6331) strain transformed with GAL-CSE4 (pMB1597) or GAL-cse4-3A (pMB1909).

Constitutive expression of histone H3 (Δ16H3) has been shown to suppress loss of RC in cdc4-1 mutant or GAL-cse416KR strain due to reduced mislocalization of Cse4 (Au et al. 2008, 2020). Hence, we examined whether loss of RC of GAL-cse4-3A would be suppressed by Δ16H3. Our results showed that RC loss was significantly reduced in GAL-cse4-3A Δ16H3 strain (Fig. 3c). Taken together, these results show that Cse4 S10, S16, and T166 prevent mislocalization of Cse4 and CIN.

Mck1 and its three potential phosphorylation sites in (Cse4 S10, S16, and T166) facilitate the interaction of Cse4 with Cdc4 in vivo

Based on our results and the role of Mck1 in promoting the interaction of Cdc4 with its substrates, we hypothesized that Mck1 facilitates the interaction of Cse4 with Cdc4. We examined whether reduced interaction of Cdc4 with Cse4 contributes to defects in ubiquitin-mediated proteolysis of Cse4 in a mck1Δ strain. Co-IP experiments were done with wild-type and mck1Δ strain co-expressing Flag-CDC4 with HA-CSE4. Immunoprecipitation with anti-HA antibody confirmed our previous results for a positive interaction between Cse4 and Cdc4 (Fig. 4a; Au et al. 2020). Consistent with our hypothesis, we observed greatly reduced interaction between Cse4 and Cdc4 in the mck1Δ strain when compared to that observed for wild-type strain (Fig. 4a).

Fig. 4.

Fig. 4.

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Mck1 and Cse4 S10, S16, and T166 facilitate the interaction of Cse4 with Cdc4 in vivo. a) The interaction of Cse4 with Cdc4 is reduced in mck1Δ strain. Co-IP experiments were performed using whole cell extracts (WCEs) from WT (BY4741) and mck1Δ (YMB10154) strains expressing Flag-CDC4 (pMB1840) with vector (pRS426) or HA-CSE4 (pMB1831). Input and IP (anti-HA) samples were analyzed by Western blot analysis and probed with anti-Flag and anti-HA antibodies. All tagged proteins are expressed from their native promoter. Percentages of remaining interaction of Flag-Cdc4 and HA-Cse4 in mck1Δ strain from two independent experiments are measured as mean ± average deviation. b) Recued interaction of Cse4-3A with Cdc4. Co-IP experiments were done using WCE from WT strain (BY4741) expressing Flag-Cdc4 (pMB1860) with vector (pMB433), GAL-HA-CSE4 (pMB1807), or GAL-HA-cse4-3A (pMB1833) prepared after transient induction in 2% galactose medium for 3 h at 25 °C. Input or IP (anti-HA beads) samples were analyzed by Western blot and probed with anti-Myc and anti-HA antibodies. Percentage of remaining interaction of Flag-Cdc4 and HA-Cse4-3A from two independent experiments is shown as mean ± average deviation. c) Model proposing a role for Mck1-mediated proteolysis of Cse4 by the SCF-Cdc4 pathway in preventing Cse4 mislocalization and CIN.

The similar phenotypes observed for GAL-CSE4mck1Δ with GAL-cse4-3A led us to examine whether Cse4-3A will also show reduced interaction with Cdc4. Co-IP experiments were done with strains co-expressing Flag-CDC4 with either HA-CSE4 or HA-cse4-3A. Reduced interaction between Cse4 and Cdc4 was observed in GAL-cse4-3A strain when compared to wild-type Cse4 strain (Fig. 4b). Taken together, these results show that Mck1 and Cse4 S10, S16, and T166 facilitate the interaction of Cse4 with Cdc4 in vivo.

