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Direct Activation of the ATM Protein Kinase by the Mre11/Rad50/Nbs1 Complex

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Science  02 Apr 2004:
Vol. 304, Issue 5667, pp. 93-96
DOI: 10.1126/science.1091496

Abstract

The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM (ataxia telangiectasia mutated). We demonstrate that MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo.

Eukaryotic cells respond to DNA damage with a rapid activation of signaling cascades that initiate from the ATR and ATM protein kinases. The response to DNA double-strand breaks (DSBs) occurs primarily through ATM and leads to phosphorylation of many targets critical for checkpoint activation, apoptosis, and DNA repair (1). One of the targets of ATM phosphorylation is the Nbs1 (nibrin) protein, which associates with the conserved DSB repair factors Mre11 and Rad50 (2). ATM phosphorylates Nbs1 on Ser-343 and other residues, modifications necessary for S-phase checkpoint activation and for survival of ionizing-radiation exposure (38).

The Nbs1 protein is not just a substrate of ATM, but also affects activation of ATM in response to DNA damage. The gene encoding Nbs1 is mutated in patients with the radiation sensitivity disorder Nijmegen breakage syndrome (NBS), characterized by chromosomal instability, radio-resistant DNA synthesis, and clinical phenotypes that include immunodeficiency and cancer (9). Cells from NBS patients do not produce full-length Nbs1 protein, and phosphorylation of Chk2, SMC1, and FANCD2 by ATM is reduced or absent in these cells (7, 8, 1014). Thus, the MRN complex may participate in activating ATM to phosphorylate multiple other downstream substrates.

To test this hypothesis, we measured the effects of MRN on ATM phosphorylation of Chk2, a protein kinase that activates S phase and mitotic checkpoints. ATM phosphorylates Chk2 on Thr-68 in response to DNA damage, which activates Chk2 to phosphorylate substrates including p53 and Cdc25C (15). Using a phospho-specific antibody specific for Chk2 Thr-68, we found that the addition of MRN to ATM stimulated Chk2 phosphorylation up to 15-fold (Fig. 1A). The activating effect of MRN was dependent on the absolute concentration of ATM in the reaction (Fig. 1B). Addition of Mre11/Rad50 (MR), lacking the Nbs1 protein, stimulated ATM phosphorylation of Chk2 only partially (Fig. 1C), indicating that Nbs1 is important for Chk2 activation.

Fig. 1.

Stimulation of ATM phosphorylation of Chk2 on Thr-68 by MRN. (A) Kinase assays contained Flag-tagged wt ATM and full-length histidine-tagged kinase-deficient Chk2, with MRN as indicated. Phosphorylation was visualized with anti-Chk2 phospho–Thr-68. Concentration of MRN = 1.9, 3.75, and 7.5 nM in lanes 3 to 5, respectively; concentration of ATM = 0.25 nM. (B) Kinase assays as in (A) except with varying ATM amounts. Concentration of MRN = 7.5 nM. (C) Kinase assays as in (A) except with wt MRN, wt MR, or MR(ATLD3/4) added at 7.5 nM. (D to G) Kinase assays as in (A) except with MRN(S343A), MRN(ATLD1/2), MRN(ATLD3/4), or MRN(p70) added instead of wt MRN. (H) Kinase assays as in (A) with 7.5 nM wt MRN, 0.25 nM wt ATM, and kd ATM added in lanes 4 to 6.

Nbs1 phosphorylation on Ser-343 is required for ATM phosphorylation of substrates including Chk2, SMC1, and FANCD2 in vivo (10, 12, 13). To test the importance of Nbs1 phosphorylation for ATM stimulation, we used an S343A mutant version of Nbs1. The MRN(S343A) complex forms similarly to the wild-type (wt) complex and exhibits DNA binding and nuclease activities identical to those of the wt enzyme (16). The MRN(S343A) mutant complex did not stimulate ATM activity toward Chk2 in vitro (Fig. 1D). The presence of Nbs1 and phosphorylation of Ser-343 by ATM is therefore essential for MRN stimulation of ATM activity on Chk2. The complete absence of ATM stimulation by MRN(S343A) suggests that the presence of Nbs1 is inhibitory when Nbs1 cannot be phosphorylated.

Ataxia telangiectasia (A-T) patients lack functional ATM protein and exhibit radiation sensitivity, genomic instability, and deficiencies in G1-S, S, and G2-M checkpoint responses. A-T–like disorder (ATLD) patients also have an A-T phenotype despite having wt alleles of the ATM gene (17). ATLD patients carry mutations in the Mre11 gene that cause a truncation of the C terminus (ATLD1/2) or a N117S missense mutation in the nuclease domain (ATLD3/4). ATLD3/4 Mre11 exhibits variability in Nbs1 association, forming complexes either with [MRN(ATLD3/4)] or without [MR(ATLD3/4)] Nbs1 (18). NBS cells express a truncated C-terminal polypeptide of Nbs1 (p70) that binds to Mre11 and forms a mutant MRN(p70) complex (19). MRN(p70), MRN(ATLD1/2), and MRN(ATLD3/4) exhibit DNA binding and nuclease activities similar to those of the wt MRN complex (18).

