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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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Interaction between NBS1 and MDC1 is mediated by the FHA domain of NBS1 and the first 500 amino acids of MDC1. (A) Endogenous MDC1 and NBS1 interact both before and after DNA damage. Lysates from U2OS cells either mock or γ irradiated (10 Gy for 1 h) were subjected to immunoprecipitation with an anti-MDC1 antibody and analyzed for the presence of NBS1 by immunoblotting. A nonimmune species-matched antibody was used as a control. Expression levels of NBS1 in whole cell extracts (bottom) indicate an equal input in each lane. (B) A schematic structure of MDC1 and NBS1. The red segment indicates the N-terminal region of MDC1 (amino acids 1–1,100) subjected to progressive deletion and interaction analysis with NBS1. (C) The N-terminal region of MDC1 spanning the first 500 amino acids efficiently interacts with cellular NBS1. The indicated HA-tagged fragments of the MDC1 N terminus were transfected into MDC1/shRNA cells (the numbers indicate the C-terminal amino acid of each fragment). After 24 h, lysates were prepared, subjected to immunoprecipitation with an anti-HA antibody, and analyzed by immunoblotting with antibodies to NBS1 (top) and HA (middle). The input into each reaction was controlled by NBS1 immunoblotting of the whole cell extracts as in A (bottom). (D) Efficient knockdown of endogenous MDC1. Lysates from U2OS cells and its derivative in which MDC1 is knocked down by stably integrated shRNA (MDC1/shRNA) were analyzed by immunoblotting with an MDC1-specific antibody (top). The NBS1 immunoblot serves as a loading control and indicates that the overall levels of NBS1 are not affected by the MDC1 knockdown (bottom). (E) The FHA domain of NBS1 interacts with the MDC1 N terminus. MDC1/shRNA cells were cotransfected with the HA-tagged MDC1 N terminus (p1100) and the following Myc-tagged variants of NBS1: WT (wild type), R28 (nonfunctional FHA domain), ΔC (C-terminal deletion without the MRE11 and ATM interaction domains), and 3A (alanine substitutions of serines 278, 343, and 397, the ATM phosphorylation sites). After 24 h, lysates were immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with anti-Myc (top) and anti-HA (middle) antibodies. The bottom panel is an immunoblot of whole cell extracts and shows the input of each of the Myc-tagged NBS1 proteins. WCE, whole cell extract; PST, Pro-Ser-Thr rich.
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fig1: Interaction between NBS1 and MDC1 is mediated by the FHA domain of NBS1 and the first 500 amino acids of MDC1. (A) Endogenous MDC1 and NBS1 interact both before and after DNA damage. Lysates from U2OS cells either mock or γ irradiated (10 Gy for 1 h) were subjected to immunoprecipitation with an anti-MDC1 antibody and analyzed for the presence of NBS1 by immunoblotting. A nonimmune species-matched antibody was used as a control. Expression levels of NBS1 in whole cell extracts (bottom) indicate an equal input in each lane. (B) A schematic structure of MDC1 and NBS1. The red segment indicates the N-terminal region of MDC1 (amino acids 1–1,100) subjected to progressive deletion and interaction analysis with NBS1. (C) The N-terminal region of MDC1 spanning the first 500 amino acids efficiently interacts with cellular NBS1. The indicated HA-tagged fragments of the MDC1 N terminus were transfected into MDC1/shRNA cells (the numbers indicate the C-terminal amino acid of each fragment). After 24 h, lysates were prepared, subjected to immunoprecipitation with an anti-HA antibody, and analyzed by immunoblotting with antibodies to NBS1 (top) and HA (middle). The input into each reaction was controlled by NBS1 immunoblotting of the whole cell extracts as in A (bottom). (D) Efficient knockdown of endogenous MDC1. Lysates from U2OS cells and its derivative in which MDC1 is knocked down by stably integrated shRNA (MDC1/shRNA) were analyzed by immunoblotting with an MDC1-specific antibody (top). The NBS1 immunoblot serves as a loading control and indicates that the overall levels of NBS1 are not affected by the MDC1 knockdown (bottom). (E) The FHA domain of NBS1 interacts with the MDC1 N terminus. MDC1/shRNA cells were cotransfected with the HA-tagged MDC1 N terminus (p1100) and the following Myc-tagged variants of NBS1: WT (wild type), R28 (nonfunctional FHA domain), ΔC (C-terminal deletion without the MRE11 and ATM interaction domains), and 3A (alanine substitutions of serines 278, 343, and 397, the ATM phosphorylation sites). After 24 h, lysates were immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with anti-Myc (top) and anti-HA (middle) antibodies. The bottom panel is an immunoblot of whole cell extracts and shows the input of each of the Myc-tagged NBS1 proteins. WCE, whole cell extract; PST, Pro-Ser-Thr rich.