Discussion

We have identified and defined a role for Mck1 kinase in proteolysis of Cse4 and propose a model (Fig. 4c) in which Mck1 kinase facilitates the interaction of Cse4 with SCF-Cdc4 for ubiquitin-mediated Cse4 proteolysis that prevents mislocalization of Cse4 and CIN. Our results also define a role for Cse4 S10, S16, and T166 as the potential phosphorylation sites of Mck1 in ubiquitin-dependent proteolysis of Cse4 to prevent mislocalization of Cse4. We were unable to provide direct evidence that Cse4 S10, S16, and T166 are phosphorylated by Mck1 in vivo and/or in vitro due to functional redundancy of multiple kinases capable of regulating Cse4 proteolysis. Hence, we cannot rule out the possibility that specific mutation(s) in the Cse4 S10A S16A T166A mutant or defective phosphorylation of Cse4 by other kinases such as Cdc5 may contribute to the phenotypes we have reported. For example, we previously showed that Cse4 S10 and S16 are phosphorylated by Cdc5 using an in vitro kinase assay (Mishra et al. 2019). In addition, structural defects of Cse4 S10A S16A T166A mutant cannot be excluded. However, our genetic, biochemical, and cell biology approaches showed that the phenotypes of GAL-cse4-3A in a wild-type strain are similar to those observed in a GAL-CSE4mck1Δ strain. Taken together, we have shown that Mck1 and Cse4 S10, S16, and T166 residues promote Cse4–Cdc4 interaction and regulate ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and CIN. Our studies are clinically relevant given the poor prognosis of CENP-A overexpressing cancers. Hence, defining pathways that prevent mislocalization of CENP-A is an area of active research. Our studies show that mislocalization of overexpressed CENP-A and defects in histone chaperone lead to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model (Shrestha et al. 2021). Since degrons of many substrates of vertebrate Fbw7 (Cdc4 homolog) are phosphorylated by GSK-3β (Mck1 homolog), it is of interest to examine if GSK-3β prevents mislocalization of CENP-A to noncentromeric regions and how defects in this pathway may contribute to aneuploidy in human cancers.

Supplementary Material

iyae108_Supplementary_Data
iyae108_supplementary_data.pdf (727.2KB, pdf)

Acknowledgments

We are grateful to members of the Basrai Laboratory for helpful discussions and Vinutha Balachandra and Makenzie Thomas for comments on the manuscript.

Contributor Information

Tianyi Zhang, Genetics Branch, Center for Cancer Research, National Cancer Institute. National Institute of Health, Bethesda, MD 20892, USA.

Wei-Chun Au, Genetics Branch, Center for Cancer Research, National Cancer Institute. National Institute of Health, Bethesda, MD 20892, USA.

Kentaro Ohkuni, Genetics Branch, Center for Cancer Research, National Cancer Institute. National Institute of Health, Bethesda, MD 20892, USA.

Roshan L Shrestha, Genetics Branch, Center for Cancer Research, National Cancer Institute. National Institute of Health, Bethesda, MD 20892, USA.

Peter Kaiser, Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, CA 92697, USA.

Munira A Basrai, Genetics Branch, Center for Cancer Research, National Cancer Institute. National Institute of Health, Bethesda, MD 20892, USA.

Data availability

All experimental data sets are available at Figshare (https://doi.org/10.6084/m9.figshare.26049877).

Supplemental material available at GENETICS online.

Funding

This research was supported by the National Institutes of Health Intramural Research Program at the National Cancer Institute to M.A.B. and the grant (R35GM148350) from the National Institutes of Health to P.K.