We tested the ability of the ATLD and p70 forms of the MRN complex to stimulate ATM phosphorylation of Chk2 (Fig. 1). The MR(ATLD3/4) complex did not stimulate ATM activity (Fig. 1C), but the MRN(ATLD3/4) form of the complex showed stimulation equivalent to that of wt MRN (Fig. 1F). The MRN(ATLD1/2) and MRN(p70) complexes stimulated Chk2 phosphorylation only partially (Fig. 1, E and G). The decreased ability of ATLD and p70 versions of MRN to stimulate ATM phosphorylation of Chk2 is consistent with the checkpoint deficiencies observed with cells from ATLD and NBS patients (20). The low levels of Chk2 phosphorylation observed in ATLD and NBS cell lines (21) are likely due to a combination of the mutations in Mre11 and Nbs1 as well as the overall reductions in intranuclear MRN levels observed in these cells (2, 17, 21).

Some missense mutations in the human ATM gene are associated with higher rates of malignancy in heterozygotes (22), and a missense allele of ATM acts as a dominant negative and increases rates of tumor formation in mice (23). We therefore tested the effects of catalytically inactive (kd) ATM (24) in reactions containing MRN and wt ATM (Fig. 1H). The kd ATM inhibited phosphorylation of Chk2 by wt ATM, indicating that kd ATM can exert dominant-negative inhibition of wt ATM activity.

We also tested the effects of MRN on ATM phosphorylation of the tumor suppressor p53, using amino acids 1 to 102 of p53 coupled to glutathione S-transferase as a substrate (GST-p53), and an antibody to phosphorylated Ser-15. Phosphorylation of p53 on Ser-15 is required for p53 stabilization and transactivation (25, 26). Addition of MRN to ATM increased p53 phosphorylation up to 12-fold over phosphorylation by ATM alone (Fig. 2A). As with Chk2, the effect of MRN on p53 phosphorylation by ATM was very sensitive to the concentration of ATM in the reaction (Fig. 2B).

Fig. 2.

Stimulation of ATM phosphorylation of p53 on Ser-15 by MRN. (A) Kinase assays as in Fig. 1 except with GST-p53 (amino acids 1 to 102) as substrate and anti-p53 phospho–Ser-15. Concentration of ATM = 0.125 nM. (B) Kinase assays as in (A) except with varying ATM amounts, with or without MRN (7.5 nM). (C) Kinase assays as in (A) with MRN (7.5 nM), Mre11 (M), Nbs1 (N), or Mre11 and Nbs1 (M + N). Mre11 and Nbs1 concentrations were equivalent to the amounts present in wt MRN. (D) Kinase assays as in (A) except with wt MRN, wt MR, or MR(ATLD3/4) added at 7.5 nM. (E to H) Kinase assays as in (A) except with MRN(S343A), MRN(ATLD1/2), MRN(ATLD3/4N), or MRN(p70) added instead of wt MRN. (I) Kinase assays with wt ATM (0.25 nM) and wt MRN (0.75 nM) on a GST-H2AX (amino acids 133 to 143) substrate with [γ-32P]ATP, visualized by PhosphorImager.

Subcomplexes of MRN were also tested for stimulation of ATM phosphorylation activity, showing that Mre11 and Nbs1, either alone or added together, were insufficient to induce p53 phosphorylation (Fig. 2C). However, Nbs1 appeared not to be required for p53 phosphorylation, because a complex of Mre11 and Rad50 (MR) stimulated ATM activity to the same extent as did the complete MRN complex (Fig. 2D). The MRN(S343A) complex exhibited nearly wt levels of activity in this assay (Fig. 2E), as did the MRN(ATLD1/2), MRN(ATLD3/4), and MRN(p70) complexes (Fig. 2, F to H). The ability of the mutant complexes to stimulate ATM phosphorylation of p53 is consistent with the normal p53 response in cell lines derived from ATLD and NBS patients (17).

One of the first targets of ATM in vivo is the C-terminal tail of histone H2AX, which is phosphorylated rapidly at the sites of chromosomal DSBs (27, 28). As with Chk2 and p53, ATM phosphorylation of a GST-H2AX substrate was also stimulated fivefold by the addition of MRN (Fig. 2I).

