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ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance.

McKerlie M, Lin S, Zhu XD - Nucleic Acids Res. (2012)

Bottom Line: In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers.Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis.Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S4K1, Canada.

ABSTRACT
Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

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(A) Nuclear localization of overexpressed various Myc-tagged TRF1 proteins. Dual indirect IF with anti-pS367 in conjunction with anti-Myc antibody was performed on HeLa cells transiently overexpressing Myc-TRF1, Myc-TRF1-S367A or Myc-TRF1-S367D. Cell nuclei were stained with DAPI shown in blue. (B) Overexpressed TRF1-S367D colocalizes with nuclear proteasomes. Dual IF was conducted on HeLaII cells transiently expressing Myc-S367D with anti-Myc antibody (red) in conjunction with either rabbit anti-proteasome (anti-PT) antibody (green) or no primary antibody (no 1° Ab). Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. GM847 cells were treated with either DMSO or MG132 (12.5 µM) for 4 hr prior to IF analysis with anti-pS367 antibody. Cell nuclei were stained with DAPI shown in blue.
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gks035-F6: (A) Nuclear localization of overexpressed various Myc-tagged TRF1 proteins. Dual indirect IF with anti-pS367 in conjunction with anti-Myc antibody was performed on HeLa cells transiently overexpressing Myc-TRF1, Myc-TRF1-S367A or Myc-TRF1-S367D. Cell nuclei were stained with DAPI shown in blue. (B) Overexpressed TRF1-S367D colocalizes with nuclear proteasomes. Dual IF was conducted on HeLaII cells transiently expressing Myc-S367D with anti-Myc antibody (red) in conjunction with either rabbit anti-proteasome (anti-PT) antibody (green) or no primary antibody (no 1° Ab). Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. GM847 cells were treated with either DMSO or MG132 (12.5 µM) for 4 hr prior to IF analysis with anti-pS367 antibody. Cell nuclei were stained with DAPI shown in blue.

Mentions: Analysis of dual indirect IF with anti-pS367 antibody in conjunction with anti-Myc antibody revealed that when transiently expressed in HeLa cells, both Myc-TRF1 and Myc-TRF1-S367A exhibited punctate nuclear staining (Figure 6A), indicative of their association with telomeric DNA (Figure 6A). While overexpressed Myc-TRF1 or Myc-TRF1-S367A did not exhibit any overlap with endogenous phosphorylated (pS367)TRF1 (Figure 6A), overexpressed Myc-TRF1-S367D was found to co-localize with phosphorylated (pS367)TRF1 (Figure 6A). Earlier we have shown that a very small amount (1–5%) of TRF1 is phosphorylated at S367 (Figure 2E) and therefore it is likely that the vast majority of overexpressed Myc-TRF1 is not phosphorylated at S367, which may account for the apparent lack of an overlap between anti-Myc and anti-pS367 staining in Myc-TRF1-overexpressing HeLaII cells. In addition, we observed an overlap between Myc-TRF1-S367D-containing foci and proteasome-containing nuclear centers (Figure 6B), suggesting that Myc-TRF1-S367D-containing foci are part of nuclear proteasome centers. Consistent with this notion, we found that treatment of cells with MG132, a potent proteasome inhibitor, completely abrogated the formation of phosphorylated (pS367)TRF1-containing foci (Figure 6C). Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent the nuclear proteolytic sites for TRF1 degradation.Figure 6.


ATM regulates proteasome-dependent subnuclear localization of TRF1, which is important for telomere maintenance.

McKerlie M, Lin S, Zhu XD - Nucleic Acids Res. (2012)