Mentions: Endogenous MDC1 and NBS1 proteins efficiently interact both before and after DNA damage (Fig. 1 A) and contain numerous structural motifs and potential targets for posttranslational modifications (Fig. 1 B) that might be involved in this interaction. To elucidate the requirements for the MDC1–MRN interaction, we first constructed a series of HA-tagged fragments progressively spanning the N-terminal part of MDC1 (Fig. 1 C). We transiently expressed each of these fragments in a U2OS cell line in which the endogenous MDC1 was down-regulated by stably integrated short hairpin RNA (shRNA; Fig. 1 D; Bekker-Jensen et al., 2006) and tested their ability to interact with endogenous NBS1. The MDC1 fragments were constructed so that they were not affected by the shRNA to endogenous MDC1 (see Materials and methods), and the robust down-regulation of MDC1 in this cell line (designated U2OS/shMDC1) allowed us to assess the binding efficiency of each fragment unbiased by higher-order complexes containing the endogenous protein. Under these conditions, a fragment spanning the first 500 amino acids of MDC1 appeared to be necessary and sufficient to bind cellular NBS1 (Fig. 1 C, lane 6), and this binding did not further increase after including the more C-terminal sequences (Fig. 1 C, lanes 7 and 8). Interestingly, although MDC1 also contains an FHA domain at the very N terminus, this domain (either isolated or in the context of a larger fragment spanning up to the first 200 amino acids of MDC1) appeared to be entirely dispensable for NBS1 interaction (Fig. 1 C, lanes 1 and 2).


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)

Interaction between NBS1 and MDC1 is mediated by the FHA domain of NBS1 and the first 500 amino acids of MDC1. (A) Endogenous MDC1 and NBS1 interact both before and after DNA damage. Lysates from U2OS cells either mock or γ irradiated (10 Gy for 1 h) were subjected to immunoprecipitation with an anti-MDC1 antibody and analyzed for the presence of NBS1 by immunoblotting. A nonimmune species-matched antibody was used as a control. Expression levels of NBS1 in whole cell extracts (bottom) indicate an equal input in each lane. (B) A schematic structure of MDC1 and NBS1. The red segment indicates the N-terminal region of MDC1 (amino acids 1–1,100) subjected to progressive deletion and interaction analysis with NBS1. (C) The N-terminal region of MDC1 spanning the first 500 amino acids efficiently interacts with cellular NBS1. The indicated HA-tagged fragments of the MDC1 N terminus were transfected into MDC1/shRNA cells (the numbers indicate the C-terminal amino acid of each fragment). After 24 h, lysates were prepared, subjected to immunoprecipitation with an anti-HA antibody, and analyzed by immunoblotting with antibodies to NBS1 (top) and HA (middle). The input into each reaction was controlled by NBS1 immunoblotting of the whole cell extracts as in A (bottom). (D) Efficient knockdown of endogenous MDC1. Lysates from U2OS cells and its derivative in which MDC1 is knocked down by stably integrated shRNA (MDC1/shRNA) were analyzed by immunoblotting with an MDC1-specific antibody (top). The NBS1 immunoblot serves as a loading control and indicates that the overall levels of NBS1 are not affected by the MDC1 knockdown (bottom). (E) The FHA domain of NBS1 interacts with the MDC1 N terminus. MDC1/shRNA cells were cotransfected with the HA-tagged MDC1 N terminus (p1100) and the following Myc-tagged variants of NBS1: WT (wild type), R28 (nonfunctional FHA domain), ΔC (C-terminal deletion without the MRE11 and ATM interaction domains), and 3A (alanine substitutions of serines 278, 343, and 397, the ATM phosphorylation sites). After 24 h, lysates were immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with anti-Myc (top) and anti-HA (middle) antibodies. The bottom panel is an immunoblot of whole cell extracts and shows the input of each of the Myc-tagged NBS1 proteins. WCE, whole cell extract; PST, Pro-Ser-Thr rich.
