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DeubiKuitylation A novel DUB enzymatic activity for the DNA repair protein, Ku70 Moran Rathaus, Batya Lerrer and Haim Y. Cohen* The Mina and Everard Goodman Faculty of Life Sciences; Bar-Ilan University; Ramat-Gan, Israel

Abbreviations: ATM, ataxia telangiectasia mutated; DUB, deubiquitylation; DNA-PKcs, DNA dependent protein kinase; DSBs, DNA double strands breaks; HR, homologous recombination; MRN, Mre11-Rad50-Nbs1; NHEJ, non homologous end joining repair; PI3K, phosphatidylinositol 3-kinase; PTM, post translational modification Key words: Ku70, deubiquitylation (DUB), Bax, apoptosis

The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Yet, despite this wide spectrum of pathways, until recently the enzymatic activity of Ku70 was elusive. Recent findings demonstrate that Ku70 is associated with the proapoptotic protein Bax and possesses a deubiquitin enzyme (DUB) activity on it. These data suggest a dual role for Ku70 in apoptotic regulation; on one hand the association with Ku70 sequestered Bax away from the mitochondria and plays an antiapoptotic function. On the other hand, this association mediates and promotes Bax deubiquitylation which will block its labeling for proteasomal degradation and in this manner Ku70 will have a proapoptotic role. The exciting finding of Ku70’s DUB activity opens numerous avenues for future research. Here we suggest candidate substrate proteins and indicate how the DUB activity of Ku70 on these, might affect the known Ku70’s related pathways.

Introduction Ku70 is a 70 kDa protein that was shown to be involved in multiple cellular pathways, mainly involving DNA repair and recombination. Among these are Non Homologous End Joining repair (NHEJ) of DNA double strands breaks (DSBs), V(D)J recombination, telomere maintenance, and the regulation of Bax-mediated apoptosis. For all these functions, except for its apoptotic one, Ku70 acts as part of a heterodimer with an approximately 80 kDa protein, Ku80. In contrast to the significant amount of data on the biological functions of Ku70, our knowledge of its biochemistry and enzymatic activity is limited. *Correspondence to: Haim Y. Cohen; Faculty of Life Sciences; Bar-Ilan University; Ramat-Gan 52900 Israel; Tel.: 972.3.531.8383; Fax: 972.3.738.4058; Email: [email protected] Submitted: 04/26/09; Accepted: 04/27/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8864

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Recently, however, a study from our group revealed that Ku70 possesses deubiquitylation (DUB) activity.1 This initial biochemical characterization should further our understanding of the roles of this molecule in varied cellular processes. In this review, we will summarize these findings and suggest possible pathways through which Ku70’s DUB activity may mediate its various other functions. Ku70 and Ku80 were first identified as autoantigens in the serum of a patient with Scleroderma Polymyositis Syndrome.2 Since then, auto antibodies against Ku70/80 were found in other autoimmune diseases, such as Scleroderma and Systemic Lupus Erythematosus. Ku70 and Ku80 show only 14% homology to each other. However, structural analysis of these proteins bound to double stranded DNA, demonstrated higher levels of structural similarity between the proteins. Both Ku70 and Ku80 have a central β barrel domain that binds nonspecifically to the sugarphosphate backbone of the DNA. Two other important shared domains are the N terminal α/β regions, and the helical carboxyl terminal arm domains. These domains are important for the association with the catalytic subunit of the protein DNA dependent protein kinase (DNA-PKcs).

The Different Roles of Ku70 As briefly mentioned above, Ku70 is a versatile protein, known to be involved in a variety of cellular pathways. The following section will review several of its diverse roles.

Ku70 in DNA Repair A large number of studies have described the role of the Ku70/80 complex in DNA repair.3,4 DSBs are probably the most dangerous threat to the stability of the genome. DSBs can be formed due to gamma radiation, due to unrepaired single strand breaks or at stalled DNA replication forks. In contrast to these, DSBs can also be formed as part of intentional events that promote genome variation during physiological processes such as meiosis and rearrangement at the V(D)J site to generate immunoglobulin diversity.

