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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

CMA blockage alters Chk1 dynamicsa,b. Immunoblot of nuclear fractions (a) and immunofluorescence of Chk1 (b) in the same cells 12h after treatment with 100μM etoposide. Dashed lines: nuclear profiles. Scale bar: 10μm. Right: Quantification of the percentage of cells with nuclear Chk1 signal, n>40 cells counted from three different experiments. c. Immunofluorescence for pChk1 in the same cells after 12h treatment with 100μM etoposide. Right: Quantification of the percentage of nuclear pChk1 in >25 cells/condition and experiment (n=4 independent experiments). Nuclei were highlighted with DAPI. d. Immunofluorescence for Chk1 in cells control and knock-down for Chk1 (shChk1). Single channel and merge with DAPI are shown. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Scale bar: 10μm Full gels are shown in Supplementary Fig. 8.
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Figure 5: CMA blockage alters Chk1 dynamicsa,b. Immunoblot of nuclear fractions (a) and immunofluorescence of Chk1 (b) in the same cells 12h after treatment with 100μM etoposide. Dashed lines: nuclear profiles. Scale bar: 10μm. Right: Quantification of the percentage of cells with nuclear Chk1 signal, n>40 cells counted from three different experiments. c. Immunofluorescence for pChk1 in the same cells after 12h treatment with 100μM etoposide. Right: Quantification of the percentage of nuclear pChk1 in >25 cells/condition and experiment (n=4 independent experiments). Nuclei were highlighted with DAPI. d. Immunofluorescence for Chk1 in cells control and knock-down for Chk1 (shChk1). Single channel and merge with DAPI are shown. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Scale bar: 10μm Full gels are shown in Supplementary Fig. 8.

Mentions: Immunoblot in nuclear fractions (Fig. 5a) and immunofluorescence for Chk1 (Fig. 5b) and pChk1 (Fig. 5c) confirmed that a higher portion of cellular Chk1 was retained in the nucleus of L2A(−) cells treated with etoposide and that this nuclear Chk1 was phosphorylated. Similar experiments in Chk1 knock-down cells confirmed the specificity of the Chk1 staining (Fig. 5d).


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

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

CMA blockage alters Chk1 dynamicsa,b. Immunoblot of nuclear fractions (a) and immunofluorescence of Chk1 (b) in the same cells 12h after treatment with 100μM etoposide. Dashed lines: nuclear profiles. Scale bar: 10μm. Right: Quantification of the percentage of cells with nuclear Chk1 signal, n>40 cells counted from three different experiments. c. Immunofluorescence for pChk1 in the same cells after 12h treatment with 100μM etoposide. Right: Quantification of the percentage of nuclear pChk1 in >25 cells/condition and experiment (n=4 independent experiments). Nuclei were highlighted with DAPI. d. Immunofluorescence for Chk1 in cells control and knock-down for Chk1 (shChk1). Single channel and merge with DAPI are shown. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Scale bar: 10μm Full gels are shown in Supplementary Fig. 8.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4400843&req=5

Figure 5: CMA blockage alters Chk1 dynamicsa,b. Immunoblot of nuclear fractions (a) and immunofluorescence of Chk1 (b) in the same cells 12h after treatment with 100μM etoposide. Dashed lines: nuclear profiles. Scale bar: 10μm. Right: Quantification of the percentage of cells with nuclear Chk1 signal, n>40 cells counted from three different experiments. c. Immunofluorescence for pChk1 in the same cells after 12h treatment with 100μM etoposide. Right: Quantification of the percentage of nuclear pChk1 in >25 cells/condition and experiment (n=4 independent experiments). Nuclei were highlighted with DAPI. d. Immunofluorescence for Chk1 in cells control and knock-down for Chk1 (shChk1). Single channel and merge with DAPI are shown. All values are mean+s.e.m. (unpaired two-tailed t-test). *P<0.05, **P <0.005 or ***P <0.0005. Scale bar: 10μm Full gels are shown in Supplementary Fig. 8.
Mentions: Immunoblot in nuclear fractions (Fig. 5a) and immunofluorescence for Chk1 (Fig. 5b) and pChk1 (Fig. 5c) confirmed that a higher portion of cellular Chk1 was retained in the nucleus of L2A(−) cells treated with etoposide and that this nuclear Chk1 was phosphorylated. Similar experiments in Chk1 knock-down cells confirmed the specificity of the Chk1 staining (Fig. 5d).

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