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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 undergoes lysosomal degradationa. Immunoblot in mouse fibroblasts untreated (−) or treated with NH4Cl and leupeptin (N/L), lactacystin (Lact) or 3-methyladenine (3MA). b. Immunofluorescence for LAMP1 and Chk1 in mouse fibroblasts treated or not with etoposide. Inset in merged show co-localization as white pixels. Percentage of co-localization is indicated, n>80 cells counted per condition in two independent experiments. c. Representative immunoblot of homogenates (Hom), cytosol (Cyt) and lysosomes with high (CMA+) and low (CMA−) CMA activity from livers of mice untreated (−) or injected with leupeptin (Leup). d. Co-immunoprecipitation (IP) of hsc70 with Chk1 in fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)). Inp: 1/10 input; FT: flow through. e–h, Immunoblot of fibroblasts treated or not with etoposide in the presence of the indicated protease and kinase inhibitors. The residues recognized by the two anti-pChk1 antibodies are indicated (h). Isogranulatimide (ISOgram), Wortmaninin (Wortman) and ATR inhibitor II (ATRinh II). In f, samples were dephosphorylated with λ protein phosphatase (λ PP) before electrophoresis. i. Immunofluorescence for pChk1 and LAMP1 in Ctr cells. Full field (left) and high magnification (right). Arrows: co-localization. Percentage of co-localization is indicated, n>80 cells in two independent experiments. The experiments in a,e,f,g and h were repeated 5 times and in c and d 3 times. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
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Figure 6: Chk1 undergoes lysosomal degradationa. Immunoblot in mouse fibroblasts untreated (−) or treated with NH4Cl and leupeptin (N/L), lactacystin (Lact) or 3-methyladenine (3MA). b. Immunofluorescence for LAMP1 and Chk1 in mouse fibroblasts treated or not with etoposide. Inset in merged show co-localization as white pixels. Percentage of co-localization is indicated, n>80 cells counted per condition in two independent experiments. c. Representative immunoblot of homogenates (Hom), cytosol (Cyt) and lysosomes with high (CMA+) and low (CMA−) CMA activity from livers of mice untreated (−) or injected with leupeptin (Leup). d. Co-immunoprecipitation (IP) of hsc70 with Chk1 in fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)). Inp: 1/10 input; FT: flow through. e–h, Immunoblot of fibroblasts treated or not with etoposide in the presence of the indicated protease and kinase inhibitors. The residues recognized by the two anti-pChk1 antibodies are indicated (h). Isogranulatimide (ISOgram), Wortmaninin (Wortman) and ATR inhibitor II (ATRinh II). In f, samples were dephosphorylated with λ protein phosphatase (λ PP) before electrophoresis. i. Immunofluorescence for pChk1 and LAMP1 in Ctr cells. Full field (left) and high magnification (right). Arrows: co-localization. Percentage of co-localization is indicated, n>80 cells in two independent experiments. The experiments in a,e,f,g and h were repeated 5 times and in c and d 3 times. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.

Mentions: Accumulation of both pChk1 and Chk1 in L2A(−) cells led us to investigate the possible contribution of CMA to Chk1 degradation. Inhibition of lysosomal proteolysis by treatment with ammonium chloride and leupeptin led to a marked increase in levels of phosphorylated Chk1 comparable to the one observed upon proteasome inhibition, already described to contribute to Chk1 degradation17 (Fig. 6a; positive controls of the efficiency of the inhibitors are shown in Supplementary Fig. 5a,b (for p62 and LC3-II (lysosomal substrates) and K48-Ubiquitinated proteins (proteasome substrates)). In contrast, inhibition of macroautophagy with 3-methyladenine did not have an effect on intracellular levels of Chk1, suggesting that a fraction of cellular Chk1 is normally degraded in lysosomes even in the absence of genotoxic stress, but not via macroautophagy (Fig. 6a; Supplementary Fig. 5c,d shows a positive control of the efficiency of 3-methyladenine to inhibit the autophagic flux in cells expressing the mCherry-GFP-LC3 reporter). Immunofluorescence in fibroblasts revealed Chk1 association with lysosomal/endosomal compartments (positive for LAMP1) (Fig. 6b). We confirmed a similar association in vivo upon isolation from mouse liver of two populations of lysosomes that participate in CMA (CMA+, enriched in lysosomal hsc70) or in macroautophagy (CMA−, deficient in hsc70)24. Chk1 demonstrated preferential association of Chk1 with CMA-active-lysosomes from mouse and rat liver (Fig. 6c and Supplementary Fig. 6a). Blockage of lysosomal proteolysis by intraperitoneal injection of mice with leupeptin increased Chk1 levels in CMA-active lysosomes confirming its degradation in this compartment in vivo (Fig. 6c; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-characterized CMA substrate25 is shown as positive control). Lastly, in further support of Chk1 degradation by CMA, we also found that a fraction of cellular Chk1 interacts with hsc70, the chaperone responsible for lysosomal targeting of CMA substrates12, and that the net amount of hsc70-bound Chk1 increased in L2A(−) cells, a feature common to bona fide CMA substrate proteins (Fig. 6d).