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Novel DUB activity of Ku70

If the cell fails to repair the break, sequence information can be lost, which can lead either to aberrant function or to programmed cell death (apoptosis). Inaccurate repair can also lead to chromosomal rearrangements that might promote carcinogenesis. The DSB repair process requires the coordination of several cellular events. To this end, two highly conserved mechanisms have evolved: NHEJ and homologous recombination (HR). Generally, in both repair pathways, sensor protein(s) first detect the lesion. This is followed by the activation of kinase proteins (usually referred as transducers) that phosphorylate and thereby activate effector proteins. These effector proteins can either arrest the cell cycle to enable DNA repair or execute apoptotic pathways.5,6 The activation of DSB signal transduction requires one of the phosphatidylinositol 3-kinase (PI3K) proteins; Ataxia Telangiectasia Mutated (ATM) or Ataxia Telangiectasia and RAD3-related (ATR). ATM is the main PI3K that responds to DSBs. Mutations in this protein cause the hereditary disease, Ataxia Telangiectasia. One of the earlier steps in the induction of the DSB repair is the recruitment of ATM to the DNA break by the Mre11-Rad50-Nbs1 (MRN) complex, which is probably the primary DSB sensor.7 When ATM or ATR are recruited to the damaged site, they target various substrates, including the checkpoint kinases 1 and 2 (CHK1 &2).8 HR has the capacity to accurately replace the damaged or missing sequence information at the DSB site. This information is provided by a template sequence located in another locus, not necessarily adjacent to the damaged site. Such sequence information would preferably be derived from the sister chromatid; its 3' end is inserted to the break point so it could be used as a template for DNA synthesis across the break (Fig. 1A). All homologuedependent repair pathways first require the resection of the 5' end of the break to generate a free 3' end. The resection is performed by the MRN complex.9 Several possible subsequent sub-pathways can then lead to annealing; these include the synthesis-dependent strand annealing pathway, the classical double-Holiday junction model, and the single strand annealing pathway.10 Unlike the complex machinery of HR, DNA repair by NHEJ directly rejoins the two free DNA ends irrespective of the presence or lack of homologous sequences.11 When the two ends are blunt, the repair is fairly precise. Yet, if the DNA ends are not compatible, the resulting repair will cause alterations at the break point. Although in rare cases, break repair by NHEJ can occur even in the absence of sequence homology, in most cases, a 1–6 bp overlap facilitates the rejoining of the DNA ends.12 In contrast, in HR, a stretch of at least 100 bp is required for guiding the repair. Thus, one major difference between HR and NHEJ is the length of the DNA sequence that is required for the repair process. Another important difference is the association between the timing of the repair along the cell cycle, and the actual mechanism of repair. DSBs occurring during the S or G2 phases are readily repaired by HR using the intact sister chromatid. However as the cell progresses into the G2-M phase, the chromatin becomes more condensed, and the search for the homologous template becomes more difficult. Nevertheless, at these time points of the cell cycle, DSBs will still be predominantly repaired by HR. During G0 1844