To investigate the mechanism underlying MRN stimulation of ATM activity, we analyzed associations between the recombinant purified proteins by gel filtration (Fig. 3). Incubation of ATM with MRN caused a subset of ATM to elute with the MRN complex, indicating an interaction between the factors (Fig. 3A). kd ATM also appeared to interact with MRN. Unlike wt ATM, however, kd ATM induced a change in the mobility of the MRN components into a range of smaller complexes, suggesting dissociation of the complex. This effect on MRN may contribute to the dominant-negative activity of kd ATM.

Fig. 3.

Multiple direct associations between ATM and the MRN complex. (A) wt MRN was incubated with wt ATM or kd ATM either separately (top), or together (bottom) for 30 min before gel filtration (Superose 6), SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting with antibodies to ATM, Rad50, and Nbs1. Molecular mass: ATM = 3.5 × 105 daltons, MRN = ∼1.2 × 106 daltons (18). (B to E) wt ATM incubated with MRN(ATLD1/2) complex (B), MRN(p70) complex (C), MRN(S343A) complex (D), or wt MR (E) and analyzed as in (A). (F) wt ATM and Nbs1 protein incubated separately (top) or together (bottom), separated by gel filtration, and analyzed as in (A) with antibodies to ATM or Nbs1.

The MRN(ATLD1/2) and MRN(p70) mutant complexes also associated with ATM (Fig. 3, B and C), although the percentage of ATM observed in the larger complex was less than that observed with similar amounts of wt MRN, particularly with the p70 complex. In contrast, ATM associated with the MRN(S343A) complex as it did with the wt complex (Fig. 3D), indicating that an interaction between the complexes occurs even in the absence of Nbs1 phosphorylation.

The stimulation of p53 phosphorylation by MR (Fig. 2) suggests that ATM must make contacts with the MRN complex that are completely independent of Nbs1. Consistent with this result, gel filtration showed that a subset of ATM comigrated with MR even in the absence of Nbs1 (Fig. 3E). ATM also associated directly with Nbs1 in the absence of MR, however (Fig. 3F), indicating that ATM makes multiple direct contacts with the MRN complex.

To determine the mechanism of MRN stimulation of ATM kinase activity, we examined the effect of the complex on the binding of specific substrates by ATM. ATM was incubated with or without MRN, then isolated by immunoprecipitation with anti-Flag beads. GST-Chk2 or GST-p53 substrates were then incubated with the beads, and bound proteins were detected with an antibody to GST (29). The presence of MRN during the first incubation stimulated binding of both p53 and Chk2 to ATM during the second incubation (Fig. 4, A to D). The MRN(S343A), MRN(ATLD1/2), and MRN(p70) complexes showed lower stimulation of GST-Chk2 association (Fig. 4B), consistent with reduced ability of the complexes in stimulating kinase activity. Mre11 did not increase association of GST-p53 with ATM, whereas MRN stimulated association by more than fivefold and MR showed an intermediate level of stimulation. These effects on substrate binding were not seen when adenosine 5′-triphosphate (ATP) and divalent cations were present during the second incubation (16), suggesting that substrates are released from ATM after phosphorylation. We propose that MRN induces a conformational change in ATM that increases its affinity for its substrates (Fig. 4E).

Fig. 4.

MRN stimulates binding of substrates to ATM. (A and C) ATM was incubated with or without MRN in kinase buffer, and ATM complexes were isolated using beads conjugated to anti-Flag (recombinant ATM contains N-terminal Flag tag). Complexes were incubated with GST-Chk2 (amino acids 2 to 107) (A) or GST-p53 (amino acids 1 to 102) (C), without ATP, and ATM was isolated again with anti-Flag beads. Associated material was eluted from the beads, separated by SDS-PAGE, and analyzed with anti-GST. (B) Association of GST-Chk2 with ATM stimulated by wt MRN, MRN(S343A), MRN(ATLD1/2), and MRN(p70) determined as in (A) and represented as the fold increase in GST-Chk2 association. (D) Association of GST-p53 with ATM stimulated by wt MRN, wt MR, and Mre11 determined as in (A) and represented as the fold increase in GST-p53 association. (E) Schematic model of MRN stimulation of ATM kinase activity. ATM is postulated to bind substrates in low-affinity mode (left), or in high-affinity mode (right) when bound to MRN.

The addition of DNA to ATM kinase assays, with or without MRN, did not stimulate phosphorylation (16), similar to findings of other reports (30). Our experiments were performed in the absence of DNA and indicate that DNA is not absolutely required for the associations between ATM, MRN, and ATM substrates. The requirement for MRN in ATM activation shown in this study may explain many aspects of the NBS and ATLD phenotypes, and it illustrates how a single complex can facilitate spatial and temporal coordination of DNA repair with signaling pathways that mediate checkpoint activation.

Supporting Online Material

www.sciencemag.org/cgi/content/full/304/5667/93/DC1

Materials and Methods

Fig. S1

References

References and Notes

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