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, 47 (13), 6871-6884

Casein Kinase 2 Regulates Telomere Protein Complex Formation Through Rap1 Phosphorylation

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Casein Kinase 2 Regulates Telomere Protein Complex Formation Through Rap1 Phosphorylation

Haruna Inoue et al. Nucleic Acids Res.

Abstract

Telomeres located at the ends of linear chromosomes play important roles in the maintenance of life. Rap1, a component of the shelterin telomere protein complex, interacts with multiple proteins to perform various functions; further, formation of shelterin requires Rap1 binding to other components such as Taz1 and Poz1, and telomere tethering to the nuclear envelope (NE) involves interactions between Rap1 and Bqt4, a nuclear membrane protein. Although Rap1 is a hub for telomere protein complexes, the regulatory mechanisms of its interactions with partner proteins are not fully understood. Here, we show that Rap1 is phosphorylated by casein kinase 2 (CK2) at multiple sites, which promotes interactions with Bqt4 and Poz1. Among the multiple CK2-mediated phosphorylation sites of Rap1, phosphorylation at Ser496 was found to be crucial for both Rap1-Bqt4 and Rap1-Poz1 interactions. These mechanisms mediate proper telomere tethering to the NE and the formation of the silenced chromatin structure at chromosome ends.

Figures

Figure 1.
Figure 1.
Rap1 is phosphorylated at seven serine residues by CK2 in vitro. (A) Rap1 phosphorylation by CK2 in vitro. In vitro kinase assays using GST-Rap1 proteins (fragments A–E) as substrates. A, 1–310 a.a.; B, 300–370 a.a.; C, 360–520 a.a.; D, 510–580 a.a.; E, 570–693 a.a. GST-Rap1 proteins were incubated with Cka1-Flag purified from S. pombe wild-type or ckb1 cells in the presence of γ-[32P]ATP. Proteins were analyzed by SDS-PAGE. Autoradiography and Coomassie Brilliant Blue (CBB) staining of the gel are shown at the top (32P) and the bottom (CBB). The asterisk indicates Cka1-Flag. (B) Mutation analyses of GST-Rap1-B. GST-Rap1-B proteins harbored single or double mutations at Ser317 and Ser329. An in vitro kinase assay was performed as in (A). Note that the upper and lower bands represent phosphorylation at Ser317 and Ser329, respectively. The double mutation at Ser317 and Ser329 completely abolished phosphorylation. (C) Mutation analyses of GST-Rap1-C. An in vitro kinase assay was performed as in (A). Note that the quadruple mutation at Ser484, Ser487, Ser496, and Ser497 completely abolished phosphorylation. (D) Mutation analysis of GST-Rap1-D. An in vitro kinase assay was performed as in (A). Note that the single mutation at Ser538 completely abolished phosphorylation. (E) Amino acid sequences of CK2 in vitro phosphorylation sites of Rap1. The phosphorylated serine residues are shown in red. Negatively charged amino acids (aspartate, glutamate, or phosphorylated serine) following the phosphorylated residues are shown in blue.
Figure 2.
Figure 2.
Rap1 is phosphorylated by CK2 throughout the cell cycle in vivo. (A) Band-shift analysis of Rap1-7A and Rap1-7E proteins. The whole cell extracts were prepared from wild-type (WT), rap1Δ, rap1-7A, and rap1-7E cells grown in YES at 32°C and were analyzed by western blotting using anti-Rap1, anti-phospho-Rap1, and anti-PSTAIR antibodies for Rap1 and Cdc2 (loading control), respectively. (B) Rap1 is phosphorylated at Ser496 and Ser497 in vivo. Immunoprecipitated Rap1-Flag proteins were incubated with or without CIAP for 1 h at 32°C and were analyzed by western blotting using anti-Flag and anti-phospho-Ser496/Ser497 antibodies. Note that the anti-phospho-Ser496/Ser497 antibody barely recognized the Rap1 protein that was dephosphorylated by CIAP. (C) Rap1 is phosphorylated at Ser496 and Ser497 in a CK2-dependent manner. Wild-type (WT) and orb5-19 cells were grown at 25°C (the permissive temperature) until mid-log phase (0 h) and incubated at 35.5°C (the restrictive temperature) for 24 h. The whole cell extracts were analyzed by western blotting using anti-Rap1 and anti-phospho-Ser496/Ser497 antibodies. (D) Rap1 is constitutively phosphorylated at Ser496/Ser497 during the cell cycle. The wild-type strain was grown in YES at 32°C to mid-log phase (asynchronous, As). Strains cdc10-129, cdc22-m45 and cdc25-22 were grown in YES at 25°C to early log phase and then arrested in G1, S or G2 phase, respectively, by a temperature shift to 35.5°C for 4 h. Strain nda3-KM311 was grown in YPD at 32°C and arrested in early M phase by a temperature shift to 20°C for 12 h. Rap1-2HA6His proteins were affinity purified and analyzed by immunoblotting using anti-Rap1 and anti- phospho-Ser496/Ser497 antibodies. Note that Rap1 is highly phosphorylated by Cdc2 in M phase, which appears as the shifted bands.
