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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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Inhibition of CK2 reduces phosphorylation of the SDT repeats in vivo. (A) Treatment of cells with a CK2 inhibitor reduces phosphate incorporation into the SDT-containing fragment of MDC1. U2OS cells were transfected with the HA-tagged fragment of MDC1 spanning the entire SDT region (amino acids 210–460) and labeled with 32P for 4 h. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the culture medium for the entire labeling period. Lysates from labeled cells were immunoprecipitated with an anti-HA antibody and, after resolving the proteins on SDS-PAGE, were analyzed by the phosphorimager for the extent of 32P incorporation (top). The equal input of proteins in each lane was subsequently verified by immunoblotting with the anti-HA antibody (bottom). (B) Inhibition of CK2 impairs phosphate incorporation into the dominant SDT repeats. The bands corresponding to the labeled SDT fragments from A were excised from the gel and subjected to tryptic digestion. The resulting phosphopeptides were separated by electrophoresis followed by thin-layer chromatography and analyzed by phosphorimager. Note that the 32P incorporation into two prominent spots representing Ser299/Thr301 (thin arrows) and Ser453/Thr455 (bold arrows) is reduced to ∼50% in cells treated with CK2 inhibitor (CK2i). Dotted circles indicate spots whose labeling did not appreciably change after CK2 inhibition; the intensity of the 32P signal associated with these spots was taken as a baseline to estimate the decrease in phosphate incorporation into the SDT repeats. Numbers indicate the charge of the phosphopeptides.
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fig5: Inhibition of CK2 reduces phosphorylation of the SDT repeats in vivo. (A) Treatment of cells with a CK2 inhibitor reduces phosphate incorporation into the SDT-containing fragment of MDC1. U2OS cells were transfected with the HA-tagged fragment of MDC1 spanning the entire SDT region (amino acids 210–460) and labeled with 32P for 4 h. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the culture medium for the entire labeling period. Lysates from labeled cells were immunoprecipitated with an anti-HA antibody and, after resolving the proteins on SDS-PAGE, were analyzed by the phosphorimager for the extent of 32P incorporation (top). The equal input of proteins in each lane was subsequently verified by immunoblotting with the anti-HA antibody (bottom). (B) Inhibition of CK2 impairs phosphate incorporation into the dominant SDT repeats. The bands corresponding to the labeled SDT fragments from A were excised from the gel and subjected to tryptic digestion. The resulting phosphopeptides were separated by electrophoresis followed by thin-layer chromatography and analyzed by phosphorimager. Note that the 32P incorporation into two prominent spots representing Ser299/Thr301 (thin arrows) and Ser453/Thr455 (bold arrows) is reduced to ∼50% in cells treated with CK2 inhibitor (CK2i). Dotted circles indicate spots whose labeling did not appreciably change after CK2 inhibition; the intensity of the 32P signal associated with these spots was taken as a baseline to estimate the decrease in phosphate incorporation into the SDT repeats. Numbers indicate the charge of the phosphopeptides.

Mentions: To explore the emerging cross talk between CK2 and the N terminus of MDC1, we tested the impact of CK2 inhibition on SDT phosphorylation in cells. Indeed, preincubation of U2OS cells with a CK2 inhibitor significantly reduced the overall incorporation of radioactive phosphate to the ectopically expressed SDT fragment (Fig. 5 A). More importantly in this context, the ensuing phosphopeptide analysis revealed that this was accompanied by an ∼50% reduction of phosphate incorporation into two prominent SDT repeats (Ser299/Thr301 and Ser453/Ser455; Fig. 5 B). Thus, CK2 can at least partially phosphorylate the SDT repeats in vivo. To further support this conclusion, we immunopurified HA-tagged MDC1 N terminus (Fig. 6 A) or endogenous MDC1 (Fig. 6 B) from cells and subjected these immunocomplexes to an in vitro kinase assay (without any additional substrate). In both cases, MDC1 copurified with an autocatalytic kinase activity that was strongly attenuated by a specific CK2 inhibitor (Fig. 6, A and B). Interestingly, this MDC1-associated CK2 activity was clearly present in undamaged cells (Fig. 6, A and B; lane 1) and did not appreciably increase after exposing the cells to IR (Fig. 6 A, lane 2). Collectively, the data in Figs. 4–6 show that CK2 can phosphorylate the SDT repeats and suggest that this may contribute to trigger a productive interaction between the MDC1 N terminus and the FHA domain of NBS1.


