The data presented above demonstrate that the two pairs of C-terminal BRCT domains of hPTIP are capable of at least two modes of phospho-epitope recognition and that they control at least two important, but independent, facets of hPTIP function: interaction with phosphorylated 53BP1 and translocation to sites of DNA damage.
At the outset of this study, it was known that hPTIP interacts with 53BP1 after DNA damage in an ATM-dependent manner (3
) but the mechanisms or significance of this interaction were unclear. In this study, we showed that a single ATM-phosphorylated residue in 53BP1—Ser25—is required for interaction with hPTIP in cells. Mutation of Ser25 did not grossly perturb 53BP1 function since the 53BP1 Ser25Ala mutant protein formed foci after DNA damage in a manner indistinguishable from the wild-type protein (31
) (data not shown). Phosphorylated Ser25 was shown to interact with the two C-terminal pairs (Pair C1 + C2) of hPTIP (). We found that, surprisingly, both of these BRCT pairs are required to bind to the phospho-Ser25 peptide and neither domain alone could bind to this peptide (B). Consistent with these data, mutations of conserved residues in Pair C1 (Trp676) or in Pair C2 (Arg910 or Trp929) severely reduced binding of hPTIP to phospho-Ser25 in vitro
. Furthermore, mutation of any of these residues abolished the binding of hPTIP to 53BP1 after DNA damage in vivo
. This is the first reported example of a requirement of two pairs of BRCT domains for binding to a single phospho-epitope. It is not yet clear why two BRCT pairs are required to recognize 53BP1 phospho-Ser25, especially when in BRCA1 and MDC1 (and in other BRCT-proteins), a single BRCT pair in each case is sufficient for interacting with phospho-H2AX and phospho-BACH1, respectively (12
). We predict that the small conserved basic patch containing Arg910 in hPTIP pair C2 contacts the phospho-Ser25 of 53BP1 and that pair C1 makes contact with residues nearby, possibly helping to determine the specificity of the interaction. Solving the crystal structure of hPTIP BRCT domain pairs C1 + C2 in complex with the 53BP1 phospho-Ser25 peptide should provide valuable insight into the detailed mechanism of this interaction.
The requirement for two pairs of BRCT domains for hPTIP to recognize a single phospsho-epitope—Ser25 of 53BP1—is somewhat surprising since isolated BRCT domain pair C2 of hPTIP was shown previously, in peptide selection experiments, to interact with synthetic phospho-peptides that lie in an pS/T-Q-V-F motif. Ser25 does not lie in this type of motif but there are two serine residues in 53BP1—Ser29 and Ser105—that do conform to the pS/T-Q-V-F motif, are not required for 53BP1to bind hPTIP after DNA damage (C). The observation that hPTIP BRCT pairs C1 + C2 recognize a phospho-peptide different from the pS/T-Q-V-F peptides bound by pair C2 in isolation suggests that there are two modes of phospho-epitope recognition resident in pairs C1 + C2. Again, it would be interesting to compare the crystal structure of hPTIP BRCT pairs C1 + C2 in complex with the two types of phospho-peptide to ascertain whether different modes of interaction are at play. It would be interesting to know if two types of phosphopeptide bind in a mutually exclusive manner or if they can bind to hPTIP simultaneously.
We showed previously that both BRCT pairs C1 and C2 are required for translocation of hPTIP to sites of DNA damage. In this study, we showed that mutation of Trp676 in hPTIP BRCT pair C1 abolished formation of IR-induced nuclear foci by hPTIP, as did mutation of Trp929 in pair C2 (). However, the R910Q mutation that also abolished binding of hPTIP to phospho-Ser25 and to 53BP1 in cells, did not affect formation of nuclear foci by hPTIP after IR. Therefore, both C-terminal pairs of BRCT domains in hPTIP appear to be essential for binding of sites of DNA damage but this appears to be independent of their ability to bind phospho-Ser25 of 53BP1. This is consistent with previous reports that ATM, the Ser25 kinase, is not required for binding of hPTIP or 53BP1 to sites of DNA damage (3
). At present, the molecular mechanisms governing translocation of hPTIP are not clear and this will be interesting to investigate. Whatever the case, it is clear both C-terminal BRCT domains of hPTIP are required.
Mutation of 53BP1 Ser25, that abolished interaction with hPTIP, and mutation of Arg910 of hPTIP that prevents interaction with phospho-Ser25, prevented phosphorylation of BRCA1 and Chk2 by ATM. Thus, 53BP1 must interact with hPTIP to exercise its role as adaptor and to protect cells against DSBs. At present, it is not clear how 53BP1 or hPTIP functions to assist ATM phosphorylation but may involve recruitment of ATM substrates to sites of DNA damage. Mutation of Ser25 also caused cells to become hypersensitive to IR, probably indicative of a DNA repair defect, since 53BP1 are defective in NHEJ of a subset of DSBs in cells (22
). It would be interesting to know if mutation of Ser25 in mice recapitulates the same spectrum of tumours seen in 53BP1 null mice, and if mutation of Ser25 affects immunoglobulin class switching. Neither 53BP1 nor hPTIP appear to have catalytic activity and probably act as ‘scaffolding’ proteins to recruit and direct ‘effector’ polypeptides to sites of DNA damage. Ultimately it will be vital to identify the effector molecules that hPTIP and 53BP1 bring to sites of DNA damage that facilitate DNA repair and that enable phosphorylation of ATM targets.