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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

Changes in the MRN complex components in cells with compromised CMAa. Immunoblot for Mre11 and Nbs1 in cells control (Ctr) or knock-down for L2A (L2A−) untreated (unt) or treated with 100μM for 12h and including samples treated with λ protein phosphatase (λ PPase) before electrophoresis. Arrows: levels of shift in the protein molecular weight. b. Immunoblot for L2A(−) cells treated with etoposide and caffeine as indicated. c. Immunoblot for Mre11 and phosphorylated Chk1 (S345: pChk1) in L2A(−) cells treated with 100μM etoposide for 12h without additions (none) or in the presence of the indicated kinase inhibitors (KU: ATM inhibitor KU55933; ATRi: ATR inhibitor II; Caff: caffeine; Chk1i: Chk1 inhibitor; Tor1: mTor inhibitor Torin 1. Untreated L2A(−) and Ctr cells are shown for reference. Left: Image of a similar experiment resolved by a longer electrophoresis run to appreciate the molecular weight shift of Mre1. Discontinued lines indicate center of untreated (blue) or etoposide treated (red) bands the highest molecular weight Mre11 signal (green). d. Immunoblot for the indicated proteins in cytosolic and nuclear fractions in Ctr and L2A(−) cells untreated or etoposide-treated (24h). Lamin A/C and GAPDH are shown as markers for nuclear and cytosolic fraction purity, respectively. e–g. Immunoprecipitation (IP) of Mre11 (e), Nbs1 (f) or Rad50 (g) in cellular extracts from Ctr or L2A(−) cells untreated (None) or treated with etoposide for 24h. Inp, input; FT, flow-through. h,i. Nuclear fractions from etoposide-treated Ctr and L2A(−) cells treated with etoposide were subjected to continuous sucrose density gradients. Representative immunoblots (h) and plots of the distribution of proteins along the gradient (i). j, k. Immunoblots in cells Ctr expressing the indicated Chk1 variants (j) or L2A(−) cells upon Chk1 knock-down (shChk1) (k). The experiments in a–d were repeated 4 times and in e–j 3 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
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Figure 9: Changes in the MRN complex components in cells with compromised CMAa. Immunoblot for Mre11 and Nbs1 in cells control (Ctr) or knock-down for L2A (L2A−) untreated (unt) or treated with 100μM for 12h and including samples treated with λ protein phosphatase (λ PPase) before electrophoresis. Arrows: levels of shift in the protein molecular weight. b. Immunoblot for L2A(−) cells treated with etoposide and caffeine as indicated. c. Immunoblot for Mre11 and phosphorylated Chk1 (S345: pChk1) in L2A(−) cells treated with 100μM etoposide for 12h without additions (none) or in the presence of the indicated kinase inhibitors (KU: ATM inhibitor KU55933; ATRi: ATR inhibitor II; Caff: caffeine; Chk1i: Chk1 inhibitor; Tor1: mTor inhibitor Torin 1. Untreated L2A(−) and Ctr cells are shown for reference. Left: Image of a similar experiment resolved by a longer electrophoresis run to appreciate the molecular weight shift of Mre1. Discontinued lines indicate center of untreated (blue) or etoposide treated (red) bands the highest molecular weight Mre11 signal (green). d. Immunoblot for the indicated proteins in cytosolic and nuclear fractions in Ctr and L2A(−) cells untreated or etoposide-treated (24h). Lamin A/C and GAPDH are shown as markers for nuclear and cytosolic fraction purity, respectively. e–g. Immunoprecipitation (IP) of Mre11 (e), Nbs1 (f) or Rad50 (g) in cellular extracts from Ctr or L2A(−) cells untreated (None) or treated with etoposide for 24h. Inp, input; FT, flow-through. h,i. Nuclear fractions from etoposide-treated Ctr and L2A(−) cells treated with etoposide were subjected to continuous sucrose density gradients. Representative immunoblots (h) and plots of the distribution of proteins along the gradient (i). j, k. Immunoblots in cells Ctr expressing the indicated Chk1 variants (j) or L2A(−) cells upon Chk1 knock-down (shChk1) (k). The experiments in a–d were repeated 4 times and in e–j 3 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.

Mentions: The increase in molecular weight of Mre11 and Nbs1 in L2A(−) cells is likely due to enhanced phosphorylation, as phosphatase treatment of the samples prior to electrophoresis eliminated the difference (Fig. 9a). This etoposide-induced hyperphosphorylation of Mre11 and Nbs1 in L2A(−) cells was prevented using caffeine or an ATR specific inhibitor but not ATM or Chk1 inhibitors (Fig. 9b, c), suggesting a possible disruption of the Chk1-ATR regulatory circuit upon CMA blockage that results in abnormal ATR-dependent phosphorylation of MRN proteins.


