HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation.Almost 30 yr ago, proliferating cell nuclear antigen (PCNA) was first identified in dividing cells using sera derived from patients suffering from systemic lupus erythematosus (Takasaki et al., 1981). A few years later, the “mother” of all cancer markers had been associated with DNA synthesis (Madsen and Celis, 1985), but it wasn’t until 1988 that Bauer and Burgers (1988) and Prelich and Stillman (1988) discovered that the homotrimeric clamp served as a processivity factor for DNA polymerases. In 1992, Shivji et al. (1992) showed that PCNA was required for DNA repair, and 10 yr later, it was identified as a target of ubiquitin and SUMO (small ubiquitin-like modifier) conjugation after exposure to ultraviolet light (Hoege et al., 2002). For a protein that has been in the spotlight of modern biochemistry, it is quite remarkable that almost nothing is known about its normal cellular turnover.Insight into this process comes now from the study of an unlikely regulator. In this issue, Groehler and Lannigan (2010) demonstrate that the relatively poorly characterized ERK8 (extracellular signal-regulated kinase 8) takes center stage in the regulation of PCNA stability in primary mammary epithelial cells. The ERK family of kinases belongs to the mitogen-activated protein kinase superfamily and carries a Thr-Glu-Tyr (T-E-Y) activation motif that needs to be phosphorylated to enable kinase activity (Abe et al., 2002). Interestingly, ERK8 also needs to bind to chromatin to become active. The authors identified a highly conserved PXXXP motif in the C-terminal half of ERK8 that appeared to confer autoinhibition, an activity which is relieved upon chromatin binding. Relatively close by, in the middle of ERK8, resides a PCNA-interacting peptide (PIP) box required for the interaction with PCNA (Warbrick, 1998). Curiously, only the chromatin-bound fraction of ERK8 bound to the chromatin-bound fraction of PCNA. However, a functional PIP box was not required for ERK8 to associate with nuclear DNA in the cell. These results argue that ERK8 is not anchored to chromatin by PCNA but associates with it independently. Moreover, they strongly suggest that ERK8’s PIP box binds to PCNA only when the kinase is associated with chromatin. Importantly, overexpression of an ERK8 PIP box mutant resulted in destabilization of PCNA. The effect on PCNA stability seemed to be highly specific, as depletion of ERK8 caused codepletion of PCNA but did not lead to a decrease in steady-state levels of a variety of other cell cycle regulators.Why is the interaction with PCNA confined to chromatin? The reason is likely due to the fact that ERK8’s PIP box is buried in the middle of the protein. Most PCNA-interacting proteins carry their PIP box either at the N or C terminus (Vivona and Kelman, 2003). One other well-studied example for a protein with an internal PIP box is the essential replication factor MCM10 (minichromosome maintenance protein 10). MCM10 undergoes cell cycle–regulated modification, which probably induces a conformational switch that is necessary for the PIP box–mediated interaction with PCNA (Das-Bradoo et al., 2006). In the same vein, it is conceivable that chromatin association and the accompanying relief of autoinhibition of ERK8 cause the middle portion of the kinase to change its configuration, thereby assuming a functional PIP box domain that can be recognized by PCNA. In situations in which the rapid unloading of PCNA is required, regulation of ERK8 may be the most effective way to dispose of chromatin-bound PCNA, which is known to have an exceedingly low exchange rate (Sporbert et al., 2002). Despite the fact that interaction with ERK8 is necessary to stabilize chromatin-bound PCNA, it remains unclear whether PCNA is a direct target of ERK8-mediated phosphorylation.The next goal of Groehler and Lannigan (2010) was to dissect the mechanism underlying the ERK8-regulated degradation of PCNA. Based on the consideration that physical contact between the kinase and PCNA was an integral part of the protection, they hypothesized that ERK8 might compete with an E3 ubiquitin ligase that may target PCNA via its own PIP box. This turned out to be a smart guess because the only candidate to test was the E3 ligase HDM2, the human homologue of murine double minute 2 (Momand et al., 1992). In a set of well-controlled experiments, the authors not only demonstrate that HDM2 interacts directly with and degrades PCNA when ERK8 is absent, but they also exclude indirect effects by p53 and retinoblastoma (Rb) on this process. p53 is a direct target of HDM2 and is stabilized when their interaction is inhibited (Tao and Levine, 1999). Elevated levels of p53 trigger cell cycle arrest concomitant with hypophosphorylation of Rb, but none of these changes affect the stability of PCNA. It is not hard to imagine that the loss of chromatin-bound PCNA has severe consequences for the functionality of DNA replication and repair, resulting in chromosome breakage. The authors argued that a similar level of genome instability should be visible in ERK8-depleted cells. This was indeed the case as visualized by the accumulation of γ-H2AX foci and broken DNA (Rogakou et al., 1998). Importantly, Groehler and Lannigan (2010) observed similar effects in the ERK8 PIP box mutant, further lending credence to their model. It is worthwhile pointing out that the turnover of PCNA expands the spectrum of replication factors whose degradation is tightly linked to chromatin. CDT1, a member of the prereplication complex (Cook, 2009), is rapidly degraded in the face of DNA damage. Its degradation occurs exclusively on the chromatin-associated fraction of the protein pool and is dependent on CDT1 binding to PCNA (Arias and Walter, 2005; Hu and Xiong, 2006; Senga et al., 2006).An important question that this study raises is of course to what extent, if at all, is PCNA turnover deregulated in cancer cells? The commonly high levels of PCNA in transformed cells would be most compatible with a deregulation of ERK8 and/or HDM2 to provide a significant growth advantage. Indeed, the authors show in the last part of their study that in at least two transformed cell lines, PCNA is rendered inert to the presence of ERK8. They speculate that the underlying reason is a defect in HDM2, and although this is the most likely explanation, it still needs to be validated. It will be interesting to see how common the misregulation of PCNA turnover is in cancer tissues. At this point, it is intriguing to envision a dynamic scenario in which a two-step mechanism facilitates cell transformation (Fig. 1). Initially, deregulation of ERK8 may cause PCNA levels to decrease. This would contribute to genome instability and the accumulation of new mutations, including those affecting proper function of HDM2. In step two, deregulation of HDM2 may turn things around and result in an increase of PCNA, supporting rapid proliferation.Open in a separate windowFigure 1.Role of ERK8 in maintaining genome stability. (A) In normal cells, chromatin-bound ERK8 interacts with the chromatin fraction of PCNA, which resides at the replication fork (here just shown at the leading strand for simplicity). ERK8 binding inhibits the E3 ubiquitin ligase HDM2 from interacting with PCNA. (B) In cancer cells, inactivation of ERK8 enables HDM2 to interact with and ubiquitinate PCNA, targeting it for degradation. A decrease in PCNA levels causes an increase in DNA damage, resulting in the accumulation of new mutations. These new mutations may render HDM2 nonfunctional (rectangular form), which ultimately results in an increase of PCNA stability and facilitates cell proliferation. The homotrimeric PCNA structure (Protein Data Bank ID 2OD8) was generated using the Chimera software program (Pettersen et al., 2004).     

