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Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin.

Melander F, Bekker-Jensen S, Falck J, Bartek J, Mailand N, Lukas J - J. Cell Biol. (2008)

Bottom Line: This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1.Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells.Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Biology and 2Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.

ABSTRACT
DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.

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Phosphorylation of the SDT clusters by CK2 promotes MDC1–NBS1 interaction in vitro. (A) CK2 phosphorylates the SDT repeats of MDC1 in vitro. GST-MDC1 fragment spanning the entire SDT region (WT; amino acids 181–480) or its derivative in which all 12 potential CK2 sites were mutated to alanines (12A) was purified from E. coli and subjected to an in vitro kinase assay using a recombinant CK2 and 32P-labeled ATP. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the reaction. After separation by SDS-PAGE, the gel was stained with Coomassie blue to validate the equal input of the GST-MDC1 fragments (bottom) and was dried and analyzed by phosphorimager (top). (B) The purified SDT region of MDC1 interacts with NBS1 in a phosphorylation- and CK2-dependent manner. GST-MDC1 fragments (as in A) were phosphorylated or not phosphorylated with recombinant CK2 (using nonradioactive ATP), captured on glutathione–Sepharose beads, and incubated with in vitro–translated 35S-labeled NBS1 WT or its derivative with a point mutation within the FHA domain (R28) for 2 h. Bound complexes were resolved by SDS-PAGE. The gel was stained with Coomassie blue to validate equal input of the GST-MDC1 proteins (bottom), and the bound NBS1 was analyzed by phosphorimager (top). CB, Coomassie blue.
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fig4: Phosphorylation of the SDT clusters by CK2 promotes MDC1–NBS1 interaction in vitro. (A) CK2 phosphorylates the SDT repeats of MDC1 in vitro. GST-MDC1 fragment spanning the entire SDT region (WT; amino acids 181–480) or its derivative in which all 12 potential CK2 sites were mutated to alanines (12A) was purified from E. coli and subjected to an in vitro kinase assay using a recombinant CK2 and 32P-labeled ATP. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the reaction. After separation by SDS-PAGE, the gel was stained with Coomassie blue to validate the equal input of the GST-MDC1 fragments (bottom) and was dried and analyzed by phosphorimager (top). (B) The purified SDT region of MDC1 interacts with NBS1 in a phosphorylation- and CK2-dependent manner. GST-MDC1 fragments (as in A) were phosphorylated or not phosphorylated with recombinant CK2 (using nonradioactive ATP), captured on glutathione–Sepharose beads, and incubated with in vitro–translated 35S-labeled NBS1 WT or its derivative with a point mutation within the FHA domain (R28) for 2 h. Bound complexes were resolved by SDS-PAGE. The gel was stained with Coomassie blue to validate equal input of the GST-MDC1 proteins (bottom), and the bound NBS1 was analyzed by phosphorimager (top). CB, Coomassie blue.

Mentions: To test this hypothesis, we first tried to identify the kinase that can phosphorylate the SDT repeats. Sequence analysis by the recently introduced NetworKIN approach (Linding et al., 2007) revealed that the SDT clusters strongly resemble consensus sites for the acidophilic protein kinase CK2. Therefore, from Escherichia coli, we purified a GST-tagged SDT fragment (spanning amino acids 181–480) and its variant in which the Ser/Thr residues in all six SDT repeats were changed to Ala (designated as 12A) and subjected these fragments to phosphorylation by CK2 in an in vitro kinase assay. Indeed, CK2 efficiently phosphorylated the WT SDT fragment in vitro (Fig. 4 A, lane 5), and this phosphorylation was inhibited by adding the CK2 inhibitor into the kinase reaction (Fig. 4 A, lane 8). Significantly, phosphorylation of the SDT-12A mutant was very inefficient (Fig. 4 A, lane 6), suggesting that the bulk of the CK2-mediated phosphorylations within the MDC1 N terminus was indeed targeted to the SDT repeats.


Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin.