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)

Inhibition of CK2 reduces phosphorylation of the SDT repeats in vivo. (A) Treatment of cells with a CK2 inhibitor reduces phosphate incorporation into the SDT-containing fragment of MDC1. U2OS cells were transfected with the HA-tagged fragment of MDC1 spanning the entire SDT region (amino acids 210–460) and labeled with 32P for 4 h. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the culture medium for the entire labeling period. Lysates from labeled cells were immunoprecipitated with an anti-HA antibody and, after resolving the proteins on SDS-PAGE, were analyzed by the phosphorimager for the extent of 32P incorporation (top). The equal input of proteins in each lane was subsequently verified by immunoblotting with the anti-HA antibody (bottom). (B) Inhibition of CK2 impairs phosphate incorporation into the dominant SDT repeats. The bands corresponding to the labeled SDT fragments from A were excised from the gel and subjected to tryptic digestion. The resulting phosphopeptides were separated by electrophoresis followed by thin-layer chromatography and analyzed by phosphorimager. Note that the 32P incorporation into two prominent spots representing Ser299/Thr301 (thin arrows) and Ser453/Thr455 (bold arrows) is reduced to ∼50% in cells treated with CK2 inhibitor (CK2i). Dotted circles indicate spots whose labeling did not appreciably change after CK2 inhibition; the intensity of the 32P signal associated with these spots was taken as a baseline to estimate the decrease in phosphate incorporation into the SDT repeats. Numbers indicate the charge of the phosphopeptides.
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Related In: Results  -  Collection

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fig5: Inhibition of CK2 reduces phosphorylation of the SDT repeats in vivo. (A) Treatment of cells with a CK2 inhibitor reduces phosphate incorporation into the SDT-containing fragment of MDC1. U2OS cells were transfected with the HA-tagged fragment of MDC1 spanning the entire SDT region (amino acids 210–460) and labeled with 32P for 4 h. Where indicated, 10 μM of the DMAT CK2 inhibitor (CK2i) was added to the culture medium for the entire labeling period. Lysates from labeled cells were immunoprecipitated with an anti-HA antibody and, after resolving the proteins on SDS-PAGE, were analyzed by the phosphorimager for the extent of 32P incorporation (top). The equal input of proteins in each lane was subsequently verified by immunoblotting with the anti-HA antibody (bottom). (B) Inhibition of CK2 impairs phosphate incorporation into the dominant SDT repeats. The bands corresponding to the labeled SDT fragments from A were excised from the gel and subjected to tryptic digestion. The resulting phosphopeptides were separated by electrophoresis followed by thin-layer chromatography and analyzed by phosphorimager. Note that the 32P incorporation into two prominent spots representing Ser299/Thr301 (thin arrows) and Ser453/Thr455 (bold arrows) is reduced to ∼50% in cells treated with CK2 inhibitor (CK2i). Dotted circles indicate spots whose labeling did not appreciably change after CK2 inhibition; the intensity of the 32P signal associated with these spots was taken as a baseline to estimate the decrease in phosphate incorporation into the SDT repeats. Numbers indicate the charge of the phosphopeptides.
Mentions: To explore the emerging cross talk between CK2 and the N terminus of MDC1, we tested the impact of CK2 inhibition on SDT phosphorylation in cells. Indeed, preincubation of U2OS cells with a CK2 inhibitor significantly reduced the overall incorporation of radioactive phosphate to the ectopically expressed SDT fragment (Fig. 5 A). More importantly in this context, the ensuing phosphopeptide analysis revealed that this was accompanied by an ∼50% reduction of phosphate incorporation into two prominent SDT repeats (Ser299/Thr301 and Ser453/Ser455; Fig. 5 B). Thus, CK2 can at least partially phosphorylate the SDT repeats in vivo. To further support this conclusion, we immunopurified HA-tagged MDC1 N terminus (Fig. 6 A) or endogenous MDC1 (Fig. 6 B) from cells and subjected these immunocomplexes to an in vitro kinase assay (without any additional substrate). In both cases, MDC1 copurified with an autocatalytic kinase activity that was strongly attenuated by a specific CK2 inhibitor (Fig. 6, A and B). Interestingly, this MDC1-associated CK2 activity was clearly present in undamaged cells (Fig. 6, A and B; lane 1) and did not appreciably increase after exposing the cells to IR (Fig. 6 A, lane 2). Collectively, the data in Figs. 4–6 show that CK2 can phosphorylate the SDT repeats and suggest that this may contribute to trigger a productive interaction between the MDC1 N terminus and the FHA domain of NBS1.

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