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fig1: Interaction between NBS1 and MDC1 is mediated by the FHA domain of NBS1 and the first 500 amino acids of MDC1. (A) Endogenous MDC1 and NBS1 interact both before and after DNA damage. Lysates from U2OS cells either mock or γ irradiated (10 Gy for 1 h) were subjected to immunoprecipitation with an anti-MDC1 antibody and analyzed for the presence of NBS1 by immunoblotting. A nonimmune species-matched antibody was used as a control. Expression levels of NBS1 in whole cell extracts (bottom) indicate an equal input in each lane. (B) A schematic structure of MDC1 and NBS1. The red segment indicates the N-terminal region of MDC1 (amino acids 1–1,100) subjected to progressive deletion and interaction analysis with NBS1. (C) The N-terminal region of MDC1 spanning the first 500 amino acids efficiently interacts with cellular NBS1. The indicated HA-tagged fragments of the MDC1 N terminus were transfected into MDC1/shRNA cells (the numbers indicate the C-terminal amino acid of each fragment). After 24 h, lysates were prepared, subjected to immunoprecipitation with an anti-HA antibody, and analyzed by immunoblotting with antibodies to NBS1 (top) and HA (middle). The input into each reaction was controlled by NBS1 immunoblotting of the whole cell extracts as in A (bottom). (D) Efficient knockdown of endogenous MDC1. Lysates from U2OS cells and its derivative in which MDC1 is knocked down by stably integrated shRNA (MDC1/shRNA) were analyzed by immunoblotting with an MDC1-specific antibody (top). The NBS1 immunoblot serves as a loading control and indicates that the overall levels of NBS1 are not affected by the MDC1 knockdown (bottom). (E) The FHA domain of NBS1 interacts with the MDC1 N terminus. MDC1/shRNA cells were cotransfected with the HA-tagged MDC1 N terminus (p1100) and the following Myc-tagged variants of NBS1: WT (wild type), R28 (nonfunctional FHA domain), ΔC (C-terminal deletion without the MRE11 and ATM interaction domains), and 3A (alanine substitutions of serines 278, 343, and 397, the ATM phosphorylation sites). After 24 h, lysates were immunoprecipitated with an anti-HA antibody and analyzed by immunoblotting with anti-Myc (top) and anti-HA (middle) antibodies. The bottom panel is an immunoblot of whole cell extracts and shows the input of each of the Myc-tagged NBS1 proteins. WCE, whole cell extract; PST, Pro-Ser-Thr rich.
Mentions: Endogenous MDC1 and NBS1 proteins efficiently interact both before and after DNA damage (Fig. 1 A) and contain numerous structural motifs and potential targets for posttranslational modifications (Fig. 1 B) that might be involved in this interaction. To elucidate the requirements for the MDC1–MRN interaction, we first constructed a series of HA-tagged fragments progressively spanning the N-terminal part of MDC1 (Fig. 1 C). We transiently expressed each of these fragments in a U2OS cell line in which the endogenous MDC1 was down-regulated by stably integrated short hairpin RNA (shRNA; Fig. 1 D; Bekker-Jensen et al., 2006) and tested their ability to interact with endogenous NBS1. The MDC1 fragments were constructed so that they were not affected by the shRNA to endogenous MDC1 (see Materials and methods), and the robust down-regulation of MDC1 in this cell line (designated U2OS/shMDC1) allowed us to assess the binding efficiency of each fragment unbiased by higher-order complexes containing the endogenous protein. Under these conditions, a fragment spanning the first 500 amino acids of MDC1 appeared to be necessary and sufficient to bind cellular NBS1 (Fig. 1 C, lane 6), and this binding did not further increase after including the more C-terminal sequences (Fig. 1 C, lanes 7 and 8). Interestingly, although MDC1 also contains an FHA domain at the very N terminus, this domain (either isolated or in the context of a larger fragment spanning up to the first 200 amino acids of MDC1) appeared to be entirely dispensable for NBS1 interaction (Fig. 1 C, lanes 1 and 2).

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