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Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.

Park C, Suh Y, Cuervo AM - Nat Commun (2015)

Bottom Line: Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing.Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability.We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA [2] Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA [3] Institute for Aging Research, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

ABSTRACT
Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

No MeSH data available.


Related in: MedlinePlus

Chk1 is a bona fide CMA substratea. Immunofluorescence for Chk1 and LAMP1 in Ctr cells untreated (none) or treated with etoposide for 6h in presence or not of 20nM leptomycin B. Full fields shown in Fig. S8. Right: Percentage of colocalization in >50 cells/condition Scale bar: 5μm. (n=3 independent experiments). b. Immunofluorescence for pChk1 in control (Ctr) and LAMP-2A knock-down cells L2A(−) treated as in a. Scale bar: 10μm. Right: Percentage of nuclear pChk1 in >25 cells/condition (n=4 independent experiments). c. Immunofluorescence for hsc70 in the same cells treated with 100μM etoposide for 12h. Left: representative images. Insets: higher magnification images. Arrows: nuclear hsc70. Scale bar: 10μm. Right: Average hsc70 nuclear signal per cell in >50 cells/condition (n=3 independent experiments). d. Scheme of Chk1 CMA-targeting motif (top) and mutagenesis performed to disrupt it (bottom). e–g. Immunoblot of cell extracts (e, g) or cytosol and nuclear fractions (f) from cells expressing wild-type (GFP-Chk1) or mutant (GFP-Chk1-AA) Chk1 after the indicated treatments. N/L: ammonium chloride and leupeptin. Right in g: Percentage of Chk1 degradation per hour. n=2 independent experiments. Values are average and range. h,i. Percentage of cells with nuclear γH2AX foci (h) or levels of γH2AX (i) in mouse fibroblasts untransfected or transfected as indicated and treated with 100 μM Etoposide, n >100 cells in >5 fields from n = 3 independent experiments. All values are mean+s.e.m. (ANOVA plus Bonferroni for a and b and unpaired two-tailed t-test for the rest) *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
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Figure 7: Chk1 is a bona fide CMA substratea. Immunofluorescence for Chk1 and LAMP1 in Ctr cells untreated (none) or treated with etoposide for 6h in presence or not of 20nM leptomycin B. Full fields shown in Fig. S8. Right: Percentage of colocalization in >50 cells/condition Scale bar: 5μm. (n=3 independent experiments). b. Immunofluorescence for pChk1 in control (Ctr) and LAMP-2A knock-down cells L2A(−) treated as in a. Scale bar: 10μm. Right: Percentage of nuclear pChk1 in >25 cells/condition (n=4 independent experiments). c. Immunofluorescence for hsc70 in the same cells treated with 100μM etoposide for 12h. Left: representative images. Insets: higher magnification images. Arrows: nuclear hsc70. Scale bar: 10μm. Right: Average hsc70 nuclear signal per cell in >50 cells/condition (n=3 independent experiments). d. Scheme of Chk1 CMA-targeting motif (top) and mutagenesis performed to disrupt it (bottom). e–g. Immunoblot of cell extracts (e, g) or cytosol and nuclear fractions (f) from cells expressing wild-type (GFP-Chk1) or mutant (GFP-Chk1-AA) Chk1 after the indicated treatments. N/L: ammonium chloride and leupeptin. Right in g: Percentage of Chk1 degradation per hour. n=2 independent experiments. Values are average and range. h,i. Percentage of cells with nuclear γH2AX foci (h) or levels of γH2AX (i) in mouse fibroblasts untransfected or transfected as indicated and treated with 100 μM Etoposide, n >100 cells in >5 fields from n = 3 independent experiments. All values are mean+s.e.m. (ANOVA plus Bonferroni for a and b and unpaired two-tailed t-test for the rest) *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.

Mentions: We found that although a fraction of cytosolic Chk1 undergoes degradation in lysosomes in basal conditions, most of the Chk1 degraded in this compartment after etoposide treatment originates from the nucleus, since treatment with the nuclear export blocker leptomycin B significantly reduced lysosomal Chk1 levels and eliminated the etoposide-induced increase in Chk1 lysosomal delivery (Fig. 7a and Supplementary Fig. 7). In agreement with a nuclear origin of lysosomal Chk1, blockage of CMA led to increased nuclear content of Chk1 (Fig. 5a and b) and leptomycin B failed to further increase nuclear levels of Chk1 in these cells (Fig. 7b), indicating that their higher nuclear Chk1 content was due mostly to its reduced export rather than to increased import.


Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.