Literature cited

  1. Amato A, Schillaci T, Lentini L, Di Leonardo A. 2009. CENPA overexpression promotes genome instability in pRb-depleted human cells. Mol Cancer. 8(1):119. doi: 10.1186/1476-4598-8-119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Athwal RK, Walkiewicz MP, Baek S, Fu S, Bui M, Camps J, Ried T, Sung M-H, Dalal Y. 2015. CENP-A nucleosomes localize to transcription factor hotspots and subtelomeric sites in human cancer cells. Epigenetics Chromatin. 8(1):2. doi: 10.1186/1756-8935-8-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Au WC, Crisp MJ, DeLuca SZ, Rando OJ, Basrai MA. 2008. Altered dosage and mislocalization of histone H3 and Cse4p lead to chromosome loss in Saccharomyces cerevisiae. Genetics. 179(1):263–275. doi: 10.1534/genetics.108.088518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Au WC, Dawson AR, Rawson DW, Taylor SB, Baker RE, Basrai MA. 2013. A novel role of the N terminus of budding yeast histone H3 variant Cse4 in ubiquitin-mediated proteolysis. Genetics. 194(2):513–518. doi: 10.1534/genetics.113.149898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Au WC, Zhang T, Mishra PK, Eisenstatt JR, Walker RL, Ocampo J, Dawson A, Warren J, Costanzo M, Baryshnikova A, et al. 2020. Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in budding yeast. PLoS Genet. 16(2):e1008597. doi: 10.1371/journal.pgen.1008597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baker RE, Rogers K. 2006. Phylogenetic analysis of fungal centromere H3 proteins. Genetics. 174(3):1481–1492. doi: 10.1534/genetics.106.062794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Basrai MA, Kingsbury J, Koshland D, Spencer F, Hieter P. 1996. Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae. Mol Cell Biol. 16(6):2838–2847. doi: 10.1128/MCB.16.6.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Biggins S. 2013. The composition, functions, and regulation of the budding yeast kinetochore. Genetics. 194(4):817–846. doi: 10.1534/genetics.112.145276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng H, Bao X, Gan X, Luo S, Rao H. 2017. Multiple E3s promote the degradation of histone H3 variant Cse4. Sci Rep. 7(1):8565. doi: 10.1038/s41598-017-08923-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheng H, Bao X, Rao H. 2016. The F-box protein Rcy1 is involved in the degradation of histone H3 variant Cse4 and genome maintenance. J Biol Chem. 291(19):10372–10377. doi: 10.1074/jbc.M115.701813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Choy JS, Mishra PK, Au WC, Basrai MA. 2012. Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae. Biochim Biophys Acta. 1819(7):776–783. doi: 10.1016/j.bbagrm.2012.02.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ciftci-Yilmaz S, Au WC, Mishra PK, Eisenstatt JR, Chang J, Dawson AR, Zhu I, Rahman M, Bilke S, Costanzo M, et al. 2018. A genome-wide screen reveals a role for the HIR histone chaperone complex in preventing mislocalization of budding yeast CENP-A. Genetics. 210(1):203–218. doi: 10.1534/genetics.118.301305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Collins KA, Furuyama S, Biggins S. 2004. Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol. 14(21):1968–1972. doi: 10.1016/j.cub.2004.10.024. [DOI] [PubMed] [Google Scholar]
