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Making sense of ubiquitin ligases that regulate p53

Making sense of ubiquitin ligases that regulate p53 review review Cancer Biology & Therapy 10:7, 665-672; October 1, 2010; © 2010 Landes Bioscience Making sense of ubiquitin ligases that regulate p53 Abhinav K. Jain and Michelle Craig Barton* Department of Biochemistry and Molecular Biology; Center for Stem Cell and Development Biology; Graduate Program in Genes and Development; UT-Houston Graduate School of Biomedical Sciences; UT MD Anderson Cancer Center; Houston, TX USA Key words: tumor suppressor, proteasome, protein degradation, tissue-specificity, developmental expression, cancer, E3-ligase threatens. Single-cell analyses reveal that p53 is post-translation- The functions of p53 most highly associated with the well- ally regulated not only in response to stress that threatens genomic studied tumor suppressor are its abilities to induce cell cycle stability, but also during normal DNA replication. Although the arrest and apoptosis in response to cellular stresses. r ecent majority of control is exercised at a post-translational level, p53 progress underscores that p53 is a multi-functional protein mRNA levels are also controlled at post-transcriptional levels by with activities that range beyond tumor suppression to normal micro-RNAs (miRNAs), e.g., miRNA-504, which targets p53 homeostasis, metabolism, fertility and differentiation. A message for degradation. unifying theme of these studies is that p53 is first and foremost a transcription factor; and control of p53 protein stability The major outcome of p53 regulation is alteration of its nuclear determines its ability to carry out this task. There are an concentration and, thus, its ability to interact with chromatin 6-9 expanding number of e3-ubiquitin ligase proteins that target and function as a transcription factor (Fig. 1). This is primarily p53 for ubiquitin tagging and protein degradation. This review achieved by a highly regulated balancing act between p53 protein discusses these many effectors of p53 protein degradation, degradation and protein synthesis. At the fulcrum of this balance and our task is to provide some level of understanding as to is a collection of proteins that ligate ubiquitin to lysine (K) resi- their differences and their similarities. Further, we propose dues, as a PTM of p53 and regulate protein stability. Ubiquitin how some degree of specialization may be assigned to the e3- is added at a specific amino acid residue as a growing chain or ligases, in their navigation toward a common goal of regulating monomeric unit, which flags p53 for elimination by the protea - p53 protein levels, and emphasize that better understanding 10,11 some complex or modifies activity of p53, respectively. The of the mechanisms involved in e3-ligase functions is needed to E3-ubiquitin ligase determines target protein selection and inter- further their potential as therapeutic targets. action, bringing a complex of E1/E2/ubiquitin together in asso- ciation with the target protein to complete transfer and linkage of a ubiquitin moiety. Ubiquitin polymerization versus monomer The tumor suppressor p53 receives considerable attention within addition is determined by the lysine amino acid (K48 versus K63) 13,14 and without the cell, as a key player in tumor prevention and the of ubiquitin that is covalently linked to p53. Conjugation of 1,2 most frequently mutated gene in human cancers. A majority of a single ubiquitin molecule to one or more lysines of a target human tumors harbor mutations in the TP53 gene and, in other protein is involved in a variety of cellular processes, including 1,2 15 cases, p53 is inactivated by other mechanisms. The functions protein trafficking, DNA repair and transcriptional regulation. of p53 in preventing propagation of DNA damage and genome Recent, comprehensive reviews that discuss the collection of pro- instability are of major import and multiple signaling pathways teins, which regulate p53 protein levels by ubiquitination and are 11,16 converge on p53 to control its protein stability and activities. On themselves regulated by p53, are available. Here, we update and off signals in the form of post-translational modifications this growing number of E3-ligases and further discuss a potential (PTMs), when enzymes covalently link chemical groups to the biological rationale for the large group of proteins intent on nega- amino acid structure of p53, are read into actions that dictate sta- tive regulation of p53 via ubiquitin addition. bility, protein partner and chromatin interactions, as well as sub- cellular localization, of p53. Recent studies offer an expanding The Principle Player: Mdm2 list of p53 regulatory functions that additionally impact devel- opment, differentiation and metabolism. The power of p53, to The paradigm of p53 modification by the addition of ubiquitin shut down the cell cycle or induce cellular death, is held at bay and subsequent regulation of protein stability seemed straight- during normal cellular homeostasis, but is activated when trouble forward with the discovery of Mdm2 and its RING-domain- 14,17-19 dependent ubiquitination