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The COP9 signalosome is vital for timely repair of DNA double-strand breaks.

Meir M, Galanty Y, Kashani L, Blank M, Khosravi R, Fernández-Ávila MJ, Cruz-García A, Star A, Shochot L, Thomas Y, Garrett LJ, Chamovitz DA, Bodine DM, Kurz T, Huertas P, Ziv Y, Shiloh Y - Nucleic Acids Res. (2015)

Bottom Line: The CSN is essential for the processivity of deep end-resection-the initial step in HRR.Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway.This novel branch of the DSB response thus significantly affects genome stability.

View Article: PubMed Central - PubMed

Affiliation: The David and Inez Myers Laboratory for Cancer Research, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, George S. Wise Faculty of Life sciences, Tel Aviv University, Tel Aviv, 69978 Israel.

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CSN3 is an ATM substrate in the DNA damage response. (A) Mapping of the phosphorylation site on CSN3. Potential ATM targets in this protein (serine or threonine residues followed by glutamine) were mutated in recombinant HA-tagged CSN3, and the various versions of the protein were expressed in HEK293 cells. The band-shift observed after DNA damage induction (500 ng/ml of NCS for 30 min) was specifically abolished by the S410A substitution, indicating that the corresponding modification occurred on S410. (B) Phosphorylation of S410 of CSN3 in response to DNA damage induction is ATM-dependent. The experiment described in (A) was repeated in HEK293 cells that stably expressed shRNA against ATM. Note the considerable reduction in CSN3's band-shift in cells depleted of ATM. (C) Detection of CSN3 phosphorylation in cells using a specific anti-phospho antibody. GFP-tagged CSN3 in wild-type and mutant (S410A) versions was ectopically expressed in U2-OS cells. Following treatment with 50 ng/ml of NCS for 1 h, ectopic CSN3 was immunoprecipitated using an anti-GFP antibody, and the immune complexes were blotted with the indicated antibodies. The ectopic wild-type protein, but not the mutant, reacts with the antibody after induction of DNA damage. In cellular extracts, the anti-phospho antibody detects the phosphorylation of another protein in response to DNA damage (asterisk). Note that endogenous CSN1 and CSN5 are pulled down by ectopic CSN3. (D) Depletion of CSN3 or ATM markedly reduces the pS410-CSN3 phosphorylation signal. U2-OS cells transfected with the indicated siRNAs were treated 72 h later with 50 ng/ml of NCS for 30 min and processed for immunoblotting analysis. (E) CSN3 phosphorylation is dose- and ATM-dependent. HEK293 cells expressing irrelevant (LacZ) or ATM shRNA were treated for 1 h with various doses of NCS, followed by immunoblotting with the indicated antibodies. (F) CSN3 phosphorylation is not dependent on DNA-PK. U2-OS cells were treated with the indicated inhibitors 30 min prior to treatment with 50 ng/ml of NCS. Phosphorylation of the ATM substrate KAP-1 on S824 served to monitor ATM activity in response to DNA damage induction. ATMi: the ATM inhibitor, KU60019 (41), applied at 5 μM concentration. DNA-PKi: the DNA-PK inhibitor, NU7441 (42) applied at 10 μM concentration. (G) Time course of phosphorylation of endogenous CSN3 following treatment of U2-OS cells with 50 ng/ml of NCS.
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Figure 1: CSN3 is an ATM substrate in the DNA damage response. (A) Mapping of the phosphorylation site on CSN3. Potential ATM targets in this protein (serine or threonine residues followed by glutamine) were mutated in recombinant HA-tagged CSN3, and the various versions of the protein were expressed in HEK293 cells. The band-shift observed after DNA damage induction (500 ng/ml of NCS for 30 min) was specifically abolished by the S410A substitution, indicating that the corresponding modification occurred on S410. (B) Phosphorylation of S410 of CSN3 in response to DNA damage induction is ATM-dependent. The experiment described in (A) was repeated in HEK293 cells that stably expressed shRNA against ATM. Note the considerable reduction in CSN3's band-shift in cells depleted of ATM. (C) Detection of CSN3 phosphorylation in cells using a specific anti-phospho antibody. GFP-tagged CSN3 in wild-type and mutant (S410A) versions was ectopically expressed in U2-OS cells. Following treatment with 50 ng/ml of NCS for 1 h, ectopic CSN3 was immunoprecipitated using an anti-GFP antibody, and the immune complexes were blotted with the indicated antibodies. The ectopic wild-type protein, but not the mutant, reacts with the antibody after induction of DNA damage. In cellular extracts, the anti-phospho antibody detects the phosphorylation of another protein in response to DNA damage (asterisk). Note that endogenous CSN1 and CSN5 are pulled down by ectopic CSN3. (D) Depletion of CSN3 or ATM markedly reduces the pS410-CSN3 phosphorylation signal. U2-OS cells transfected with the indicated siRNAs were treated 72 h later with 50 ng/ml of NCS for 30 min and processed for immunoblotting analysis. (E) CSN3 phosphorylation is dose- and ATM-dependent. HEK293 cells expressing irrelevant (LacZ) or ATM shRNA were treated for 1 h with various doses of NCS, followed by immunoblotting with the indicated antibodies. (F) CSN3 phosphorylation is not dependent on DNA-PK. U2-OS cells were treated with the indicated inhibitors 30 min prior to treatment with 50 ng/ml of NCS. Phosphorylation of the ATM substrate KAP-1 on S824 served to monitor ATM activity in response to DNA damage induction. ATMi: the ATM inhibitor, KU60019 (41), applied at 5 μM concentration. DNA-PKi: the DNA-PK inhibitor, NU7441 (42) applied at 10 μM concentration. (G) Time course of phosphorylation of endogenous CSN3 following treatment of U2-OS cells with 50 ng/ml of NCS.