  14. Crotti LB, Basrai MA. 2004. Functional roles for evolutionarily conserved Spt4p at centromeres and heterochromatin in Saccharomyces cerevisiae. EMBO J. 23(8):1804–1814. doi: 10.1038/sj.emboj.7600161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Crusio KM, King B, Reavie LB, Aifantis I. 2010. The ubiquitous nature of cancer: the role of the SCF(Fbw7) complex in development and transformation. Oncogene. 29(35):4865–4873. doi: 10.1038/onc.2010.222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deyter GM, Biggins S. 2014. The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A. Genes Dev. 28(16):1815–1826. doi: 10.1101/gad.243113.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Doble BW, Woodgett JR. 2003. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci. 116(7):1175–1186. doi: 10.1242/jcs.00384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Edenberg ER, Vashisht AA, Topacio BR, Wohlschlegel JA, Toczyski DP. 2014. Hst3 is turned over by a replication stress-responsive SCF(Cdc4) phospho-degron. Proc Natl Acad Sci U S A. 111(16):5962–5967. doi: 10.1073/pnas.1315325111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Eisenstatt JR, Boeckmann L, Au WC, Garcia V, Bursch L, Ocampo J, Costanzo M, Weinreich M, Sclafani RA, Baryshnikova A, et al. 2020. Dbf4-dependent kinase (DDK)-mediated proteolysis of CENP-A prevents mislocalization of CENP-A in Saccharomyces cerevisiae. G3 (Bethesda). 10(6):2057–2068. doi: 10.1534/g3.120.401131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Eisenstatt JR, Ohkuni K, Au WC, Preston O, Gliford L, Suva E, Costanzo M, Boone C, Basrai MA. 2021. Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae. Genetics. 218(1):iyab033. doi: 10.1093/genetics/iyab033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fiol CJ, Mahrenholz AM, Wang Y, Roeske RW, Roach PJ. 1987. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem. 262(29):14042–14048. doi: 10.1016/S0021-9258(18)47901-X. [DOI] [PubMed] [Google Scholar]
  22. Gonzalez M, He H, Dong Q, Sun S, Li F. 2014. Ectopic centromere nucleation by CENP-A in fission yeast. Genetics. 198(4):1433–1446. doi: 10.1534/genetics.114.171173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hao B, Oehlmann S, Sowa ME, Harper JW, Pavletich NP. 2007. Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol Cell. 26(1):131–143. doi: 10.1016/j.molcel.2007.02.022. [DOI] [PubMed] [Google Scholar]
  24. Henikoff S, Furuyama T. 2012. The unconventional structure of centromeric nucleosomes. Chromosoma. 121(4):341–352. doi: 10.1007/s00412-012-0372-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, Karpen GH. 2006. Mislocalization of the Drosophila centromere-specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell. 10(3):303–315. doi: 10.1016/j.devcel.2006.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hewawasam GS, Mattingly M, Venkatesh S, Zhang Y, Florens L, Workman JL, Gerton JL. 2014. Phosphorylation by casein kinase 2 facilitates Psh1 protein-assisted degradation of Cse4 protein. J Biol Chem. 289(42):29297–29309. doi: 10.1074/jbc.M114.580589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hewawasam G, Shivaraju M, Mattingly M, Venkatesh S, Martin-Brown S, Florens L, Workman JL, Gerton JL. 2010. Psh1 is an E3 ubiquitin ligase that targets the centromeric histone variant Cse4. Mol Cell. 40(3):444–454. doi: 10.1016/j.molcel.2010.10.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ikui AE, Rossio V, Schroeder L, Yoshida S. 2012. A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to inhibit DNA re-replication. PLoS Genet. 8(12):e1003099. doi: 10.1371/journal.pgen.1003099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jiang W, Lim MY, Yoon HJ, Thorner J, Martin GS, Carbon J. 1995. Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5. Mol Gen Genet. 246(3):360–366. doi: 10.1007/BF00288609. [DOI] [PubMed] [Google Scholar]
  30. Kishi T, Ikeda A, Nagao R, Koyama N. 2007. The SCFCdc4 ubiquitin ligase regulates calcineurin signaling through degradation of phosphorylated Rcn1, an inhibitor of calcineurin. Proc Natl Acad Sci U S A. 104(44):17418–17423. doi: 10.1073/pnas.0704951104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lacoste N, Woolfe A, Tachiwana H, Garea AV, Barth T, Cantaloube S, Kurumizaka H, Imhof A, Almouzni G. 2014. Mislocalization of the centromeric histone variant CenH3/CENP-A in human cells depends on the chaperone DAXX. Mol Cell. 53(4):631–644. doi: 10.1016/j.molcel.2014.01.018. [DOI] [PubMed] [Google Scholar]