of p53. In fact, deletion of Mdm2 from the mouse genome illustrates the consequences of unleash- *Correspondence to: Michelle Craig Barton; Email: [email protected] ing p53 from regulatory control by Mdm2. Knockout of Mdm2 Submitted: 08/13/10; Accepted: 08/29/10 Previously published online: is early embryonic lethal, a phenotype that is rescued by loss 20,21 www.landesbioscience.com/journals/cbt/article/13445 of Trp53 expression. Mdm2 has its homologue in humans, DOI: 10.4161/cbt.10.7.13445 where the protein is referred to as HDM2 while the gene remains www.landesbioscience.com Cancer Biology & Therapy 665 Figure 1. Complex control of p53 protein levels. Mdm2 as an example: An intricate regulatory mechanism controls ubiquitination of p53 and dictates its activity. MDM2 in partnership with MDMX poly-ubiquitinates p53, which is targeted for proteasomal degradation. MDM2 also directs self- destruction and MDMX degradation, mediated by its ri NG-domain. Mono-ubiquitination of p53 by MDM2 leads to its nuclear export. Direct binding with MDMX may also render p53 transcriptionally inactive. p53 upregulates transcriptional activation of MDM2 to complete a negative feedback loop. MDM2, but no Mdm2 homologue exists in invertebrates even contain single point mutations within the DBD site of Mdm2 where p53 is conserved. Efforts of a number of labs define an interaction, have increased levels of Mdm2-dependent ubiquit- autoregulatory circuit between the E3-ligase Mdm2 and p53; ination but are not degraded. Mdm2 has a highly similar part- where p53 activates expression of MDM2, Mdm2/HDM2 in ner called MdmX or Mdm4, which is a RING-domain protein turn associates with p53, p53 is ubiquitinated and degraded by defective in ubiquitin ligase activity. MdmX has considerable the proteasome complex and MDM2 expression declines; the inu fl ence on Mdm2 and p53: MdmX inhibits p53 transcrip- loop continues to maintain both Mdm2/HDM2 and p53 at low tional functions by direct binding and interacts with Mdm2 via 23 31,32 levels in normal cells (Fig. 1). Further studies revealed how their respective RING domains (Fig. 1). Multiple upstream Mdm2-directed stability of p53 is regulated in response to stress modifying enzymes regulate both p53-Mdm2 interactions and signaling, which temporarily breaks the Mdm2-p53 circuitry and protein levels during the activation of p53. During this process, 24 24,34 allows p53 protein levels to increase. Mdm2 levels are them- Mdm2 may ubiquitinate itself and its partner, MdmX. selves highly regulated and finely tuned; aberrant Mdm2 may Further investigations show that Mdm2-mediated regulation either contribute to oncogenesis or tumor suppression, as recently of p53 is more complex than initially thought, as Mdm2 also reviewed in ref. 25. inu fl ences subcellular localization and transcriptional potential Repression of p53 activity by Mdm2 may be achieved by two of p53 (Fig. 1). By conjugating a single ubiquitin onto one or major mechanisms: (1) by promoting p53 degradation and (2) more lysines within the carboyx (C)-terminus and DBD of p53, 35,36 by disrupting the ability of p53 to function as a transcription Mdm2 enhances the nuclear export of p53. How Mdm2 17,19,26 factor. Mdm2 binds p53 at its amino (N)-terminus to block activity is regulated to effect mono- versus poly-ubiquitination the transactivation domain of p53; additionally a second bind- of p53 is not well understood. Several reports suggest that other 37 38 39 40 ing site for Mdm2 within the DNA-binding domain (DBD) of proteins, including p300, YY1, Gankyrin, KAP1 and 28,29 41 p53 is known. Interestingly, some mutant forms of p53, which Siva1, may modulate Mdm2-mediated poly-ubiquitination of 666 Cancer Biology & Therapy volume 10 issue 7 Table 1. Ubiquitin e3-ligases that regulate p53 activity p53- Ligase Type E2 Ub p53-Lysine residue p53 status Mode responsive K370, K372, K373, Mdm2 r iNG Yes UbcH5b Mono/Poly Degradation Direct K381, K382, K386 Pirh2 r iNG Yes UbcH5b Poly - Degradation Direct Cop1 r iNG Yes UbcH5b Poly - Degradation Direct Tr iM24 r iNG - UbcH8 Poly - Degradation Direct Ar F-BP1 HeCT No UbcH5c Poly - Degradation Direct CAr P1/2 r iNG - - Poly - Degradation Direct TOPOr S r iNG - UbcH5a/c, UbcH6 Poly - Degradation Direct Synoviolin r iNG - UbcH5c Poly - Degradation Direct CHiP - - UbcH5b Poly - Degradation Direct Skp1-Cul1- JFK r iNG - - Poly - Degradation r bx1-F-box MKr N1 r iNG No - - K291, K292 Degradation Direct e4orf6 and elonginB-Cul5- r iNG - UbcH5a Poly - Degradation e1B55K r bx1 Translocation to iCP0 r iNG - UbcH5a, UbcH6 Poly - Direct Nuclear Foci MSL2 r iNG - - Poly K351, K357 Nuclear export Direct Transcriptional CUL7 - - - Mono/Di - Direct inactivation w w P1 HeCT Yes UbcH5c Mono/Poly - Nuclear export Direct Prevent Ubc13 - r epressed Ubc13 Poly - Direct Tetramerization Translocation to e4F1 - - - Poly K320 Direct Chromatin elonginB/c- BZLF1 - - UbcH5a/c Poly - Degradation Cul2/5-SOCS p53, while others support a model where the levels of Mdm2 endogenous p53 mouse model that allows rapid and reversible determine the type of ubiquitin conjugated to p53. Mdm2 also toggling of endogenous p53 between nucleus and cytoplasm, inhibits p53 by promoting its