Figure 3.
Figure 3.
Rap1 phosphorylation by CK2 promotes the Rap1–Bqt4 interaction. (A) Schematic illustration of the Rap1 protein. Orange circles indicate seven CK2 phosphorylation sites, whereas blue circles indicate three Cdc2 phosphorylation sites (17). Black bars indicate binding domains to partner proteins revealed by yeast two-hybrid assays (9,17). Blue bars indicate the interaction surfaces with Poz1 and Bqt4 (27–29). Abbreviations for structural domains are as follows: BRCT, BRACA1 C terminus; Myb, Myb-related HLH (helix-loop-helix) motif; RCT, Rap1 C-terminal. (B) Rap1-7A is defective in its interactions with Bqt4. A budding yeast strain (Y190) was transformed with the plasmids indicated. β-galactosidase activity assays were performed using X-gal as substrates. (C) The S496A mutation has the strongest effect on Rap1–Bqt4 interaction. Yeast two-hybrid assays were performed as in (B) using the pACT2-rap1+ plasmid containing a series of single mutations. (D) Rap1-7A, but not Rap1-7E, has a lower affinity for Bqt4 compared to wild-type Rap1. GST pull-down assays were performed using GST or GST-Bqt4 (2–181 a.a.) incubated with S. pombe cell extracts prepared from the wild-type, rap1-7A, or rap1-7E strains. The protein samples purified with glutathione beads were analyzed by western blotting using anti-Rap1 antibody (upper) and by CBB gel staining (lower).
Figure 4.
Figure 4.
Rap1 phosphorylation by CK2 facilitates telomere tethering to the NE. (A) Schematic illustration of the method used to measure distances between telomeres and the NE. Telomeres, the NE, and microtubules were visualized with Taz1-mCherry, Ish1-GFP, and GFP-Atb2, respectively. Optical section data (13 focal planes with 0.3 μm spacing) were collected using a DeltaVision microscope system (Applied Precision) and were processed by a three-dimensional deconvolution method (37). Only the two-dimensional distance on a focal plane near the nuclear mid-plane was analyzed to ensure accurate measurements. Note that the telomeres of chromosome 3 (weak signals of Taz1-mCherry) are located in the nucleolus, and thus only the telomeres of chromosomes 1 and 2 (bright signals of Taz1-mCherry due to the clustering of telomeres) were analyzed. (B) Examples of images of wild-type, rap1Δ, rap1-7A, rap1-7E and rap1-S496A cells in G2 phase. Shown are deconvolved images of the nuclear mid-plane. Bar, 10 μm. (C) Scatterplots of the distances between telomeres and the NE during G2 phase. Cells were grown in EMM medium at 26°C. Bars in the graph indicate the median distances. p, Mann–Whitney U test. NS (not significant), p> 0.05; **p < 0.01. (D) Examples of images of wild-type and orb5-19 cells in G2 phase. Shown are deconvolved images of the mid-plane of the nucleus. Bar, 10 μm. (E) Scatterplots of the relative distances between telomeres and the NE during G2 phase normalized with each nuclear diameter. Cells were grown in YES medium at 25°C (the permissive temperature) or 35.5°C (the restrictive temperature) for 8 h. Bars in the graph indicate the median distances (0.037 [WT 25°C], 0.039 [orb5-19 25°C], 0.047 [WT 35.5°C] and 0.066 [orb5-19 35.5°C]. p, Mann–Whitney U test. NS, p > 0.05; **p < 0.01.
Figure 5.
Figure 5.
Rap1 phosphorylation by CK2 promotes the Rap1-Poz1 interaction at telomeres. (A) Rap1 phosphorylation by CK2 promotes the Rap1–Poz1 interaction in vivo. Poz1-Flag was immunoprecipitated from cell extracts of the wild-type or poz1-flag strains harbouring rap1+, rap1-7A and rap1-7E. Immunoprecipitated samples were analyzed by immunoblotting using anti-Rap1 and anti-Flag antibodies. An asterisk indicates the position of non-specific bands. (B) Substantial Rap1-Taz1 interaction is maintained in rap1-7A. Taz1-Flag was immunoprecipitated from cell extracts of the wild-type or taz1-flag strains harbouring rap1+, rap1-7A, and rap1-7E. Immunoprecipitated samples were analyzed by immunoblotting using anti-Rap1 and anti-Flag antibodies. (C) Rap1 phosphorylation promotes the localization of Poz1 at telomeres. Poz1-Flag localization at telomeres was determined by ChIP analyses followed by q-PCR. Fold enrichment at telomeres (detected by the primer set: jk1333 and jk1334) relative to the his1 locus is shown. Error bars indicate the s.d. N > 3. p, Student's t-test (versus wild type or between 7A and S496A). NS, p > 0.05; 0.01 < *p < 0.05; **p < 0.01.