  32. Li Y, Zhu Z, Zhang S, Yu D, Yu H, Liu L, Cao X, Wang L, Gao H, Zhu M. 2011. ShRNA-targeted centromere protein A inhibits hepatocellular carcinoma growth. PLoS One. 6(3):e17794. doi: 10.1371/journal.pone.0017794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Liu Q, Larsen B, Ricicova M, Orlicky S, Tekotte H, Tang X, Craig K, Quiring A, Le Bihan T, Hansen C, et al. 2011. SCFCdc4 enables mating type switching in yeast by cyclin-dependent kinase-mediated elimination of the Ash1 transcriptional repressor. Mol Cell Biol. 31(3):584–598. doi: 10.1128/MCB.00845-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Longtine MS, McKenzie A III, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 14(10):953–961. [DOI] [PubMed] [Google Scholar]
  35. Lyons NA, Fonslow BR, Diedrich JK, Yates JR III, Morgan DO. 2013. Sequential primed kinases create a damage-responsive phosphodegron on Eco1. Nat Struct Mol Biol. 20(2):194–201. doi: 10.1038/nsmb.2478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McGovern SL, Qi Y, Pusztai L, Symmans WF, Buchholz TA. 2012. Centromere protein-A, an essential centromere protein, is a prognostic marker for relapse in estrogen receptor-positive breast cancer. Breast Cancer Res. 14(3):R72. doi: 10.1186/bcr3181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mishra PK, Au WC, Choy JS, Kuich PH, Baker RE, Foltz DR, Basrai MA. 2011. Misregulation of Scm3p/HJURP causes chromosome instability in Saccharomyces cerevisiae and human cells. PLoS Genet. 7(9):e1002303. doi: 10.1371/journal.pgen.1002303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mishra PK, Olafsson G, Boeckmann L, Westlake TJ, Jowhar ZM, Dittman LE, Baker RE, D’Amours D, Thorpe PH, Basrai MA. 2019. Cell cycle-dependent association of polo kinase Cdc5 with CENP-A contributes to faithful chromosome segregation in budding yeast. Mol Biol Cell. 30(8):1020–1036. doi: 10.1091/mbc.E18-09-0584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mizunuma M, Hirata D, Miyaoka R, Miyakawa T. 2001. GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast. EMBO J. 20(5):1074–1085. doi: 10.1093/emboj/20.5.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mok J, Kim PM, Lam HY, Piccirillo S, Zhou X, Jeschke GR, Sheridan DL, Parker SA, Desai V, Jwa M, et al. 2010. Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs. Sci Signal. 3(109):ra12. doi: 10.1126/scisignal.2000482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Moreno-Moreno O, Medina-Giro S, Torras-Llort M, Azorin F. 2011. The F box protein partner of paired regulates stability of Drosophila centromeric histone H3, CenH3(CID). Curr Biol. 21(17):1488–1493. doi: 10.1016/j.cub.2011.07.041. [DOI] [PubMed] [Google Scholar]
  42. Moreno-Moreno O, Torras-Llort M, Azorín F. 2006. Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res. 34(21):6247–6255. doi: 10.1093/nar/gkl902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ohkuni K, Abdulle R, Kitagawa K. 2014. Degradation of centromeric histone H3 variant Cse4 requires the Fpr3 peptidyl-prolyl cis-trans isomerase. Genetics. 196(4):1041–1045. doi: 10.1534/genetics.114.161224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ohkuni K, Au WC, Kazi AZ, Villamil M, Kaiser P, Basrai MA. 2024. Interaction of histone H4 with Cse4 facilitates conformational changes in Cse4 for its sumoylation and mislocalization. Nucleic Acids Res. 52(2):643–659. doi: 10.1093/nar/gkad1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ohkuni K, Gliford L, Au WC, Suva E, Kaiser P, Basrai MA. 2022. Cdc48Ufd1/Npl4 segregase removes mislocalized centromeric histone H3 variant CENP-A from non-centromeric chromatin. Nucleic Acids Res. 50(6):3276–3291. doi: 10.1093/nar/gkac135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ohkuni K, Levy-Myers R, Warren J, Au WC, Takahashi Y, Baker RE, Basrai MA. 2018. N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin. G3 (Bethesda). 