Mentions: Early studies of the ATM protein in our lab included a search for ATM-interacting proteins using the two-hybrid assay. A bait spanning ATM residues 1184–1583, which contain a leucine zipper—a protein-protein interaction motif—identified CSN subunit 8 (CSN8) as prey (Supplementary Figure S1A). Co-immunoprecipitation of endogenous ATM and CSN8 supported the notion of a physical interaction between them (Supplementary Figure S1B), raising the possibility of functional interaction between ATM and CSN and possibly rendering CSN an ATM target. In order to search for DNA damage-induced phosphorylation of CSN subunits in cells, we expressed these subunits in HEK293 cells as ectopic HA-tagged proteins, and treated the cells with the radiomimetic drug neocarzinostatin (NCS) concurrently with a protein phospho-labeling pulse. This experiment revealed marked enhancement of phospho-labeling of CSN subunit 3 (CSN3) following NCS treatment (Supplementary Figure S1C). We further noticed that in response to DNA damage induction, a portion of CSN3 exhibited altered electrophoretic migration (‘gel shift’) (Figure 1A and B). Since this band-shift was largely abolished upon knockdown of ATM (Figure 1B), we assumed that it represented ATM-mediated phosphorylation of CSN3 in cells. In order to map the phosphorylation site, we expressed mutant versions of CSN3 in cells. In each mutant, one of its four S/TQ sequences—potential ATM target sites—was abolished by Ser→Ala substitutions. Only the S410A substitution eliminated the band-shift (Figure 1A), suggesting that the presumed phosphorylation occurred on Ser410. A polyclonal phospho-specific antibody raised to detect this assumed phosphorylation reacted strongly with ectopic wild-type CSN3 expressed following NCS treatment, but not with an S410A mutant version of this protein (Figure 1C). This result indicated that phosphorylation of CSN3 on Ser410 occurred in cells in response to DNA damage and was detected by the antibody. The antibody also detected the phosphorylation of endogenous CSN3, which was ATM- and dose-dependent (Figure 1D–G), and DNA-PK independent (Figure 1F); it peaked within 30 min of damage induction and subsided several hours later—a time course typical of many ATM-mediated protein phosphorylations (Figure 1G). These results established that Ser410 of CSN3 is an ATM target in response to DSB induction and suggested a role for CSN in the ATM-mediated DSB response.


The COP9 signalosome is vital for timely repair of DNA double-strand breaks.