L2DTL/CDT2 and PCNA Interact with p53 and Regulate p53 Polyubiquitination and Protein Stability through MDM2 and CUL4A/DDB1 Complexes

The CUL4-ROC1 E3 ligase complex regulates genome stability, replication, and cell cycle progression. A novel WD40 domain-containing protein, L2DTL, and PCNA were identified as proteins associated with CUL4/DDB1 complexes. Inactivation of CUL4A, L2DTL, PCNA, DDB1, or ROC1 induced p53 stabilization and growth arrest. L2DTL, PCNA, and DDB1/CUL4A complexes were found to physically interact with p53 tumor suppressor and its regulator MDM2/HDM2. The isolated CUL4A complexes display potent and robust polyubiquitination activity towards p53 and this activity is dependent on L2DTL, PCNA, DDB1, ROC1, and MDM2/HDM2. We also found that the interaction between p53 and CUL4 complex is regulated by DNA damage. Our data further showed that MDM2/HDM2 is rapidly proteolyzed in response to UV irradiation and this process is regulated by CUL4/DDB1 and PCNA. Our studies demonstrate that PCNA, L2DTL, and the DDB1-CUL4A complex play critical and differential roles in regulating the protein stability of p53 and MDM2/HDM2 in unstressed and stressed cells.     

The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4^Cdt2)–PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2–proteasome pathway to facilitate TLS pathway.     

Characterization of human Spartan/C1orf124, an ubiquitin-PCNA interacting regulator of DNA damage tolerance

Unrepaired DNA damage may arrest ongoing replication forks, potentially resulting in fork collapse, increased mutagenesis and genomic instability. Replication through DNA lesions depends on mono- and polyubiquitylation of proliferating cell nuclear antigen (PCNA), which enable translesion synthesis (TLS) and template switching, respectively. A proper replication fork rescue is ensured by the dynamic ubiquitylation and deubiquitylation of PCNA; however, as yet, little is known about its regulation. Here, we show that human Spartan/C1orf124 protein provides a higher cellular level of ubiquitylated-PCNA by which it regulates the choice of DNA damage tolerance pathways. We find that Spartan is recruited to sites of replication stress, a process that depends on its PCNA- and ubiquitin-interacting domains and the RAD18 PCNA ubiquitin ligase. Preferential association of Spartan with ubiquitin-modified PCNA protects against PCNA deubiquitylation by ubiquitin-specific protease 1 and facilitates the access of a TLS polymerase to the replication fork. In concert, depletion of Spartan leads to increased sensitivity to DNA damaging agents and causes elevated levels of sister chromatid exchanges. We propose that Spartan promotes genomic stability by regulating the choice of rescue of stalled replication fork, whose mechanism includes its interaction with ubiquitin-conjugated PCNA and protection against PCNA deubiquitylation.     