(A) Nuclear localization of overexpressed various Myc-tagged TRF1 proteins. Dual indirect IF with anti-pS367 in conjunction with anti-Myc antibody was performed on HeLa cells transiently overexpressing Myc-TRF1, Myc-TRF1-S367A or Myc-TRF1-S367D. Cell nuclei were stained with DAPI shown in blue. (B) Overexpressed TRF1-S367D colocalizes with nuclear proteasomes. Dual IF was conducted on HeLaII cells transiently expressing Myc-S367D with anti-Myc antibody (red) in conjunction with either rabbit anti-proteasome (anti-PT) antibody (green) or no primary antibody (no 1° Ab). Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. GM847 cells were treated with either DMSO or MG132 (12.5 µM) for 4 hr prior to IF analysis with anti-pS367 antibody. Cell nuclei were stained with DAPI shown in blue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gks035-F6: (A) Nuclear localization of overexpressed various Myc-tagged TRF1 proteins. Dual indirect IF with anti-pS367 in conjunction with anti-Myc antibody was performed on HeLa cells transiently overexpressing Myc-TRF1, Myc-TRF1-S367A or Myc-TRF1-S367D. Cell nuclei were stained with DAPI shown in blue. (B) Overexpressed TRF1-S367D colocalizes with nuclear proteasomes. Dual IF was conducted on HeLaII cells transiently expressing Myc-S367D with anti-Myc antibody (red) in conjunction with either rabbit anti-proteasome (anti-PT) antibody (green) or no primary antibody (no 1° Ab). Cell nuclei were stained with DAPI shown in blue. (C) Phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. GM847 cells were treated with either DMSO or MG132 (12.5 µM) for 4 hr prior to IF analysis with anti-pS367 antibody. Cell nuclei were stained with DAPI shown in blue.
Mentions: Analysis of dual indirect IF with anti-pS367 antibody in conjunction with anti-Myc antibody revealed that when transiently expressed in HeLa cells, both Myc-TRF1 and Myc-TRF1-S367A exhibited punctate nuclear staining (Figure 6A), indicative of their association with telomeric DNA (Figure 6A). While overexpressed Myc-TRF1 or Myc-TRF1-S367A did not exhibit any overlap with endogenous phosphorylated (pS367)TRF1 (Figure 6A), overexpressed Myc-TRF1-S367D was found to co-localize with phosphorylated (pS367)TRF1 (Figure 6A). Earlier we have shown that a very small amount (1–5%) of TRF1 is phosphorylated at S367 (Figure 2E) and therefore it is likely that the vast majority of overexpressed Myc-TRF1 is not phosphorylated at S367, which may account for the apparent lack of an overlap between anti-Myc and anti-pS367 staining in Myc-TRF1-overexpressing HeLaII cells. In addition, we observed an overlap between Myc-TRF1-S367D-containing foci and proteasome-containing nuclear centers (Figure 6B), suggesting that Myc-TRF1-S367D-containing foci are part of nuclear proteasome centers. Consistent with this notion, we found that treatment of cells with MG132, a potent proteasome inhibitor, completely abrogated the formation of phosphorylated (pS367)TRF1-containing foci (Figure 6C). Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent the nuclear proteolytic sites for TRF1 degradation.Figure 6.

Bottom Line: In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers.Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis.Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S4K1, Canada.

ABSTRACT
Ataxia telangiectasia mutated (ATM), a PI-3 kinase essential for maintaining genomic stability, has been shown to regulate TRF1, a negative mediator of telomerase-dependent telomere extension. However, little is known about ATM-mediated TRF1 phosphorylation site(s) in vivo. Here, we report that ATM phosphorylates S367 of TRF1 and that this phosphorylation renders TRF1 free of chromatin. We show that phosphorylated (pS367)TRF1 forms distinct non-telomeric subnuclear foci and that these foci occur predominantly in S and G2 phases, implying that their formation is cell cycle regulated. We show that phosphorylated (pS367)TRF1-containing foci are sensitive to proteasome inhibition. We find that a phosphomimic mutation of S367D abrogates TRF1 binding to telomeric DNA and renders TRF1 susceptible to protein degradation. In addition, we demonstrate that overexpressed TRF1-S367D accumulates in the subnuclear domains containing phosphorylated (pS367)TRF1 and that these subnuclear domains overlap with nuclear proteasome centers. Taken together, these results suggest that phosphorylated (pS367)TRF1-containing foci may represent nuclear sites for TRF1 proteolysis. Furthermore, we show that TRF1 carrying the S367D mutation is unable to inhibit telomerase-dependent telomere lengthening or to suppress the formation of telomere doublets and telomere loss in TRF1-depleted cells, suggesting that S367 phosphorylation by ATM is important for the regulation of telomere length and stability.

Show MeSH
Related in: MedlinePlus