BRCA1 ubiquitylation of CtIP: Just the tIP of the iceberg?

d n a r e p a i r 5 ( 2 0 0 6 ) 1499–1504

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BRCA1 ubiquitylation of CtIP: Just the tIP of the iceberg? Louise J. Barber, Simon J. Boulton ∗

a r t i c l e

i n f o

a b s t r a c t

Article history:

Ubiquitylation is an important regulatory mechanism of many cellular processes. The breast and

Received 11 August 2006

ovarian cancer-specific tumour suppressor BRCA1 is well acknowledged to be a RING/E3 ubiquitin

Received in revised form

ligase, however, identification of its physiological substrates has proved elusive. Recently published

24 August 2006

data have shown that the BRCA1-interacting protein CtIP is in fact ubiquitylated by BRCA1, and

Accepted 29 August 2006

opens new avenues for the isolation of other substrate proteins.

Published on line 5 October 2006

© 2006 Elsevier B.V. All rights reserved.

Keywords: CtIP BRCA1 Ubiquitylation E3-ubiquitin ligase

Ubiquitin is a highly conserved 76-amino acid protein modifier that has a well-established role in the targeting of proteins for proteasome-mediated proteolysis. However, in recent years, it has become apparent that this small protein tag is also a crucial regulator of other non-proteolytic cellular processes including DNA repair, transcription, cell cycle progression, gene silencing, and protein trafficking [1–3]. A cascade of enzymes is required for the covalent modification of a substrate protein by ubiquitylation, providing the means for specification within the cell [4]. The first step is the activation of ubiquitin through the formation of a high-energy thioester link to the E1 ubiquitin-activating enzyme. This activated ubiquitin is then transferred to one of a number of E2 ubiquitin-conjugating enzymes, which, in cooperation with a substrate-specifying E3 ubiquitin ligase, forms a covalent isopeptide linkage between the terminal carboxyl group of ubiquitin and the ␧-amino group of a lysine residue on the target protein. Ubiquitin contains seven lysine residues that can be conjugated to further ubiquitin moieties to generate polyubiquitin chains. It is believed that Lys48-linked polyubiquitin chains are specifically recognized by the proteasome and consequently target substrate proteins for degradation [2]. However, alternative polyubiquitin chains are also observed 1568-7864/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2006.08.009

as distinct signals in the regulation of other processes. Recent work has shown that Lys63-linked polyubiquitylation of the replication sliding clamp PCNA is essential for the initiation of RAD51-independent error-free DNA damage bypass, whereas monoubiquitylation of PCNA stimulates error-prone translesion synthesis [5,6]. Ubiquitylation is also a reversible process, and a number of deubiquitylation enzymes (DUBs) have been identified to date [1].

1.

BRCA1

For the most part, substrate specificity for ubiquitin modification is provided by the large and expanding family of E3 ubiquitin ligases [2,4]. The tumour suppressor gene BRCA1 contains an N-terminal RING-finger domain responsible for heterodimerisation with BARD1, a structurally related protein that also possesses a RING-domain. These proteins exist as an obligate heterodimer in vivo and together possess E3-ubiquitin ligase activity with specificity for UbcH5c as its E2-Ub conjugating enzyme [7–13]. The existence of a tumour-associated BRCA1 mutation affecting the RING domain (C61G), implicates E3 ubiquitin ligase activity as a key cellular function of BRCA1