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

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

Chk1 undergoes lysosomal degradationa. Immunoblot in mouse fibroblasts untreated (−) or treated with NH4Cl and leupeptin (N/L), lactacystin (Lact) or 3-methyladenine (3MA). b. Immunofluorescence for LAMP1 and Chk1 in mouse fibroblasts treated or not with etoposide. Inset in merged show co-localization as white pixels. Percentage of co-localization is indicated, n>80 cells counted per condition in two independent experiments. c. Representative immunoblot of homogenates (Hom), cytosol (Cyt) and lysosomes with high (CMA+) and low (CMA−) CMA activity from livers of mice untreated (−) or injected with leupeptin (Leup). d. Co-immunoprecipitation (IP) of hsc70 with Chk1 in fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)). Inp: 1/10 input; FT: flow through. e–h, Immunoblot of fibroblasts treated or not with etoposide in the presence of the indicated protease and kinase inhibitors. The residues recognized by the two anti-pChk1 antibodies are indicated (h). Isogranulatimide (ISOgram), Wortmaninin (Wortman) and ATR inhibitor II (ATRinh II). In f, samples were dephosphorylated with λ protein phosphatase (λ PP) before electrophoresis. i. Immunofluorescence for pChk1 and LAMP1 in Ctr cells. Full field (left) and high magnification (right). Arrows: co-localization. Percentage of co-localization is indicated, n>80 cells in two independent experiments. The experiments in a,e,f,g and h were repeated 5 times and in c and d 3 times. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
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Figure 6: Chk1 undergoes lysosomal degradationa. Immunoblot in mouse fibroblasts untreated (−) or treated with NH4Cl and leupeptin (N/L), lactacystin (Lact) or 3-methyladenine (3MA). b. Immunofluorescence for LAMP1 and Chk1 in mouse fibroblasts treated or not with etoposide. Inset in merged show co-localization as white pixels. Percentage of co-localization is indicated, n>80 cells counted per condition in two independent experiments. c. Representative immunoblot of homogenates (Hom), cytosol (Cyt) and lysosomes with high (CMA+) and low (CMA−) CMA activity from livers of mice untreated (−) or injected with leupeptin (Leup). d. Co-immunoprecipitation (IP) of hsc70 with Chk1 in fibroblasts control (Ctr) or knock-down for LAMP-2A (L2A(−)). Inp: 1/10 input; FT: flow through. e–h, Immunoblot of fibroblasts treated or not with etoposide in the presence of the indicated protease and kinase inhibitors. The residues recognized by the two anti-pChk1 antibodies are indicated (h). Isogranulatimide (ISOgram), Wortmaninin (Wortman) and ATR inhibitor II (ATRinh II). In f, samples were dephosphorylated with λ protein phosphatase (λ PP) before electrophoresis. i. Immunofluorescence for pChk1 and LAMP1 in Ctr cells. Full field (left) and high magnification (right). Arrows: co-localization. Percentage of co-localization is indicated, n>80 cells in two independent experiments. The experiments in a,e,f,g and h were repeated 5 times and in c and d 3 times. Scale bar: 10μm. Full gels are shown in Supplementary Fig. 8.
Mentions: Accumulation of both pChk1 and Chk1 in L2A(−) cells led us to investigate the possible contribution of CMA to Chk1 degradation. Inhibition of lysosomal proteolysis by treatment with ammonium chloride and leupeptin led to a marked increase in levels of phosphorylated Chk1 comparable to the one observed upon proteasome inhibition, already described to contribute to Chk1 degradation17 (Fig. 6a; positive controls of the efficiency of the inhibitors are shown in Supplementary Fig. 5a,b (for p62 and LC3-II (lysosomal substrates) and K48-Ubiquitinated proteins (proteasome substrates)). In contrast, inhibition of macroautophagy with 3-methyladenine did not have an effect on intracellular levels of Chk1, suggesting that a fraction of cellular Chk1 is normally degraded in lysosomes even in the absence of genotoxic stress, but not via macroautophagy (Fig. 6a; Supplementary Fig. 5c,d shows a positive control of the efficiency of 3-methyladenine to inhibit the autophagic flux in cells expressing the mCherry-GFP-LC3 reporter). Immunofluorescence in fibroblasts revealed Chk1 association with lysosomal/endosomal compartments (positive for LAMP1) (Fig. 6b). We confirmed a similar association in vivo upon isolation from mouse liver of two populations of lysosomes that participate in CMA (CMA+, enriched in lysosomal hsc70) or in macroautophagy (CMA−, deficient in hsc70)24. Chk1 demonstrated preferential association of Chk1 with CMA-active-lysosomes from mouse and rat liver (Fig. 6c and Supplementary Fig. 6a). Blockage of lysosomal proteolysis by intraperitoneal injection of mice with leupeptin increased Chk1 levels in CMA-active lysosomes confirming its degradation in this compartment in vivo (Fig. 6c; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a well-characterized CMA substrate25 is shown as positive control). Lastly, in further support of Chk1 degradation by CMA, we also found that a fraction of cellular Chk1 interacts with hsc70, the chaperone responsible for lysosomal targeting of CMA substrates12, and that the net amount of hsc70-bound Chk1 increased in L2A(−) cells, a feature common to bona fide CMA substrate proteins (Fig. 6d).

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