and G1 phases of the cell cycle, when sister chromatids are absent and repair of DSB with an apparently homologous sequence on another chromosomal locus might be harmful, DSBs are mainly repaired by NHEJ. Several proteins are required for efficient repair of DSB by NHEJ. The core complex consists of the DNA-PK complex and the ligase IV/XRCC4/XLF complex (Fig. 1B). In practice, NHEJ initiates upon the binding of two ring-shaped Ku70/Ku80 heterodimers to both DNA broken ends within seconds of the creation of the DNA damage. The serine/threonine kinase, DNA-PKcs, is also recruited to this DNA-Ku scaffold and probably enables the formation of a synaptic complex. In the synaptic complex, the DNA broken ends are positioned next to each other. Depending on the properties of the lesion, some DNA ends must be processed before the final ligation step can take place. For example, a damaged DNA end can contain an aberrant 5' hydroxyl group, aberrant 3' phosphate, damaged base and/or damaged backbone sugar residue. Several enzymes can process such lesions: Polynucleotide kinase, which is associated with XRCC4, can polish DNA carrying 5' hydroxyl or 3' phosphate groups;13,14 Werner helicase, which was shown to be associated with Ku70, may also prepare DNA ends with its exonuclease activity.15 Finally, Artemis, a structure-specific nuclease, can cleave DNA hairpin structures and remove 3' overhang DNA.16 When the end processing step has been accomplished, ligase IV/ XRCC4 can catalyze the final ligation reaction. XLF/Cernunnos is a recently discovered protein that probably collaborates with ligase IV/XRCC4 to enhance the ligation reaction.17 An additional role for Ku70 in DNA repair is during the process of V(D)J recombination, the diversity generating assembly process, affecting the variable domain of immunoglobulin and TCR genes. In this process, different segments are joined to each other via genetic recombination, and as a result, the immune repertoire is enriched. The ability to join different segments is achieved by introducing intended DSBs in the genomic locus encoding the variable region of the heavy chain. These breaks are later joined by the NHEJ protein members.18 Thus, in mice, deletion of Ku70 results in a severe combined immunodeficiency phenotype.

Telomere Maintenance In addition to their involvement in DNA repair, Ku70 and Ku80 were also shown to be important for telomere maintenance and telomeric silencing. Telomeres are unique DNA-chromatin structures located at the ends of eukaryotic chromosomes. In vertabrates, they are comprised of tandem DNA repeats of the sequence TTAGGG, terminated with a long (130–270 base pairs) 3' single-stranded G-rich overhang coated with heterochromatin. This single strand (ssDNA) overhang is capable of invading the telomeric sequence, yielding a structure known as a T-loop.19-22 One of the main roles of telomeres is to protect the chromosome tips from being treated by the cell as broken DNA ends. One way to protect the chromosome ends and prevent execution of DNA repair pathways on these ends is to form a telomeric “cap”. This “cap” consists of telomeric-binding proteins and inhibits end fusion events. Among these proteins are the Telomeric Repeat binding Factor proteins, TRF1 and TRF2, which can specifically

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Figure 1. Simplified overview of double strand break repair. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent two important double strand break (DSB) repair pathways. (A) During HR, DSB is recognized by the Mre11-Rad50-Nbs1 (MRN) complex. Processing of the DSB ends by the MRN complex results in the formation of 3' single stranded (ss) DNA overhangs. The ssDNA is coated with Rad51 protein generating a nucleofilament. Additional proteins, such as Rad52, Rad54 and BRCA1&2 are also recruited to assist the search for the homologous DNA sequence in the undamaged sister chromatid. Once a homology is found, a strand invasion and exchange results in joint molecule formation. Finally, DNA synthesis based on the homologous sequence is followed by ligation and resolution of the two double helices. (B) In NHEJ the ends of a DSB are detected and bound by the ring shaped Ku70/Ku80 heterodimer, which, together with the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), generates the DNA-PK complex. The nuclease activity of Artemis leads to ends processing, and the process is accomplished by break sealing through the XRCC4-ligase IV heterodimer with the aid of the XLF/Cernunnos protein.

bind telomeric DNA.23-26 Both TRF proteins can recruit various DSB repair proteins, such as the Ku heterodimer.27-29 Similarly, in yeast, Ku70 and Ku80 are localized to the telomeres, and deletion of either one of these proteins leads to www.landesbioscience.com

defective telomere silencing, shortening by about 60% of their normal length, and deregulation of G-strand overhang.30,31 It was suggested that the Ku heterodimer provides protection against the activity of either nucleases or recombinases.32