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[9,10,14,15]. BRCA1 and BARD1 both possess two tandem BRCT domains at their C termini, a motif that has been shown to facilitate protein–protein interactions by binding to phosphopeptides [16,17]. It is therefore possible that the BRCT motifs may allow for simultaneous binding to multiple phosphorylated proteins, or increase the affinity for a single interactor [18]. BRCA1 is believed to play a role in DNA damage response pathways, although its exact function is largely unknown. The extensive genome instability, DNA damage sensitivity, and defective DNA double-strand break (DSB) repair of BRCA1 or BARD1 mutant cell lines have suggested a role in homologous recombination (HR) and checkpoint signalling [18,19]. BRCA1 also co-localises in nuclear repair foci with the HR proteins Rad51 and BRCA2, the Fanconi anaemia repair factor FANCD2, and the repair and checkpoint MRN complex [20]. However direct interactions have not been identified, other than the existence of a damage-specific association with MRN as part of a large multi-factorial complex [21]. Two recent reports have established that ubiquitylation is rapidly induced at repair foci in response to DNA damage and can be detected using an antibody that specifically detects conjugated ubiquitin [13,22]. Importantly, ubiquitylation events at damage sites co localize extensively with BRCA1/BARD1 and are abolished in cells deficient for BRCA1, BARD1 or the E2-Ub conjugating enzyme UbcH5c. Over-expression of a K6R mutant of Ub in cells also abolishes damage-induced ubiquitylation events, indicating that Lys6 poly-Ub chain formation is important for this process [13,22]. Intriguingly, auto-activation of the E3-Ub ligase activity of BRCA1 is dependent on Lys6 of Ub [12]. These observations established that BRCA1/BARD1 in conjunction with UbcH5c are responsible for the majority of ubiquitylation events that are induced at repair foci that also rely on a rare form of Ub-chain linkage. It was shown that BRCA1mediated ubiquitylation also depends on recruitment of BRCA1 to repair foci by the MRN complex, as well as on the kinase activities of the checkpoint proteins ATM and ATR and on ␥-H2AX [13]. The identity of the substrate(s) ubiquitylated at repair foci and the impact of this modification on the function of the substrate(s) remains the critical unanswered questions arising from these studies [18].

2.

CtIP

The nuclear protein CtIP (RBBP8) was originally identified by independent yeast two-hybrid analyses as an interactor of the transcriptional repressor CtBP [23], the retinoblastoma protein RB [24], and also BRCA1 [25,26] (Fig. 1). An association with tumour suppressor genes suggested a role in tumour progression, and correspondingly, a number of tumours exhibit missense mutations in CtIP, and truncations have been detected in colon cancers [27,28]. CtIP has since been observed to interact with other proteins involved in development, such as LMO2 and LMO4 [29], and also transcription, such as TFIIB and members of the Ikaros family that bind CtBP and repress transcription through the recruitment of histone deacetylatases [30,31]. Consistent with a role in nuclear processes such as cell cycle control, CtIP has three putative nuclear localisation sequences [32]. However, only two known structural motifs have been observed for CtIP, and these are a pair of putative coiled-coil domains that lie at each terminus of the protein (Fig. 1). The N terminal coiled coil has been shown to facilitate homodimerisation of CtIP, but is dispensable for the interaction with either BRCA1 or LMO4 in mammalian cells [32]. CtIP is thought to interact with RB and its related protein p130 through a consensus LECEE motif at the end of the N-terminal coiled coil [24,33], and with CtBP through the centrally sited PLDLS binding motif [23] (Fig. 1). These two motifs are both required for the complete transcriptional repression activity of CtIP, as determined using a Gal4 reporter assay [33]. Recent studies have shown a critical role for CtIP in the transcriptional repression of a number of genes including the angiogenic factor ANG1 [34], cyclin D1 and other E2F-regulated genes [35], and the transmembrane receptor Notch [36]. CtIP binds to the BRCT domains of BRCA1 in vivo, and three tumour-associated mutations (A1708E, M1775R, Y1853D) in the BRCA1 BRCT abolish this interaction, suggesting a crucial physiological role [25,26]. As already discussed, the BRCT domain is a binding site for phosphopeptides. CtIP has been shown to be phosphorylated at three distinct serine residues (S327, S664, S745) with differential regulation [37,38] (Fig. 1). Phosphorylation of S327 occurs at a Ser-Pro consensus sequence suggesting a requirement for a cell cycle regulated cyclin-dependent kinase. Furthermore, S327 modification

Fig. 1 – CtIP schematic showing the N and C terminal coiled coil domains (residues 22–160 and 695–778), RB-binding motif LECEE (residues 153–157), CtBP-binding motif PLDLS (residues 490–494), and BRCA1-BRCT binding region (residues 133–282). The positions of the known phosphorylated serine resides (S327, S664, S745) are also shown.