Melander F, Bekker-Jensen S, Falck J, Bartek J, Mailand N, Lukas J - J. Cell Biol. (2008)

Phosphorylation of the SDT clusters by CK2 promotes MDC1–NBS1 interaction in vitro. (A) CK2 phosphorylates the SDT repeats of MDC1 in vitro. GST-MDC1 fragment spanning the entire SDT region (WT; amino acids 181–480) or its derivative in which all 12 potential CK2 sites were mutated to alanines (12A) was purified from E. coli and subjected to an in vitro kinase assay using a recombinant CK2 and 32P-labeled ATP. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the reaction. After separation by SDS-PAGE, the gel was stained with Coomassie blue to validate the equal input of the GST-MDC1 fragments (bottom) and was dried and analyzed by phosphorimager (top). (B) The purified SDT region of MDC1 interacts with NBS1 in a phosphorylation- and CK2-dependent manner. GST-MDC1 fragments (as in A) were phosphorylated or not phosphorylated with recombinant CK2 (using nonradioactive ATP), captured on glutathione–Sepharose beads, and incubated with in vitro–translated 35S-labeled NBS1 WT or its derivative with a point mutation within the FHA domain (R28) for 2 h. Bound complexes were resolved by SDS-PAGE. The gel was stained with Coomassie blue to validate equal input of the GST-MDC1 proteins (bottom), and the bound NBS1 was analyzed by phosphorimager (top). CB, Coomassie blue.
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fig4: Phosphorylation of the SDT clusters by CK2 promotes MDC1–NBS1 interaction in vitro. (A) CK2 phosphorylates the SDT repeats of MDC1 in vitro. GST-MDC1 fragment spanning the entire SDT region (WT; amino acids 181–480) or its derivative in which all 12 potential CK2 sites were mutated to alanines (12A) was purified from E. coli and subjected to an in vitro kinase assay using a recombinant CK2 and 32P-labeled ATP. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the reaction. After separation by SDS-PAGE, the gel was stained with Coomassie blue to validate the equal input of the GST-MDC1 fragments (bottom) and was dried and analyzed by phosphorimager (top). (B) The purified SDT region of MDC1 interacts with NBS1 in a phosphorylation- and CK2-dependent manner. GST-MDC1 fragments (as in A) were phosphorylated or not phosphorylated with recombinant CK2 (using nonradioactive ATP), captured on glutathione–Sepharose beads, and incubated with in vitro–translated 35S-labeled NBS1 WT or its derivative with a point mutation within the FHA domain (R28) for 2 h. Bound complexes were resolved by SDS-PAGE. The gel was stained with Coomassie blue to validate equal input of the GST-MDC1 proteins (bottom), and the bound NBS1 was analyzed by phosphorimager (top). CB, Coomassie blue.
Mentions: To test this hypothesis, we first tried to identify the kinase that can phosphorylate the SDT repeats. Sequence analysis by the recently introduced NetworKIN approach (Linding et al., 2007) revealed that the SDT clusters strongly resemble consensus sites for the acidophilic protein kinase CK2. Therefore, from Escherichia coli, we purified a GST-tagged SDT fragment (spanning amino acids 181–480) and its variant in which the Ser/Thr residues in all six SDT repeats were changed to Ala (designated as 12A) and subjected these fragments to phosphorylation by CK2 in an in vitro kinase assay. Indeed, CK2 efficiently phosphorylated the WT SDT fragment in vitro (Fig. 4 A, lane 5), and this phosphorylation was inhibited by adding the CK2 inhibitor into the kinase reaction (Fig. 4 A, lane 8). Significantly, phosphorylation of the SDT-12A mutant was very inefficient (Fig. 4 A, lane 6), suggesting that the bulk of the CK2-mediated phosphorylations within the MDC1 N terminus was indeed targeted to the SDT repeats.

Bottom Line: This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1.Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells.Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Biology and 2Centre for Genotoxic Stress Research, Danish Cancer Society, DK-2100 Copenhagen, Denmark.

ABSTRACT
DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.

Show MeSH
Related in: MedlinePlus