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Hsu et al.27 showed that in mammals, the Ku 70/80 heterodimer is localized to the telomeric repeats by a high affinity interaction with TRF1, in a DNA-PKcs independent manner. Thus, the Ku dimer can prevent telomeric end joining. This heterodimer was also shown to bind mammalian telomeric sequences in vitro33 and to prevent chromosomal end-to-end fusions.27 In addition, Ku 70/80 was shown to physically associate with the catalytic subunit of telomerase.34 In analogy to the Ku70/80 deficiency in yeast, di Faganga and colleagues found telomeric shortening in mouse embryonic fibroblast (MEFs), embryonic stem cells and adult tissues from Ku70-/- mice.35 Yet, these findings remain controversial, since cells derived from Ku80-deficient mice do not demonstrate G-strand overhang or telomeric shortening.36 Taken together, these results demonstrate that Ku70 and Ku80 are important for telomere maintenance and the chromatin structure surrounding the telomeres. Yet, the mechanisms by which these proteins regulate the chromatin structure are still unknown.

Regulation of Bax-Mediated Apoptosis by Ku70 In addition to its role in regulating DNA-repair, V(D)J recombination and telomere maintenance, Ku70 was recently shown to function in regulating Bax-mediated apoptosis. Programmed cell death, or apoptosis, is a natural process of removing unnecessary or damaged cells, and is required for the proper execution of the organism’s life cycle.37,38 Apoptosis was shown to be involved in numerous processes including embryonic development,39 response to cellular damage,40-43 aging and as a mechanism of tumor suppression.44-47 Two pathways were shown to induce apoptosis, an extrinsic and an intrinsic one. The difference between these two pathways is the mechanism by which the death signal is transduced.38 Whereas the extrinsic pathway is activated by binding of ligands to a death receptor, the intrinsic pathway is activated by cellular stress, for example DNA damage. The intrinsic pathway involves the release of cytochrome c from the intermembrane space of the mitochondria. Together with apoptotic protease activating factor 1 (APAF1), cytochrome c activates caspase 9, leading to activation of downstream caspases and the induction of the death response. Key players in the regulation of the intrinsic pathway include the Bcl2 protein family, which can influence the permeability of the outer mitochondrial membrane.48 Members of the Bcl2 protein family are divided into proapoptotic proteins such as Bax, Bak and Bok, and antiapoptotic ones including Bcl2, Bcl-X, Bcl-w and Mcl-1. Proteins of a third subfamily, known as the BH3-only proteins, are thought to be initiators of apoptosis, and probably function by regulating Bcl2-like proteins from the other two subfamilies. In healthy cells, Bax exists as a monomer, either in the cytosol or weakly bound to the outer mitochondrial membrane. Upon stimulation of apoptosis, Bax undergoes a series of conformational changes and translocates to the mitochondria, where it becomes anchored into the mitochondrial membrane. Following its translocation, Bax oligomerizes into large complexes, which are essential for the permeabilization of the mitochondrial membrane.49 Given its central role in mediating apoptosis, several mechanisms have been proposed for Bax regulation and retention in the cytosol, 1846