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appears to correlate with the maximal cellular levels of CtIP in G2/M phase [38]. S327 lies within the region (residues 133–369) responsible for binding directly to BRCA1 [39] and also matches the consensus motif recognised by the BRCA1 BRCT domain, as determined through in vitro studies of the BRCT domain [16,17]. Consistent with association dependent upon a cell-cycle regulated phosphorylation event, the BRCA1/CtIP complex appears to be specific to G2, and is required for downstream Chk1 phosphorylation and the G2/M transition [38]. In contrast to the S327 modification, S664 and S745 are phosphorylated in an ATM-dependent manner after DNA damage [37,40]. It has been suggested that phosphorylation of S327 in unperturbed cells may facilitate the subsequent hyperphosphorylation by ATM, as BRCA1 is required for damageinduced phosphorylation of CtIP [41]. In addition to BRCA1, it has also been proposed that CtIP interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2 [42]. SIAH-1 expression is induced during apoptosis by p53 and p21 [43,44]. In contrast to the ubiquitylation of CtIP by BRCA1, interaction of SIAH-1 with CtIP leads to its degradation by the proteasome [42].

3. CtIP is a bone fide target of BRCA1 ubiquitylation In a recent paper by Yu et al. [45], it was shown that CtIP can be ubiquitylated in vitro by the BRCA1/BARD1 heterodimer in combination with its associated E2, UbcH5c. Correspondingly, CtIP ubiquitylation was abolished in this assay by substitution with the BRCA1 RING domain mutant I26A that has impaired E2 binding, and also ex vivo, in the BRCA1-deficient HCC1937 cell line. This implies that CtIP must be bound to BRCA1 through its BRCT domain in order for ubiquitylation to occur, as the insertion mutation carried in HCC1937 cells leads to a truncated form of BRCA1 lacking only the terminal BRCT domain [17,46]. A crystal structure of the BRCA1 BRCT repeats bound to a phosphopeptide corresponding to residues 322–333 of CtIP, obtained by Varma et al. [47], demonstrated that CtIP interacts with a cleft in the BRCA1 protein formed at the interface of the two BRCT domains. Furthermore, the tumour-associated BRCA1 mutation, M1775R, was shown to sterically conflict with the crucial CtIP anchoring residue, F330, disrupting binding within the cleft [47]. Hence, it appears that both domains are required for a stable interaction with BRCA1. In support of the observation that BRCA1-dependent ubiquitylation occurs via K6-mediated conjugation and not K48 as utilised in the targeting of proteins to the proteasome [22,48], Yu et al. failed to detect any correlation between CtIP ubiquitylation and its degradation. As BRCA1 and CtIP have been previously shown to interact during G2 and impact upon the G2/M checkpoint after DNA damage [38], they hypothesized that the ubiquitylation of CtIP may be coordinated with damage detection. Indeed, they found CtIP to be ubiquitylated in a BRCA1-dependent manner after ␥-irradiation, which resulted in the recruitment to chromatin and colocalisation of BRCA1 and CtIP in nuclear foci. Interestingly, although CtIP and BRCA1 have been shown to interact in the absence of ubiquitylation through phosphorylation of CtIP S327, colocalisation in foci after DNA damage appears to be dependent