Figure 6.
Figure 6.
Rap1 phosphorylation by CK2 promotes the localization of shelterin components at telomeres and subtelomeres. (A) Schematic illustration of the S. pombe shelterin complex. (B) The association of shelterin components with telomeres is decreased in the rap1-7A mutant. ChIP analyses of Flag-tagged shelterin proteins. Telomere DNA and rDNA were detected by the Southern analyses. Representative data of slot Southern blots are shown in Supplementary S3B. Fold enrichment at telomeres relative to the rDNA loci in rap1-7A compared with that in wild type is shown. Error bars indicate the s.d. N = 3. p, Student's t-test (versus wild type). 0.01 < *p < 0.05; **p < 0.01. (C) The association of shelterin components with subtelomeres is decreased in the rap1-7A mutant. ChIP analyses of Flag-tagged shelterin proteins followed by q-PCR. Blue dots indicate the values of wild type, whereas red dots are rap1-7A. Fold enrichment at subtelomeres relative to the his1 locus is shown. Error bars indicate the s.d. N = 3. Green bars indicate 1 on the Y axis, which indicates no enrichment. p, Student's t-test (versus wild type) for the data at 0.2, 6.5 and 11 kb from telomeres. No mark, p > 0.05; 0.01 < *p < 0.05; **p < 0.01.
Figure 7.
Figure 7.
Rap1 phosphorylation by CK2 contributes to the maintenance of normal telomere DNA length. (A) Analyses of telomere DNA length of the rap1-7A and rap1-7E mutants. Cells were grown in YES at 32°C. Genomic DNA was digested with ApaI, separated in a 1% agarose gel and subjected to Southern blot analysis using telomere DNA as a probe. An asterisk indicates the position of telomeres of chromosome 3. (B) Analyses of telomere DNA length of the rap1-7A and rap1-7E mutants with high resolution. Cells were grown in YES at 32°C. Genomic DNA was digested with ApaI, separated in a 3% agarose gel and subjected to Southern blot analysis using telomere DNA as a probe. Three independent clones for each genotype were analyzed. (C) The orb5-19 mutant shows elongated telomere DNA. Cells were grown at 25°C or at 35.5°C for 8 h. Southern blot analyses were performed as in (B).
Figure 8.
Figure 8.
Rap1 phosphorylation by CK2 facilitates the formation of silenced chromatin at chromosome ends. (A) Rap1 and Poz1 are required for normal gene silencing at subtelomeres. The RNA level of the tlh1/2+ genes was analyzed by qRT-PCR. Expression of tlh1/2+ relative to that of his1+ was normalized to that in the wild type strain. Error bars indicate the s.d. N = 3. p, Student's t-test (versus wild type). **p < 0.01. (B) Rap1 phosphorylation by CK2 is required for gene silencing at subtelomeres. RNA expression of the tlh1/2+ genes was analyzed as in (A). p, Student's t-test (versus wild type). NS, p > 0.05; 0.01 < *p < 0.05; **p < 0.01. (C) Defective subtelomeric gene silencing in the orb5-19 mutant. Wild-type and orb5-19 cells were grown in YES at 25°C or at 35.5°C for 8 h. Expression of tlh1/2+ relative to that of his1+ was normalized to that in the wild type strain at 25°C. p, Student's t-test (versus wild type or between the strains indicated by bracket lines). NS, p > 0.05; 0.01 < *p < 0.05; **p < 0.01. (D)The bqt4Δ mutant does not show a defect in subtelomeric gene silencing. RNA expression of the tlh1/2+ genes was analyzed as in (A). p, Student's t-test (versus wild type). 0.01 < *p < 0.05; **p < 0.01. (E) The rap1-7A mutant is not defective in heterochromatin formation. ChIP analyses of histone H3 methylated at K9 and Swi6 at the subtelomere of the right arm of chromosome 2 followed by qPCR. Fold enrichment relative to the his1 locus is shown. Error bars indicate the s.d. N = 3.
Figure 9.
Figure 9.
Model of the regulatory mechanisms of CK2-mediated Rap1 phosphorylation. (A) Rap1 phosphorylation by CK2 facilitates two interactions, namely Rap1–Bqt4 and Rap1–Poz1 associations. The Rap1–Bqt4 association enables telomere tethering to the NE. The Rap1–Poz1 association is required for the normal localization of shelterin components at telomeres. (B) Rap1 phosphorylation by CK2 also facilitates the normal localization of shelterin components to subtelomeres to form a specialized chromatin structure at chromosome ends. It is speculated that most Pot1 proteins at subtelomeres are not linked to the relatively short ss-telomere DNA at chromosome ends, although it is possible that a small portion of Pot1 at subtelomeres binds to ss-telomere DNA.

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