both by binding to other proteins and through posttranslational modifications. One of the major questions regarding the regulation of Bax is how this protein is retained in the cytosol under non-stress conditions. One of the first proteins that were shown to sequester Bax away from the mitochondria was Ku70.44-47 Human embryonic kidney cells overexpressing Ku70 show lower levels of cell death after apoptotic stimuli, such as Staurosporine or ultraviolet radiation. Moreover, overexpression of Ku70 also blocks apoptotic cell death induced by transfection of the cell with Bax. Similarly, cells expressing reduced levels of Ku70 or Ku70-/- MEFs are more sensitive to Bax-mediated apoptosis than wild type cells.44 Taken together, these results suggest that Ku70 inhibits Bax-mediated apoptosis, and that Ku70 has anti-apoptotic activity. Although these findings suggest one model for the retention of Bax in the cytosol, they do not explain the mechanism by which Bax is released from Ku70 upon stress conditions. The answer arose when eight acetylation sites on Ku70 were identified. In order to understand the role of these acetylations, each acetylated lysine was mutated to arginine or glutamine, to mimic constitutively deacetylated and acetylated lysine, respectively. Mimicking acetylation on Ku70 residues K539 and K542 blocks the inhibition of Bax by Ku70.44 Moreover, apoptotic stimuli lead to dissociation of the Ku70-Bax complex, resulting in cell death. These and other experiments suggested a mechanism whereby, upon apoptotic stimulus, the CBP and PCAF acetyltransferases acetylate Ku70. As a result, Bax dissociates from Ku70 and translocates to the mitochondria, leading to initiation of apoptosis (Fig. 2). Recently, at least four additional proteins, Bcl2,50 14-3-3,51 Humanin52 and Clusterin53 were also shown to negatively regulate apoptosis by binding to Bax and sequestering it from the mitochondria. These findings raised the interesting question of why so many proteins are required to sequester Bax. Moreover, it was not known whether the association with Ku70 serves any additional functions besides maintaining Bax in the cytosol. It is also not known whether different populations of Bax simultaneously associate separately with each of these proteins, or if there is a chronological order for these associations, which serve as multiple lines of defense against aberrant release. Finally, a mutation resulting in knock out of Ku70 expression in mice is not embryonic lethal, and Ku70-/- mice exhibit a significant increase in their basal level of apoptosis only in the brain54 and in gastrointestinal tissue.55 Thus, these observations suggest that additional steps subsequent to the release from Ku70 also regulate Bax activation, and that Ku70 might have other apoptosis-related functions in addition to its role in sequestering Bax. To further elucidate the anti-apoptotic function of Ku70, Amsel et al. followed the fate of Bax in the absence of Ku70 without apoptotic stimuli.1 When Ku70 protein levels were reduced in osteosarcoma cells either by siRNA or by expression of an antisense sequence, or in Ku70-/- MEFs, Bax appeared at higher molecular weight by SDS gel electrophoresis. These results suggest that Bax is modified in the absence of Ku70. Indeed, analysis of the modification revealed that Bax is ubiquitylated under these conditions. Protein poly-ubiquitylation tags proteins for proteasomal degrada-

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Figure 2. Regulation of Bax mediated apoptosis by Ku70 DUB activity. Following its synthesis, Bax undergoes ubiquitylation, which negatively regulates its proapoptotic function by labeling it for proteasomal degradation. Alternatively, ubiquitylated Bax can associates with Ku70, which mediates and promotes Bax deubiquitylation. Upon apoptotic stimulus, Ku70 is acetylated by the acetyltransferases CBP or PCAF, and releases Bax. The free cytosolic un-ubiquitylated Bax is now able to localize to the mitochondria to execute its apoptotic function.

tion, suggesting that in the absence of Ku70, Bax is tagged for degradation, in order to avoid excessive apoptosis. Similarly, when Ku70-expressing cells are exposed to apoptotic stimuli, the level of ubiquitylated Bax decreases. Treatment of these cells with the proteasomal inhibitor, MG-132, results in an increase in the levels of ubiquitylated Bax. These findings suggest that Bax ubiquitylation inhibits Bax function, and that Bax must be deubiquitylated in order to mediate apoptosis. The increase in the levels of ubiquitylated Bax in Ku70-deficient cells suggests that Bax ubiquitylation might provide backup protection to the cell from Bax-mediated apoptosis when Ku70 dissociates from Bax, releasing its sequestration from the mitochondria. If the ubiquitylation of Bax indeed confers backup protection against unwanted cell death, one would predict that during apoptosis, no ubiquitylated Bax would be found in Ku70 defiwww.landesbioscience.com

cient cells. Strikingly, time course analysis of Bax ubiquitylation after apoptotic stimulus in Ku70 deficient cells shows an increase rather than a decrease in the levels of ubiquitylated Bax. Therefore, Ku70 might mediate Bax deubiquitylation or its degradation. Adding recombinant Ku70 into Ku70 deficient cell extract resulted in decreased levels of ubiquitylated Bax even when the cells were treated with the proteasomal inhibitor, MG-132. Thus, Ku70 regulates the process of Bax deubiquitylation and not its degradation. More importantly, Ku70 itself probably possessed deubiquitylation enzymatic activity (DUB) on Bax, since in vitro assay with recombinant polyubiquitin chains and Ku70 revealed that Ku70 does possess such deubiquitylation activity. Taken together, these data suggest a novel mechanism for Bax regulation: under normal unstressed conditions, following its synthesis, Bax undergoes ubiquitylation, which negatively regulates