Post-Translational Modifications of PCNA in Control of DNA Synthesis and DNA Damage Tolerance-the Implications in Carcinogenesis

The faithful DNA replication is a critical event for cell survival and inheritance. However, exogenous or endogenous sources of damage challenge the accurate synthesis of DNA, which causes DNA lesions. The DNA lesions are obstacles for replication fork progression. However, the prolonged replication fork stalling leads to replication fork collapse, which may cause DNA double-strand breaks (DSB). In order to maintain genomic stability, eukaryotic cells evolve translesion synthesis (TLS) and template switching (TS) to resolve the replication stalling. Proliferating cell nuclear antigen (PCNA) trimer acts as a slide clamp and encircles DNA to orchestrate DNA synthesis and DNA damage tolerance (DDT). The post-translational modifications (PTMs) of PCNA regulate these functions to ensure the appropriate initiation and termination of replication and DDT. The aberrant regulation of PCNA PTMs will result in DSB, which causes mutagenesis and poor response to chemotherapy. Here, we review the roles of the PCNA PTMs in DNA duplication and DDT. We propose that clarifying the regulation of PCNA PTMs may provide insights into understanding the development of cancers.     

APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage

Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome^Cdh1 (APC/C^Cdh1), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C^Cdh1. USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C^Cdh1 inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C^Cdh1 in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry.     

Readers of PCNA modifications

The eukaryotic sliding clamp, proliferating cell nuclear antigen (PCNA), acts as a central coordinator of DNA transactions by providing a multivalent interaction surface for factors involved in DNA replication, repair, chromatin dynamics and cell cycle regulation. Posttranslational modifications (PTMs), such as mono- and polyubiquitylation, sumoylation, phosphorylation and acetylation, further expand the repertoire of PCNA’s binding partners. These modifications affect PCNA’s activity in the bypass of lesions during DNA replication, the regulation of alternative damage processing pathways such as homologous recombination and DNA interstrand cross-link repair, or impact on the stability of PCNA itself. In this review, we summarise our current knowledge about how the PTMs are “read” by downstream effector proteins that mediate the appropriate action. Given the variety of interaction partners responding to PCNA’s modified forms, the ensemble of PCNA modifications serves as an instructive model for the study of biological signalling through PTMs in general.     

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna^K164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage.     

Phosphorylation of PNKP by ATM prevents its proteasomal degradation and enhances resistance to oxidative stress

We examined the mechanism regulating the cellular levels of PNKP, the major kinase/phosphatase involved in the repair of oxidative DNA damage, and find that it is controlled by ATM phosphorylation and ubiquitylation-dependent proteasomal degradation. We discovered that ATM-dependent phosphorylation of PNKP at serines 114 and 126 in response to oxidative DNA damage inhibits ubiquitylation-dependent proteasomal degradation of PNKP, and consequently increases PNKP stability that is required for DNA repair. We have also purified a novel Cul4A-DDB1 ubiquitin ligase complex responsible for PNKP ubiquitylation and identify serine–threonine kinase receptor associated protein (STRAP) as the adaptor protein that provides specificity of the complex to PNKP. Strap^−/− mouse embryonic fibroblasts subsequently contain elevated cellular levels of PNKP, and show elevated resistance to oxidative DNA damage. These data demonstrate an important role for ATM and the Cul4A-DDB1-STRAP ubiquitin ligase in the regulation of the cellular levels of PNKP, and consequently in the repair of oxidative DNA damage.     

DNA damage induces nuclear translocation of parkin

Parkinson's disease (PD) is the second most common form of human degenerative disorder. Mutation of parkin is one of the most prevalent causes of autosomal recessive PD. Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here I show that parkin translocates into nucleus upon DNA damage. Nuclear translocation of parkin appears to be required to promote DNA repair. These findings suggest that DNA damage induces nuclear translocation of parkin leading to the PCNA interaction and possibly other nuclear proteins involved in DNA repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in parkinsonism.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

The hMsh2-hMsh6 complex acts in concert with monoubiquitinated PCNA and Pol η in response to oxidative DNA damage in human cells

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an?S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the?presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.     

Regulation of Elg1 activity by phosphorylation

ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1''s activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of Δrad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage.     

New Insights into Replication Clamp Unloading

The sliding clamp protein proliferating cell nuclear antigen (PCNA) is situated at the core of the eukaryotic replisome, where it acts as an interaction scaffold for numerous replication and repair factors and coordinates DNA transactions ranging from Okazaki fragment maturation to chromatin assembly and mismatch repair. PCNA is loaded onto DNA by a dedicated complex, the replication factor C, whose mechanism has been studied in detail. Until recently, however, it was unclear how PCNA is removed from DNA upon completion of DNA synthesis. Two complementary studies now present data strongly implicating the replication factor C-like complex, Elg1/ATAD5-RLC, in the unloading of PCNA during replication in yeast and human cells. They indicate that an appropriate control over PCNA's residence on the chromatin is important for maintaining genome stability. At the same time, they suggest that the interaction of Elg1/ATAD5 with SUMO, which was also reported to contribute to its role in genome maintenance, affects aspects of its function distinct from its unloading activity.     

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.     

Tyrosine phosphorylation controls PCNA function through protein stability

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.