Meir M, Galanty Y, Kashani L, Blank M, Khosravi R, Fernández-Ávila MJ, Cruz-García A, Star A, Shochot L, Thomas Y, Garrett LJ, Chamovitz DA, Bodine DM, Kurz T, Huertas P, Ziv Y, Shiloh Y - Nucleic Acids Res. (2015)

CSN3 is an ATM substrate in the DNA damage response. (A) Mapping of the phosphorylation site on CSN3. Potential ATM targets in this protein (serine or threonine residues followed by glutamine) were mutated in recombinant HA-tagged CSN3, and the various versions of the protein were expressed in HEK293 cells. The band-shift observed after DNA damage induction (500 ng/ml of NCS for 30 min) was specifically abolished by the S410A substitution, indicating that the corresponding modification occurred on S410. (B) Phosphorylation of S410 of CSN3 in response to DNA damage induction is ATM-dependent. The experiment described in (A) was repeated in HEK293 cells that stably expressed shRNA against ATM. Note the considerable reduction in CSN3's band-shift in cells depleted of ATM. (C) Detection of CSN3 phosphorylation in cells using a specific anti-phospho antibody. GFP-tagged CSN3 in wild-type and mutant (S410A) versions was ectopically expressed in U2-OS cells. Following treatment with 50 ng/ml of NCS for 1 h, ectopic CSN3 was immunoprecipitated using an anti-GFP antibody, and the immune complexes were blotted with the indicated antibodies. The ectopic wild-type protein, but not the mutant, reacts with the antibody after induction of DNA damage. In cellular extracts, the anti-phospho antibody detects the phosphorylation of another protein in response to DNA damage (asterisk). Note that endogenous CSN1 and CSN5 are pulled down by ectopic CSN3. (D) Depletion of CSN3 or ATM markedly reduces the pS410-CSN3 phosphorylation signal. U2-OS cells transfected with the indicated siRNAs were treated 72 h later with 50 ng/ml of NCS for 30 min and processed for immunoblotting analysis. (E) CSN3 phosphorylation is dose- and ATM-dependent. HEK293 cells expressing irrelevant (LacZ) or ATM shRNA were treated for 1 h with various doses of NCS, followed by immunoblotting with the indicated antibodies. (F) CSN3 phosphorylation is not dependent on DNA-PK. U2-OS cells were treated with the indicated inhibitors 30 min prior to treatment with 50 ng/ml of NCS. Phosphorylation of the ATM substrate KAP-1 on S824 served to monitor ATM activity in response to DNA damage induction. ATMi: the ATM inhibitor, KU60019 (41), applied at 5 μM concentration. DNA-PKi: the DNA-PK inhibitor, NU7441 (42) applied at 10 μM concentration. (G) Time course of phosphorylation of endogenous CSN3 following treatment of U2-OS cells with 50 ng/ml of NCS.
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Figure 1: CSN3 is an ATM substrate in the DNA damage response. (A) Mapping of the phosphorylation site on CSN3. Potential ATM targets in this protein (serine or threonine residues followed by glutamine) were mutated in recombinant HA-tagged CSN3, and the various versions of the protein were expressed in HEK293 cells. The band-shift observed after DNA damage induction (500 ng/ml of NCS for 30 min) was specifically abolished by the S410A substitution, indicating that the corresponding modification occurred on S410. (B) Phosphorylation of S410 of CSN3 in response to DNA damage induction is ATM-dependent. The experiment described in (A) was repeated in HEK293 cells that stably expressed shRNA against ATM. Note the considerable reduction in CSN3's band-shift in cells depleted of ATM. (C) Detection of CSN3 phosphorylation in cells using a specific anti-phospho antibody. GFP-tagged CSN3 in wild-type and mutant (S410A) versions was ectopically expressed in U2-OS cells. Following treatment with 50 ng/ml of NCS for 1 h, ectopic CSN3 was immunoprecipitated using an anti-GFP antibody, and the immune complexes were blotted with the indicated antibodies. The ectopic wild-type protein, but not the mutant, reacts with the antibody after induction of DNA damage. In cellular extracts, the anti-phospho antibody detects the phosphorylation of another protein in response to DNA damage (asterisk). Note