The human genome is under constant attack by radiation and different genotoxic chemicals. These exposures induce various DNA lesions that subsequently generate DNA damage responses to maintain genome integrity (11
). This involves a large number of proteins that are implicated in DNA damage repair, the regulation of cell cycle checkpoints and transcription, and apoptosis when damage is not properly repaired. We, and others, recently reported that RAP80 translocates to IRIF after IR treatment and plays an important role in DNA damage responses (4
). RAP80 interacts with BRCA1 and is critical for efficient repair through DNA damage-induced homology-directed recombination and in cell cycle checkpoint control.
ATM plays a key role in the activation of cell cycle checkpoints after IR-induced DSBs. Loss of ATM, as in A-T cells, results in defects in the control of all checkpoints (16
). In addition to ATM itself, ATM phosphorylates many DNA damage response proteins, including BRCA1 and H2AX. RAP80 was recently identified as a novel substrate of ATM and after IR became phosphorylated at several sites (4
was identified as one of the ATM phosphorylation sites in vitro
). In the current study, we show that exposure of cells to IR induces phosphorylation of RAP80 at Ser205
and that subsequently, phosphorylated RAP80Ser205P
translocates to DNA damage foci where it co-localizes with γ-H2AX (). This phosphorylation was demonstrated to depend on the activation of ATM. Both ATM and DNAPK have been reported to be able to phosphorylate H2AX, and in A-T cells, it is DNAPK that phosphorylates H2AX after IR (37
). Our results show that RAP80 is not phosphorylated at Ser205
in A-T cells after IR (), indicating RAP80Ser205 is not a target of DNAPK.
We further demonstrate that the phosphorylation of RAP80 at Ser205
occurs rapidly and long before RAP80Ser205P
translocates to DNA damage foci. The delayed translocation to IRIF suggests a role for RAP80 at a later stage of DNA repair whereas its rapid phosphorylation by ATM suggests that RAP80 might have a role in the regulation of an early event in the DNA damage response as well, such as control of cell cycle checkpoints. The latter hypothesis is consistent with findings showing that depletion of RAP80 causes defects in the control of the G2/M checkpoint after IR (5
). Besides Ser205
, several other ATM phosphorylation sites were identified in RAP80 (5
). Interestingly, none of the sites is highly conserved across species (5
). This raises several interesting questions: what is the function of RAP80 phosphorylation? Are all phosphorylation sites important for G2/M checkpoint control or is phosphorylation of RAP80 at different sites associated with different functions? Although RAP80 phosphorylation by ATM does not affect its association with BRCA1 or its translocation to IRIF (4
), the phosphorylation might change the conformation of RAP80 and affect the interaction of RAP80 or RAP80/BRCA1 complex with other proteins. Subsequently this might affect the activity of the complex and the roles it plays in DNA damage response signaling. In support of this hypothesis, phosphorylation of BRCA1 at distinct sites has been reported to be linked to the regulation of different checkpoints (38
). Whether phosphorylation of RAP80 at distinct sites is linked to different functions needs further investigation.
Previous studies reported that a functional BRCA1 is required for ATM- and ATR-dependent phosphorylation of several proteins, including p53, c-Jun, NBS1, CtIP, and Chk2 (25
). These proteins are required for checkpoint activation and/or apoptosis, and as observed for RAP80, appear to be part of a BRCA1 complex even before the induction of DNA damage. However, we show that the phosphorylation of RAP80 is independent of a functional BRCA1 ().
In this study, we further demonstrate that RAP80 translocates to DNA damage foci after UV irradiation and that also this migration is dependent on the UIMs of RAP80 (). We show that RAP80 is phosphorylated at Ser205
and provide evidence that this phosphorylation is mediated by ATR and not ATM. Unlike IR, which induces DSBs directly, UV treatment mainly induces cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) (39
). Although UV-induced phosphorylation of H2AX and γ-H2AX foci formation can be triggered by DSBs induced by blocked replication forks in S-phase cells (34
), recent studies demonstrated a cell cycle-independent induction of H2AX phosphorylation and γ-H2AX foci formation after UV triggered by single strand DNA repair intermediates (21
). Our observations showing that after UV most cells contained foci that were positive for both γ-H2AX and RAP80, are consistent with this and suggest a role for RAP80 in DNA damage responses triggered by other types of DNA lesions, in addition to those induced by DSBs. This was supported by studies demonstrating that RAP80 depletion caused increased sensitivity to UV irradiation (5
). Thus, RAP80 appears to play a role in several types of DNA damage responses.
BRCT motifs are found in a number of proteins with functions in DNA repair responses (29
). Mutations in the BRCT motifs of BRCA1 have been linked to elevated risk for breast and ovarian cancer (29
). Structural analysis showed that many of the cancer-related mutations in the BRCT repeats disrupt the structure of the pSer(Thr)-X-X-Phe binding pocket, abolish the interaction of BRCT with its partners, and prevents BRCA1 from translocating to DNA damage foci (26
). The BRCT motifs of BRCA1 were shown to be essential for its association with RAP80 and this interaction is required for the translocation of BRCA1 to DNA damage foci (4
). In agreement with these findings, we demonstrated that the BRCT missense mutation R1699W abolished the interaction of BRCA1 with RAP80 (). Although RAP80 has one potential pSer(Thr)-X-X-Phe motif within the region essential for its association with BRCA1 (4
), this motif is not required for this interaction () suggesting that RAP80 and BRCA1 may interact indirectly by binding an intermediary protein. This concept was supported by recent studies showing that this interaction is mediated by CCDC98 (40
In summary, our study extends previous observations and demonstrates that RAP80 becomes phosphorylated at Ser205 in an ATM-dependent manner in IR-treated cells. This phosphorylation occurs long before RAP80 translocates to IRIF and is independent of BRCA1. UV irradiation also induces phosphorylation of RAP80 at Ser205 and its translocation to DNA damage foci. This phosphorylation requires ATR, not ATM. We further show that the BRCT mutant R1699W does not interact with RAP80. Future studies have to determine what the functions are of the different phosphorylation sites of RAP80.