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

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

Changes in the MRN complex components in cells with compromised CMAa. Immunoblot for Mre11 and Nbs1 in cells control (Ctr) or knock-down for L2A (L2A−) untreated (unt) or treated with 100μM for 12h and including samples treated with λ protein phosphatase (λ PPase) before electrophoresis. Arrows: levels of shift in the protein molecular weight. b. Immunoblot for L2A(−) cells treated with etoposide and caffeine as indicated. c. Immunoblot for Mre11 and phosphorylated Chk1 (S345: pChk1) in L2A(−) cells treated with 100μM etoposide for 12h without additions (none) or in the presence of the indicated kinase inhibitors (KU: ATM inhibitor KU55933; ATRi: ATR inhibitor II; Caff: caffeine; Chk1i: Chk1 inhibitor; Tor1: mTor inhibitor Torin 1. Untreated L2A(−) and Ctr cells are shown for reference. Left: Image of a similar experiment resolved by a longer electrophoresis run to appreciate the molecular weight shift of Mre1. Discontinued lines indicate center of untreated (blue) or etoposide treated (red) bands the highest molecular weight Mre11 signal (green). d. Immunoblot for the indicated proteins in cytosolic and nuclear fractions in Ctr and L2A(−) cells untreated or etoposide-treated (24h). Lamin A/C and GAPDH are shown as markers for nuclear and cytosolic fraction purity, respectively. e–g. Immunoprecipitation (IP) of Mre11 (e), Nbs1 (f) or Rad50 (g) in cellular extracts from Ctr or L2A(−) cells untreated (None) or treated with etoposide for 24h. Inp, input; FT, flow-through. h,i. Nuclear fractions from etoposide-treated Ctr and L2A(−) cells treated with etoposide were subjected to continuous sucrose density gradients. Representative immunoblots (h) and plots of the distribution of proteins along the gradient (i). j, k. Immunoblots in cells Ctr expressing the indicated Chk1 variants (j) or L2A(−) cells upon Chk1 knock-down (shChk1) (k). The experiments in a–d were repeated 4 times and in e–j 3 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
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Figure 9: Changes in the MRN complex components in cells with compromised CMAa. Immunoblot for Mre11 and Nbs1 in cells control (Ctr) or knock-down for L2A (L2A−) untreated (unt) or treated with 100μM for 12h and including samples treated with λ protein phosphatase (λ PPase) before electrophoresis. Arrows: levels of shift in the protein molecular weight. b. Immunoblot for L2A(−) cells treated with etoposide and caffeine as indicated. c. Immunoblot for Mre11 and phosphorylated Chk1 (S345: pChk1) in L2A(−) cells treated with 100μM etoposide for 12h without additions (none) or in the presence of the indicated kinase inhibitors (KU: ATM inhibitor KU55933; ATRi: ATR inhibitor II; Caff: caffeine; Chk1i: Chk1 inhibitor; Tor1: mTor inhibitor Torin 1. Untreated L2A(−) and Ctr cells are shown for reference. Left: Image of a similar experiment resolved by a longer electrophoresis run to appreciate the molecular weight shift of Mre1. Discontinued lines indicate center of untreated (blue) or etoposide treated (red) bands the highest molecular weight Mre11 signal (green). d. Immunoblot for the indicated proteins in cytosolic and nuclear fractions in Ctr and L2A(−) cells untreated or etoposide-treated (24h). Lamin A/C and GAPDH are shown as markers for nuclear and cytosolic fraction purity, respectively. e–g. Immunoprecipitation (IP) of Mre11 (e), Nbs1 (f) or Rad50 (g) in cellular extracts from Ctr or L2A(−) cells untreated (None) or treated with etoposide for 24h. Inp, input; FT, flow-through. h,i. Nuclear fractions from etoposide-treated Ctr and L2A(−) cells treated with etoposide were subjected to continuous sucrose density gradients. Representative immunoblots (h) and plots of the distribution of proteins along the gradient (i). j, k. Immunoblots in cells Ctr expressing the indicated Chk1 variants (j) or L2A(−) cells upon Chk1 knock-down (shChk1) (k). The experiments in a–d were repeated 4 times and in e–j 3 times. All values are mean+s.e.m. (unpaired two-tailed t-test). *P <0.005. Full gels are shown in Supplementary Fig. 8.
Mentions: The increase in molecular weight of Mre11 and Nbs1 in L2A(−) cells is likely due to enhanced phosphorylation, as phosphatase treatment of the samples prior to electrophoresis eliminated the difference (Fig. 9a). This etoposide-induced hyperphosphorylation of Mre11 and Nbs1 in L2A(−) cells was prevented using caffeine or an ATR specific inhibitor but not ATM or Chk1 inhibitors (Fig. 9b, c), suggesting a possible disruption of the Chk1-ATR regulatory circuit upon CMA blockage that results in abnormal ATR-dependent phosphorylation of MRN proteins.

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