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upon CtIP ubiquitylation by BRCA1, as the CtIP-S327 mutant is not recruited to repair centres although BRCA1 foci form as normal. Consistent with a role for BRCA1 ubiquitin ligase in the G2/M checkpoint, they additionally show that the BRCA1-I26A mutant is unable to restore the checkpoint defect observed in HCC1937 cells after DNA damage. Hence BRCA1 E3 ubiquitin ligase activity is essential for the G2/M DNA damage checkpoint response, and this may be mediated through CtIP. This most recent work clearly shows that CtIP is a critical substrate of BRCA1 in the G2/M DNA damage response, however there are a number of key questions that remain unanswered. Firstly, given that CtIP and BRCA1 interact in the absence of exogenous insult during G2, yet BRCA1 does not automatically ubiquitylate this bound substrate, it suggests that there must be a further signal to trigger CtIP ubiquitylation upon detection of DNA damage. This notion is further supported by the observation that the other known BRCA1 BRCT-interacting protein, BRIP1 (BACH1), is not ubiquitylated [45]. One possibility would be that hyperphosphorylation of CtIP by ATM after damage triggers BRCA1-mediated ubiquitylation. As BRCA1 ubiquitin ligase activity is dependent upon its heterodimeric partner, BARD1, which also possesses two phosphopeptide-binding BRCT domains [7–13], it could be envisaged that the BRCT domain of BARD1 only interacts with CtIP once it is phosphorylated at the ATM-specific residues S664 and S745. Perhaps this interaction induces a conformational change in the BRCA1/BARD1 heterodimer that activates the E3 domain, resulting in ubiquitylation of CtIP (Fig. 2). It is conceivable that CtIP may not only be a substrate of BRCA1, but rather may function to initiate BRCA1 ubiquitin ligase activity. Alternative triggers of DNA damage specific ubiquitylation by BRCA1 could include the phosphorylation of BRCA1 itself by ATM [49], and/or the recruitment of BRCA1 to chromatin by the MRN complex [13,21]. It is possible that the dependence of CtIP on the E3 activity of BRCA1 for recruitment to repair foci is not a result of its own ubiquitylation, but rather that the E3 activity of BRCA1 is required to ubiquitylate a further substrate that allows BRCA1 and any interacting proteins to be recruited to chromatin. One possible candidate would be histones, as certain histone components (H2A, H2B) are known to be ubiquitylated after DNA damage [50]. Although H2A ubiquitylation after UV damage requires the E3 Ring2A [50], other types of DNA damage may induce histone ubiquitylation via alternative E3 enzymes, such as BRCA1. It could be expected that the nucleosomes in the vicinity of the damaged site would need to be removed in order to recruit the relevant repair and checkpoint signalling factors. The demonstration that the ubiquitylation of CtIP is dependent upon a phosphorylation-specific interaction with the BRCT domain of BRCA1 is reminiscent of the targeting of phosphorylated substrates for ubiquitylation by SCF (Skp1Cdc53/Cul1-F-box) E3 ubiquitin ligases [51]. In this scenario, an adapter protein of the F box family binds to phosphorylated proteins through an interaction between the F box WD40 motif and the substrate consensus site (CPD, Cdc4 phospho-degron) [51]. However, unlike the SCF system, the interaction between CtIP and BRCA1 is not alone sufficient for ubiquitylation, but rather requires another signal as discussed above. Hence it would seem that activation of BRCA1-mediated ubiquitylation

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Fig. 2 – A model depicting the phosphorylation-dependent interactions of CtIP with the BRCA1/BARD1 heterodimer before and after DNA damage. It is proposed that in G2, CDK-dependent phosphorylation of S327 causes CtIP to interact with the BRCT domains of BRCA1. After DNA damage, CtIP is hyperphosphorylated by ATM/ATR at S664 and S745, allowing binding to the BRCT domains of BARD1, resulting in the activation of BRCA1/BARD1 E3 ligase activity and subsequent CtIP ubiquitylation.