8(4):1215–1223. doi: 10.1534/g3.117.300419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ohkuni K, Suva E, Au WC, Walker RL, Levy-Myers R, Meltzer PS, Baker RE, Basrai MA. 2020. Deposition of centromeric histone H3 variant CENP-A/Cse4 into chromatin is facilitated by its C-terminal sumoylation. Genetics. 214(4):839–854. doi: 10.1534/genetics.120.303090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ohkuni K, Takahashi Y, Fulp A, Lawrimore J, Au WC, Pasupala N, Levy-Myers R, Warren J, Strunnikov A, Baker RE, et al. 2016. SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin. Mol Biol Cell. 27(9):1500–1510. doi: 10.1091/mbc.E15-12-0827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Perkins G, Drury LS, Diffley JF. 2001. Separate SCF(CDC4) recognition elements target Cdc6 for proteolysis in S phase and mitosis. EMBO J. 20(17):4836–4845. doi: 10.1093/emboj/20.17.4836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Ranjitkar P, Press MO, Yi X, Baker R, MacCoss MJ, Biggins S. 2010. An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain. Mol Cell. 40(3):455–464. doi: 10.1016/j.molcel.2010.09.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shero JH, Hieter P. 1991. A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1). Genes Dev. 5(4):549–560. doi: 10.1101/gad.5.4.549. [DOI] [PubMed] [Google Scholar]
  52. Shrestha RL, Ahn GS, Staples MI, Sathyan KM, Karpova TS, Foltz DR, Basrai MA. 2017. Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells. Oncotarget. 8(29):46781–46800. doi: 10.18632/oncotarget.18108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shrestha RL, Rossi A, Wangsa D, Hogan AK, Zaldana KS, Suva E, Chung YJ, Sanders CL, Difilippantonio S, Karpova TS, et al. 2021. CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity. J Cell Biol. 220(4):e202007195. doi: 10.1083/jcb.202007195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Spencer F, Gerring SL, Connelly C, Hieter P. 1990. Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae. Genetics. 124(2):237–249. doi: 10.1093/genetics/124.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sun X, Clermont PL, Jiao W, Helgason CD, Gout PW, Wang Y, Qu S. 2016. Elevated expression of the centromere protein-A(CENP-A)-encoding gene as a prognostic and predictive biomarker in human cancers. Int J Cancer. 139(4):899–907. doi: 10.1002/ijc.30133. [DOI] [PubMed] [Google Scholar]
  56. Tomonaga T, Matsushita K, Yamaguchi S, Oohashi T, Shimada H, Ochiai T, Yoda K, Nomura F. 2003. Overexpression and mistargeting of centromere protein-A in human primary colorectal cancer. Cancer Res. 63:3511–3516. [PubMed] [Google Scholar]
  57. Welcker M, Clurman BE. 2008. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer. 8(2):83–93. doi: 10.1038/nrc2290. [DOI] [PubMed] [Google Scholar]
  58. Yeh CH, Bellon M, Nicot C. 2018. FBXW7: a critical tumor suppressor of human cancers. Mol Cancer. 17(1):115. doi: 10.1186/s12943-018-0857-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang W, Mao JH, Zhu W, Jain AK, Liu K, Brown JB, Karpen GH. 2016. Centromere and kinetochore gene misexpression predicts cancer patient survival and response to radiotherapy and chemotherapy. Nat Commun. 7(1):12619. doi: 10.1038/ncomms12619. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

iyae108_Supplementary_Data
iyae108_supplementary_data.pdf (727.2KB, pdf)

Data Availability Statement

All experimental data sets are available at Figshare (https://doi.org/10.6084/m9.figshare.26049877).

Supplemental material available at GENETICS online.

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