Park C, Suh Y, Cuervo AM - Nat Commun (2015)

Chk1 is a bona fide CMA substratea. Immunofluorescence for Chk1 and LAMP1 in Ctr cells untreated (none) or treated with etoposide for 6h in presence or not of 20nM leptomycin B. Full fields shown in Fig. S8. Right: Percentage of colocalization in >50 cells/condition Scale bar: 5μm. (n=3 independent experiments). b. Immunofluorescence for pChk1 in control (Ctr) and LAMP-2A knock-down cells L2A(−) treated as in a. Scale bar: 10μm. Right: Percentage of nuclear pChk1 in >25 cells/condition (n=4 independent experiments). c. Immunofluorescence for hsc70 in the same cells treated with 100μM etoposide for 12h. Left: representative images. Insets: higher magnification images. Arrows: nuclear hsc70. Scale bar: 10μm. Right: Average hsc70 nuclear signal per cell in >50 cells/condition (n=3 independent experiments). d. Scheme of Chk1 CMA-targeting motif (top) and mutagenesis performed to disrupt it (bottom). e–g. Immunoblot of cell extracts (e, g) or cytosol and nuclear fractions (f) from cells expressing wild-type (GFP-Chk1) or mutant (GFP-Chk1-AA) Chk1 after the indicated treatments. N/L: ammonium chloride and leupeptin. Right in g: Percentage of Chk1 degradation per hour. n=2 independent experiments. Values are average and range. h,i. Percentage of cells with nuclear γH2AX foci (h) or levels of γH2AX (i) in mouse fibroblasts untransfected or transfected as indicated and treated with 100 μM Etoposide, n >100 cells in >5 fields from n = 3 independent experiments. All values are mean+s.e.m. (ANOVA plus Bonferroni for a and b and unpaired two-tailed t-test for the rest) *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
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Figure 7: Chk1 is a bona fide CMA substratea. Immunofluorescence for Chk1 and LAMP1 in Ctr cells untreated (none) or treated with etoposide for 6h in presence or not of 20nM leptomycin B. Full fields shown in Fig. S8. Right: Percentage of colocalization in >50 cells/condition Scale bar: 5μm. (n=3 independent experiments). b. Immunofluorescence for pChk1 in control (Ctr) and LAMP-2A knock-down cells L2A(−) treated as in a. Scale bar: 10μm. Right: Percentage of nuclear pChk1 in >25 cells/condition (n=4 independent experiments). c. Immunofluorescence for hsc70 in the same cells treated with 100μM etoposide for 12h. Left: representative images. Insets: higher magnification images. Arrows: nuclear hsc70. Scale bar: 10μm. Right: Average hsc70 nuclear signal per cell in >50 cells/condition (n=3 independent experiments). d. Scheme of Chk1 CMA-targeting motif (top) and mutagenesis performed to disrupt it (bottom). e–g. Immunoblot of cell extracts (e, g) or cytosol and nuclear fractions (f) from cells expressing wild-type (GFP-Chk1) or mutant (GFP-Chk1-AA) Chk1 after the indicated treatments. N/L: ammonium chloride and leupeptin. Right in g: Percentage of Chk1 degradation per hour. n=2 independent experiments. Values are average and range. h,i. Percentage of cells with nuclear γH2AX foci (h) or levels of γH2AX (i) in mouse fibroblasts untransfected or transfected as indicated and treated with 100 μM Etoposide, n >100 cells in >5 fields from n = 3 independent experiments. All values are mean+s.e.m. (ANOVA plus Bonferroni for a and b and unpaired two-tailed t-test for the rest) *P<0.05, **P <0.005 or ***P <0.0005. Full gels are shown in Supplementary Fig. 8.
Mentions: We found that although a fraction of cytosolic Chk1 undergoes degradation in lysosomes in basal conditions, most of the Chk1 degraded in this compartment after etoposide treatment originates from the nucleus, since treatment with the nuclear export blocker leptomycin B significantly reduced lysosomal Chk1 levels and eliminated the etoposide-induced increase in Chk1 lysosomal delivery (Fig. 7a and Supplementary Fig. 7). In agreement with a nuclear origin of lysosomal Chk1, blockage of CMA led to increased nuclear content of Chk1 (Fig. 5a and b) and leptomycin B failed to further increase nuclear levels of Chk1 in these cells (Fig. 7b), indicating that their higher nuclear Chk1 content was due mostly to its reduced export rather than to increased import.

Bottom Line: Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing.Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability.We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA [2] Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA [3] Institute for Aging Research, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

ABSTRACT
Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

No MeSH data available.


Related in: MedlinePlus