is subject to a much more intricate system of regulation than the targeting of substrates to the proteasome. Although Yu et al. speculate that the function of CtIP ubiquitylation is to target it to chromatin and sites of DNA damage in order to mediate the G2/M damage checkpoint, the published data actually demonstrates that ubiquitylation of CtIP occurs after its recruitment [45] as the ubiquitylated species is clearly only detected in the chromatin fraction and not in soluble extracts. It is not yet obvious whether BRCA1-mediated recruitment of CtIP is sufficient for ubiquitylation, or if further signals such as ATM-dependent phosphorylation are also required, as already discussed. Therefore it remains to be determined what physiological role CtIP ubiquitylation plays within the cell. A possible means to evaluate the role of CtIP ubiquitylation would be to map and point mutate the lysine residues targeted after DNA damage. However, CtIP contains 48 different lysines, and research into other ubiquitylated proteins has suggested that when the primary target lysine(s) is mutated other lysine residues in the vicinity may be ubiquitylated to compensate and elicit the same functional effect. It is likely that it would be extremely difficult, if not impossible, to mutate enough of these lysines to observe a physiological effect, without impairing the stability or conformation of the entire CtIP protein. Furthermore, there may be multiple sites of ubiquitylation in vivo, the significance of which may not be reflected in any studies conducted in vitro. It is possible that ubiquitylated CtIP may act as a scaffold to recruit other repair and checkpoint factors

to the sites of DNA damage. If so, it would be expected that such interacting proteins would possess an ubiquitin-binding domain (UBD), several classes of which have now been identified [15,52]. Therefore, identification of UBD-proteins that interact with ubiquitylated CtIP may provide an alternative means to gain new insight into the functional consequence of CtIP ubiquitylation. Yu and co-workers clearly show that the levels of endogenous CtIP vary considerably during an unperturbed cell cycle, and that they are maximal during G2/M, consistent with a role in the G2/M checkpoint. However, others have demonstrated roles for CtIP at other times, such as the G1-S transition [35,53]. It is possible that the cellular levels of CtIP might increase considerably in the presence of stalled or blocked replication forks, such as after treatment with HU, and this has not been considered in the published work. BRCA1 has also been proposed to play crucial roles in the DNA-damage induced checkpoints during both S and G2/M phases [54]. Together, BRCA1 and CtIP may be required for both checkpoints, however, if the phosphorylation of CtIP at S327 required for interaction with BRCA1 were indeed mediated by a cyclin dependent kinase, different kinases would be necessary for CtIP activation at different stages of the cell cycle. An alternative explanation would be that the phosphorylation-dependent interaction of BRCA1 and CtIP during G2 provides specific regulation for the G2/M DNA damage response, and that in S phase, BRCA1 and CtIP have independent roles in checkpoint activation. This would be supported by the recent work by Greenberg et al.

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[21] in which they observed activation of the G2/M checkpoint by BRCA1/BARD1 interaction with CtIP and the MRN complex, whereas the S phase checkpoint is instead dependent upon BRCA1/BARD1 interaction with BRIP1 and TopBP1. CtIP and BRIP1 have previously been shown to be mutually exclusive binding partners of the BRCA1 BRCT [38]. Furthermore, the role of CtIP in the G1-S checkpoint may instead involve its alternative binding partner RB, as the G1 arrest observed after CtIP depletion in mouse embryonic fibroblasts is dependent upon RB [53]. BRCA1 has been proposed to function in a number of different cellular processes, including transcriptional regulation, cell cycle progression and meiotic sex chromosome inactivation, many of which do not appear to be impacted on by CtIP. It is therefore intriguing to speculate that there may be other substrates of BRCA1 ubiquitylation that must initially interact with the BRCT domain through phosphorylation [18]. It follows that analysis of BRCT-interacting proteins in addition to screens for proteins containing putative BRCT-interacting motifs may well open up new avenues for the identification of novel substrates of BRCA1 ubiquitylation. The identification of CtIP as a bone fide physiological substrate of the BRCA1 E3 ubiquitin ligase represents a significant advance in the understanding of the downstream effects of BRCA1. It is hoped that this discovery will facilitate more rapid progress in this complex regulatory pathway.

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Louise J. Barber, Simon J. Boulton ∗ are in the DNA Damage Response Laboratory, Cancer Research UK, The London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK ∗ Corresponding

author. Tel.: +44 1707625774; fax: +44 2082693801. E-mail address: [email protected] (S.J. Boulton)