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its proapoptotic function by labeling it for proteasomal degradation. The association with Ku70 mediates and promotes Bax deubiquitylation, a process that is enhanced following apoptotic stimuli. Upon apoptotic stimulus, Ku70 is acetylated by the acetyltransferases CBP or PCAF, and releases Bax.44 The free cytosolic un-ubiquitylated Bax localizes to the mitochondria where it mediates its apoptotic function (Fig. 2). These findings also suggest a complex role for Ku70 with both pro-apoptotic (maintaining an active pool of Bax) and anti-apoptotic (sequestering Bax away from the mitochondria) elements.

Potential Novel Roles for Ku70 as a DUB Enzyme The exciting finding of Ku70’s DUB activity is the first proven enzymatic activity for Ku70. Such evidence opens numerous avenues for future research. Although the large number of ubiquitylated proteins in the cell makes it difficult to a priori identify specific substrates for the DUB activity mediated by Ku70, one can speculate on candidate substrate proteins involved in each of Ku70’s related pathways. In the next paragraphs, we will indicate how the DUB activity of Ku70 might affect the known Ku70dependent pathways.

Chromatin Structure As part of its function in DSB repair, Ku70 may affect either the chromatin structure surrounding the DNA break site or the telomere. One immediate potential substrate could be the histones. Histones were found to be heavily modified by several post translational modifications (PTMs) including acetylation, methylation, phosphorylation, sumoylation and ubiquitylation.56-60 Although identified over 30 years ago,61 histone ubiquitylation remains one of the most poorly understood histone modifications. The most highly ubiquitylated histone is H2A (about 10% of total H2A), followed by H2B, and finally, H3 and H1. Histone ubiquitylation was found in positive correlation with the expression of several genes.62 Yet there are also reports showing that ubiquitylated histones are excluded from some active promoters.63,64 Histone ubiquitylation can affect transcription by three mechanisms, a direct effect on chromatin structure, recruiting other factors, or by impacting other histone modifications. PTMs on lysine are believed to cancel the positive charge of this residue on histones, which enables euchromatin structure. For example, lysine acetylation on the N-terminal tails of histones cancels their positive charge, thereby generating a looser chromatin structure and allowing the transcription machinery easier access to a particular promoter. Thus, ubiquitylation on these lysine residues might have a similar effect as acetylation. Moreover, whereas a single ubiquitin molecule is a peptide of 76 amino acids corresponding to 8.6 kDa, the acetyl group (CH3CO) adds only 42 Da. Therefore histone ubiquitylation might also impair compact chromatin folding due to size effects. Indeed, it was shown that during the transition from G2 to M, when the chromatin becomes compact, ubiquitylated H2A and H2B disappear;65,66 these ubiquitylated species reappear as chromatin is decondensed during the transition to G1 phase.67 More recent studies showed that ubiquitylated histones might affect chromatin folding at the fiber level.68 1848