that endogenous CSN1 and CSN5 are pulled down by ectopic CSN3. (D) Depletion of CSN3 or ATM markedly reduces the pS410-CSN3 phosphorylation signal. U2-OS cells transfected with the indicated siRNAs were treated 72 h later with 50 ng/ml of NCS for 30 min and processed for immunoblotting analysis. (E) CSN3 phosphorylation is dose- and ATM-dependent. HEK293 cells expressing irrelevant (LacZ) or ATM shRNA were treated for 1 h with various doses of NCS, followed by immunoblotting with the indicated antibodies. (F) CSN3 phosphorylation is not dependent on DNA-PK. U2-OS cells were treated with the indicated inhibitors 30 min prior to treatment with 50 ng/ml of NCS. Phosphorylation of the ATM substrate KAP-1 on S824 served to monitor ATM activity in response to DNA damage induction. ATMi: the ATM inhibitor, KU60019 (41), applied at 5 μM concentration. DNA-PKi: the DNA-PK inhibitor, NU7441 (42) applied at 10 μM concentration. (G) Time course of phosphorylation of endogenous CSN3 following treatment of U2-OS cells with 50 ng/ml of NCS.
Mentions: Early studies of the ATM protein in our lab included a search for ATM-interacting proteins using the two-hybrid assay. A bait spanning ATM residues 1184–1583, which contain a leucine zipper—a protein-protein interaction motif—identified CSN subunit 8 (CSN8) as prey (Supplementary Figure S1A). Co-immunoprecipitation of endogenous ATM and CSN8 supported the notion of a physical interaction between them (Supplementary Figure S1B), raising the possibility of functional interaction between ATM and CSN and possibly rendering CSN an ATM target. In order to search for DNA damage-induced phosphorylation of CSN subunits in cells, we expressed these subunits in HEK293 cells as ectopic HA-tagged proteins, and treated the cells with the radiomimetic drug neocarzinostatin (NCS) concurrently with a protein phospho-labeling pulse. This experiment revealed marked enhancement of phospho-labeling of CSN subunit 3 (CSN3) following NCS treatment (Supplementary Figure S1C). We further noticed that in response to DNA damage induction, a portion of CSN3 exhibited altered electrophoretic migration (‘gel shift’) (Figure 1A and B). Since this band-shift was largely abolished upon knockdown of ATM (Figure 1B), we assumed that it represented ATM-mediated phosphorylation of CSN3 in cells. In order to map the phosphorylation site, we expressed mutant versions of CSN3 in cells. In each mutant, one of its four S/TQ sequences—potential ATM target sites—was abolished by Ser→Ala substitutions. Only the S410A substitution eliminated the band-shift (Figure 1A), suggesting that the presumed phosphorylation occurred on Ser410. A polyclonal phospho-specific antibody raised to detect this assumed phosphorylation reacted strongly with ectopic wild-type CSN3 expressed following NCS treatment, but not with an S410A mutant version of this protein (Figure 1C). This result indicated that phosphorylation of CSN3 on Ser410 occurred in cells in response to DNA damage and was detected by the antibody. The antibody also detected the phosphorylation of endogenous CSN3, which was ATM- and dose-dependent (Figure 1D–G), and DNA-PK independent (Figure 1F); it peaked within 30 min of damage induction and subsided several hours later—a time course typical of many ATM-mediated protein phosphorylations (Figure 1G). These results established that Ser410 of CSN3 is an ATM target in response to DSB induction and suggested a role for CSN in the ATM-mediated DSB response.

Bottom Line: The CSN is essential for the processivity of deep end-resection-the initial step in HRR.Cullin 4a (CUL4A) is recruited to DSB sites in a CSN- and neddylation-dependent manner, suggesting that CSN partners with CRL4 in this pathway.This novel branch of the DSB response thus significantly affects genome stability.

View Article: PubMed Central - PubMed

Affiliation: The David and Inez Myers Laboratory for Cancer Research, Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, George S. Wise Faculty of Life sciences, Tel Aviv University, Tel Aviv, 69978 Israel.

Show MeSH
Related in: MedlinePlus