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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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ATM phosphorylates S367 of TRF1 both in vivo and in vitro. (A) Schematic diagram of TRF1 domain structures. S367 of TRF1 in red was identified by mass spectrometry analysis to be a candidate phosphorylation site. (B) Affinity-purified anti-pS367 antibody specifically recognizes TRF1 peptide containing phosphorylated S367 (pS367-peptide). An increasing amount of peptide either carrying unmodified S367 (S367-peptide) or phosphorylated S367 (pS367-peptide) was spotted on a nitrocellulose membrane, followed by immunoblotting with affinity-purified anti-pS367 antibody or crude serum. The amount of peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (C) Peptide competition assays. Affinity-purified anti-pS367 antibody was incubated with 5.5 µg of either unmodified (S367-peptide) or phosphorylated peptide (pS367-peptide) prior to immunoblotting. Crude serum was used to show the presence of pS367-peptide on the nitrocellulose membrane. The amount of pS367-peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (D) Western analysis of phosphorylated TRF1. The whole cell extract (20 µg) from HeLaII cells was immunoblotted with affinity-purified anti-pS367 antibody. (E) Depletion of endogenous TRF1 leads to loss of phosphorylated TRF1 recognized by anti-pS367 antibody. HeLaII cells were infected with retrovirus expressing shTRF1 or the vector pRS alone. Western analysis was performed with anti-pS367 or anti-TRF1 antibody. The γ-tubulin blot was used as a loading control. (F) In vitro kinase assays. Bacterial-expressed his-tagged wild-type TRF1 (2 µg) or TRF1 mutant S367A (2 µg) was incubated with either ATM immunoprecipitated from HeLa cells or purified DNA-PKcs in the presence of γ-32P-ATP. (G) ATM inhibition leads to a diminished anti-pS367 staining. HeLaI.2.11 cells were treated with DMSO, KU55933 (an ATM inhibitor) or NU7026 (a DNA-PKcs inhibitor) for 90 min, followed by western analysis. Immunoblotting was performed with anti-pS367, anti-TRF1 or anti-γ-tubulin antibody. (H) Loss of anti-pS367 staining in ATM-deficient cells. Western analysis of cell extracts from IMR90 cells, HeLaI.2.11 cells and ATM-deficient cells (GM09607 and GM05849). Immunoblotting was performed with anti-pS367 or anti-γ-tubulin antibody. (I) NU7026 leads to a further reduction in S367 phosphorylation in ATM-deficient cells. GM09607 cells were treated with DMSO, KU55933 or NU7026 for 90 min, following by immunoblotting with anti-pS367 or anti-γ-tubulin antibody.
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gks035-F1: ATM phosphorylates S367 of TRF1 both in vivo and in vitro. (A) Schematic diagram of TRF1 domain structures. S367 of TRF1 in red was identified by mass spectrometry analysis to be a candidate phosphorylation site. (B) Affinity-purified anti-pS367 antibody specifically recognizes TRF1 peptide containing phosphorylated S367 (pS367-peptide). An increasing amount of peptide either carrying unmodified S367 (S367-peptide) or phosphorylated S367 (pS367-peptide) was spotted on a nitrocellulose membrane, followed by immunoblotting with affinity-purified anti-pS367 antibody or crude serum. The amount of peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (C) Peptide competition assays. Affinity-purified anti-pS367 antibody was incubated with 5.5 µg of either unmodified (S367-peptide) or phosphorylated peptide (pS367-peptide) prior to immunoblotting. Crude serum was used to show the presence of pS367-peptide on the nitrocellulose membrane. The amount of pS367-peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (D) Western analysis of phosphorylated TRF1. The whole cell extract (20 µg) from HeLaII cells was immunoblotted with affinity-purified anti-pS367 antibody. (E) Depletion of endogenous TRF1 leads to loss of phosphorylated TRF1 recognized by anti-pS367 antibody. HeLaII cells were infected with retrovirus expressing shTRF1 or the vector pRS alone. Western analysis was performed with anti-pS367 or anti-TRF1 antibody. The γ-tubulin blot was used as a loading control. (F) In vitro kinase assays. Bacterial-expressed his-tagged wild-type TRF1 (2 µg) or TRF1 mutant S367A (2 µg) was incubated with either ATM immunoprecipitated from HeLa cells or purified DNA-PKcs in the presence of γ-32P-ATP. (G) ATM inhibition leads to a diminished anti-pS367 staining. HeLaI.2.11 cells were treated with DMSO, KU55933 (an ATM inhibitor) or NU7026 (a DNA-PKcs inhibitor) for 90 min, followed by western analysis. Immunoblotting was performed with anti-pS367, anti-TRF1 or anti-γ-tubulin antibody. (H) Loss of anti-pS367 staining in ATM-deficient cells. Western analysis of cell extracts from IMR90 cells, HeLaI.2.11 cells and ATM-deficient cells (GM09607 and GM05849). Immunoblotting was performed with anti-pS367 or anti-γ-tubulin antibody. (I) NU7026 leads to a further reduction in S367 phosphorylation in ATM-deficient cells. GM09607 cells were treated with DMSO, KU55933 or NU7026 for 90 min, following by immunoblotting with anti-pS367 or anti-γ-tubulin antibody.

Mentions: Phosphorylation has been shown to play an important role in modulating the function of TRF1 in telomere metabolism (12,26,39–41). In an effort to identify TRF1 phosphorylation sites in vivo, we generated an HT1080 cell line stably expressing Flag-tagged TRF1. Mass spectrometry analysis of immunoprecipitated Flag-TRF1 indicated serine at position 367 of TRF1 to be a candidate phosphorylation site in vivo (Figure 1A and data not shown).Figure 1.