DNA Repair Several chromatin modifications have been associated with the repair of DSB, especially histone phosphorylation and acetylation. One of the hallmarks of the region surrounding DSBs is the phosphorylation of histone H2AX on Ser 139 (γ-H2AX). Although this modification is not required for the repair itself, it is required for two processes: the recruitment of the repair machinery along the lesion sites, and to recruit a specific H2A/ H4 histone acetyltransferase in order to relax the chromatin. In addition to histone H4 acetylation, histone H3 acetylation has also been associated with the repair of DSB. For example, while mutations in the acetylation sites of H4 block the repair process in general, mutations in the acetylation sites of H3 specifically block HR. Ubiquitin itself has several roles in DSB. It was shown that RNF8, together with the E2 ligase, UBC13, bind phosphorylated MDC1 (by ATM) and ubiquitylate histones H2A and H2AX. The K63 linked polyubiquitylated histones recruit the BRCA1/BARD1/CCDC98/RAP80 complex via the ubiquitin interacting motif of RAP80.69-76 BRCA1 was shown to localize DSB sites, and its absence increases genome instability. BRCA1 is involved in both HR and NHEJ repair, through an unclear mechanism.77-80 Thus, it is possible that Ku70 may deubiquitylate histones H2A and H2AX and regulate the association of the BRCA1 complex with the lesion sites. Another possibility is that the activity of Ku70 on these histones might regulate chromatin condensation surrounding the break, enabling access to the repair machinery, but at the same time blocking the entrance of unwanted factors such as transcription factors. In addition to the above, Ku70 might auto-regulate its ubiquitylation level or that of its associated proteins. For example, it was shown that in some cell lines, the level of Ku70 decreases due to its proteasomal degradation upon apoptotic stimuli.81 Thus, Ku70 can regulate its various activities by controlling its own ubiquitin levels. Similarly, many Ku70-associated proteins were also found to be ubiquitylated (Table 1). Therefore, Ku70 may regulate either their levels or activities. Recently it was shown that ubiquitylation of Ku80 promotes its removal from DSB sites.82 Thus, one could speculate that Ku70 might retain Ku80 in its deubiquitylated form in order to enable DSB repair. Another possibility is that dissociation of Ku70 from Ku80 allows the release of the latter from the DNA once the repair process is completed. Based on published data, we can predict some novel Ku70 substrates. Ku70 was shown to be associated with Werner helicase, which was suggested to counteract the NHEJ pathway, possibly by displacement of DNA-PKcs from DNA ends.83,84 The Werner helicase-interacting protein 1, WRNIP1, was shown to be hyper polyubiquitylated upon UV radiation, suggesting a role for polyubiquitin in regulating the role of the Werner complex in DNA repair. Although the precise role for the polyubiquitylation of WRNIP1 is not yet clear, WRNIP1 might also be regulated by the DUB activity of Ku70. Moreover, one can propose a model in which the cellular decision between different types of DNA repair modes might be controlled by the tightly regulated interaction of Ku70 DUB activity on WRN and WRNIP1.

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Table 1 Ku70 associated protein and their ubiquitylation status Function

Protein

Acetylation

Role of ubiquitylationA

Ubiquitylated

ReferenceB

SIRT1

No

-

CBP

Yes

ND

PCAF

Yes

ND

Bax

Yes

Stability

1

Ku80

Yes

Removal from DNA

82

DNA-PKcs

No

-

WRN

Yes

ND

γH2AX

Yes

Regulation and promotion of the accumulation of proteins in DSB repair

Yes (autoubiquitylation)

ND

Apoptosis DNA repair

RAG1

PCNA Yes

Facilitating and coordinating the recruitment and activation of translesional polymerases to process DNA lesions through error-prone or error-free pathways

Telomeric proteins

TRF2

No

-

Transcription factors

YY1

No

-

Tbdn100

No

98–101

-

C/EBPα Yes Others

97

Controlling both transcription-dependent and transcription-independent activities of C/EBPa

102

Securin

Yes

P40phox

No

-

EGFR

Yes

Regulation of localization and stability

103

Regulation of the growth-promoting activity of TRBP in response to the signals from increased cell-to-cell contact.

104

TRBP Yes

ND

AND, not determined. BReferences are referred to the role of ubiquitylation of this protein.

