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