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

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

ATM phosphorylates S367 of TRF1 both in vivo and in vitro. (A) Schematic diagram of TRF1 domain structures. S367 of TRF1 in red was identified by mass spectrometry analysis to be a candidate phosphorylation site. (B) Affinity-purified anti-pS367 antibody specifically recognizes TRF1 peptide containing phosphorylated S367 (pS367-peptide). An increasing amount of peptide either carrying unmodified S367 (S367-peptide) or phosphorylated S367 (pS367-peptide) was spotted on a nitrocellulose membrane, followed by immunoblotting with affinity-purified anti-pS367 antibody or crude serum. The amount of peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (C) Peptide competition assays. Affinity-purified anti-pS367 antibody was incubated with 5.5 µg of either unmodified (S367-peptide) or phosphorylated peptide (pS367-peptide) prior to immunoblotting. Crude serum was used to show the presence of pS367-peptide on the nitrocellulose membrane. The amount of pS367-peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (D) Western analysis of phosphorylated TRF1. The whole cell extract (20 µg) from HeLaII cells was immunoblotted with affinity-purified anti-pS367 antibody. (E) Depletion of endogenous TRF1 leads to loss of phosphorylated TRF1 recognized by anti-pS367 antibody. HeLaII cells were infected with retrovirus expressing shTRF1 or the vector pRS alone. Western analysis was performed with anti-pS367 or anti-TRF1 antibody. The γ-tubulin blot was used as a loading control. (F) In vitro kinase assays. Bacterial-expressed his-tagged wild-type TRF1 (2 µg) or TRF1 mutant S367A (2 µg) was incubated with either ATM immunoprecipitated from HeLa cells or purified DNA-PKcs in the presence of γ-32P-ATP. (G) ATM inhibition leads to a diminished anti-pS367 staining. HeLaI.2.11 cells were treated with DMSO, KU55933 (an ATM inhibitor) or NU7026 (a DNA-PKcs inhibitor) for 90 min, followed by western analysis. Immunoblotting was performed with anti-pS367, anti-TRF1 or anti-γ-tubulin antibody. (H) Loss of anti-pS367 staining in ATM-deficient cells. Western analysis of cell extracts from IMR90 cells, HeLaI.2.11 cells and ATM-deficient cells (GM09607 and GM05849). Immunoblotting was performed with anti-pS367 or anti-γ-tubulin antibody. (I) NU7026 leads to a further reduction in S367 phosphorylation in ATM-deficient cells. GM09607 cells were treated with DMSO, KU55933 or NU7026 for 90 min, following by immunoblotting with anti-pS367 or anti-γ-tubulin antibody.
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gks035-F1: ATM phosphorylates S367 of TRF1 both in vivo and in vitro. (A) Schematic diagram of TRF1 domain structures. S367 of TRF1 in red was identified by mass spectrometry analysis to be a candidate phosphorylation site. (B) Affinity-purified anti-pS367 antibody specifically recognizes TRF1 peptide containing phosphorylated S367 (pS367-peptide). An increasing amount of peptide either carrying unmodified S367 (S367-peptide) or phosphorylated S367 (pS367-peptide) was spotted on a nitrocellulose membrane, followed by immunoblotting with affinity-purified anti-pS367 antibody or crude serum. The amount of peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (C) Peptide competition assays. Affinity-purified anti-pS367 antibody was incubated with 5.5 µg of either unmodified (S367-peptide) or phosphorylated peptide (pS367-peptide) prior to immunoblotting. Crude serum was used to show the presence of pS367-peptide on the nitrocellulose membrane. The amount of pS367-peptide spotted from left to right is 0.7, 1.75 and 3.5 µg. (D) Western analysis of phosphorylated TRF1. The whole cell extract (20 µg) from HeLaII cells was immunoblotted with affinity-purified anti-pS367 antibody. (E) Depletion of endogenous TRF1 leads to loss of phosphorylated TRF1 recognized by anti-pS367 antibody. HeLaII cells were infected with retrovirus expressing shTRF1 or the vector pRS alone. Western analysis was performed with anti-pS367 or anti-TRF1 antibody. The γ-tubulin blot was used as a loading control. (F) In vitro kinase assays. Bacterial-expressed his-tagged wild-type TRF1 (2 µg) or TRF1 mutant S367A (2 µg) was incubated with either ATM immunoprecipitated from HeLa cells or purified DNA-PKcs in the presence of γ-32P-ATP. (G) ATM inhibition leads to a diminished anti-pS367 staining. HeLaI.2.11 cells were treated with DMSO, KU55933 (an ATM inhibitor) or NU7026 (a DNA-PKcs inhibitor) for 90 min, followed by western analysis. Immunoblotting was performed with anti-pS367, anti-TRF1 or anti-γ-tubulin antibody. (H) Loss of anti-pS367 staining in ATM-deficient cells. Western analysis of cell extracts from IMR90 cells, HeLaI.2.11 cells and ATM-deficient cells (GM09607 and GM05849). Immunoblotting was performed with anti-pS367 or anti-γ-tubulin antibody. (I) NU7026 leads to a further reduction in S367 phosphorylation in ATM-deficient cells. GM09607 cells were treated with DMSO, KU55933 or NU7026 for 90 min, following by immunoblotting with anti-pS367 or anti-γ-tubulin antibody.
Mentions: Phosphorylation has been shown to play an important role in modulating the function of TRF1 in telomere metabolism (12,26,39–41). In an effort to identify TRF1 phosphorylation sites in vivo, we generated an HT1080 cell line stably expressing Flag-tagged TRF1. Mass spectrometry analysis of immunoprecipitated Flag-TRF1 indicated serine at position 367 of TRF1 to be a candidate phosphorylation site in vivo (Figure 1A and data not shown).Figure 1.

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