V(D)J Recombination Another intriguing possibility, is that Ku70 regulates V(D)J recombination via its DUB enzymatic activity. Both RAG1 and RAG2, the V(D)J recombinases, were found to be ubiquitylated. While RAG1 possesses an autoubiquitylation activity,85 RAG2 is ubiquitylated by Skp2-SCF ubiquitin ligase.86 The ubiquitylation of RAG2 allows its degradation during S phase, and the coupling of V(D)J recombination to the G0 and G1 cell cycle phases. Thus, Ku70 might regulate the timing and efficiency of V(D) J recombination by controlling the levels of RAG1 and RAG2 ubiquitylation.

Telomere Maintenance Increasing data suggest that ubiquitylation might be involved in telomere maintenance. Yet, our knowledge of this pathway is still limited and it is too early to speculate how Ku70 DUB enzymatic activity may contribute to telomere length and maintenance.

Apoptosis Regarding apoptosis, it would be fascinating to reveal the full breadth of the involvement of Ku70. Given the complex role of Ku70 acting either as a positive or negative regulator of apoptosis, this protein might monitor the expression or activity of various pro- and anti-apoptotic proteins. It is possible that in addition to www.landesbioscience.com

Bax, Ku70 associates with and deubiquitylates other members of the Bcl-2 family of proteins. Within this family, many proteins have been shown to be ubiquitylated, including the anti-apoptotic Bcl-2 and Mcl-1, and the pro-apoptotic proteins Bax, Bak, Bad, Bid and Bim. Ubiquitylation was shown to regulate the levels of each of these proteins, suggesting that the polyubiquitin moiety marks these proteins for degradation.87-96 Yet, we cannot rule out the possibility that some members are modified by mono or multiubiquitylation and that this ubiquitylation also regulates other functions of these proteins besides degradation.

Perspective The finding that Ku70 possesses a DUB enzymatic activity suggests many new routes of investigation. Ku70 was shown to be involved in genome stability, apoptosis and telomere maintenance and in generating antibody variation. Thus, it is likely that its DUB enzymatic activity is involved in the regulation of these pathways (Fig. 3). The DUB activity of KU70 also provides a new perspective on the versatility of DNA repair proteins. Our view of Ku70 has evolved, from that of a “common industrial worker” with a specific and defined function, to a sophisticated protein that regulates a multitude of pathways and proteins. The complex phenotype resulting from the mutation of many DNA repair proteins is difficult to understand simply on the basis of their repair function, but could be better explained by this novel

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Figure 3. The possible involvement of Ku70 in different cellular pathways.

Ku70 enzymatic activity. The model of Ku70 as a versatile protein with diverse functions raises an interesting questions: Do other DNA repair proteins actually posses more than a single activity, and are these proteins as versatile in their control of various cellular pathways as Ku70? We predict that in the near future, a plethora of additional functions will be found for these proteins. Acknowledgements

We thank Shelley Schwarzbaum and Shoshana Naiman (BIU, Israel) and members of the Cohen lab for their helpful comments on the manuscript. This study was supported by grants from the Israeli Academy of Sciences, German-Israeli Foundation, Binational US-Israel Foundation, the Israel Cancer Association, Israel Cancer Research Fund, the Koret Foundation and the Israel Health Ministry. References 1. Amsel AD, Rathaus M, Kronman N, Cohen HY. Regulation of the proapoptotic factor Bax by Ku70-dependent deubiquitylation. Proc Natl Acad Sci USA 2008; 105:5117-22. 2. Mimori T, Akizuki M, Yamagata H, Inada S, Yoshida S, Homma M. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest 1981; 68:611-20. 3. Karran P. DNA double strand break repair in mammalian cells. Curr Opin Genet Dev 2000; 10:144-50. 4. Kanaar R, Hoeijmakers JH, van Gent DC. Molecular mechanisms of DNA double strand break repair. Trends Cell Biol 1998; 8:483-9. 5. Zhou BB, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000; 408:433-9. 6. Kobayashi J, Iwabuchi K, Miyagawa K, Sonoda E, Suzuki K, Takata M, et al. Current topics in DNA double-strand break repair. J Radiat Res 2008; 49:93-103.

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