conjugation to the ubiquitin-like Evan and colleagues showed that, although p53 is simultaneously molecule NEDD8. Modifications of C-terminal lysines of p53 active in all tested tissue of Mdm2-deficient mice, its levels fall by ubiquitination, neddylation, sumoylation or acetylation affect rapidly after functional restoration post DNA-damage, suggest- interactions between p53 and Mdm2 to compete for Mdm2- ing that a negative feedback loop remains operable even in absence 43,44 mediated ubiquitination. of Mdm2. Other reports of in vivo, mouse knock-in models that express mutant forms of p53 (p53-6KR or p53-7KR), lacking the Nothing is Simple about Regulation of p53 majority of ubiquitination sites for Mdm2, show that mutant p53 47,48 has a normal half-life and is stabilized and activated by stress. With further studies of p53 regulation, the model of Mdm2 as Thus, alternative pathways for p53-degradation likely exist inde- sole arbiter of p53 protein levels began to erode. Genetic knock-in pendent of Mdm2. methodology was used to create mice that express E3-ligase defi - Benchimol and colleagues were the first to show that Mdm2 cient Mdm2 (a C462A mutant), which retains the ability to bind is not alone as an E3-ligase capable of controlling p53 protein to p53 and theoretically inhibit p53 transcriptional activity with- levels by ubitiquin-tagging p53 for degradation. The identifi - out ubiquitination. Surprisingly, these mice exhibit p53-depen- cation of Pirh2, another RING-domain E3-ligase that mediates dent embryonic lethality, similar to Mdm2-null mice, and also degradation of p53 by direct interaction, was soon followed by retain ubiquitination and degradation of Mdm2. These results discoveries of several other proteins that regulate p53 at the level suggest that protein-protein interactions between p53 and Mdm2 of protein stability. Table 1 presents a summary of numerous are insufficient to block p53-mediated apoptosis during embry - E3-ligases that target p53 and underscores the major premise of onic development and, more importantly, Mdm2-independent this review: Why are there so many E3-ligases that negatively reg- pathways must exist that regulate protein stability of both p53 ulate p53? We will address this question in a teleological manner and Mdm2 by ubiquitin modification. Using a switchable, by assuming there is specificity among these negative regulators www.landesbioscience.com Cancer Biology & Therapy 667 of p53. We offer the multi-component What, When and Where Protein-1 (MKRN1) displays expression in developing neural tis- Hypothesis of specificity: (1) E3-ligase proteins are expressed in sues, including the forebrain and the ear at 10.5 dpc. ARF-BP1 distinct tissues or cell types at specific times of development. A appears to be ubiquitously expressed but available data are com- corollary to this is that tissue- or stage-specific expression of the piled at a later stage of 14.5 dpc. At this same stage, Synoviolin1 required E2 or co-regulators dictates function of an E3-ligase. is primarily expressed in selected bone and cartilaginous sites of (2) Interactions between p53 and an E3-ligase are determined by the embryo. These examples of embryonic expression support readout of a p53 “PTM code”, e.g., situational signaling to p53 the hypothesis that the many E3-ligases of p53 display distinct leads to modification of specific residues, which likely determine expression patterns during embryonic development. protein-protein interactions, including those with E3-ligases. (3) Further analyses are needed to encompass adult tissues and An E3-ligase mediates ubiquitination of a defined residue of p53 expression levels, in order to address a major question of the that determines a specific outcome or timing of delayed versus roles that E3-ligases may play in specific tumor development immediate degradation. (4) An E3-ligase itself acquires specific and potential treatments based on these functions. An intrigu- PTMs under a specific condition that determines its interaction ing aspect of p53 regulation, which is less studied than most, with p53 and ultimately the status of p53 activity. (5) Targeting is that the p53 gene encodes other expressed p53 protein iso- p53 for ubiquitination by an E3-ligase is specific to the isoform of forms, which have been associated with specific cancers. These p53 under different situations. isoforms are expressed as a result of secondary promoter use and alternative splicing, which create a form of p53 truncated at the Embryonic Expression of E3-Ligases that Target p53 N-terminus. As the N-terminus is the site of interaction with Mdm2, additional E3-ligases might provide a “failsafe” mecha- None of the regulators, listed in Table 1, has been scrutinized nism to keep these truncated p53 proteins in check and regulate at the level of Mdm2, especially in development of mouse mod- their protein stability. Additionally, work with mouse models els of function. Lack of Mdm2 causes embryonic lethality and, suggests that specificity in regulation of full-length p53 occurs in the category of embryonic regulation of p53, Mdm2 is the at a tissue-specific level. When normal mice are exposed to dam - 20,21 principle player. Its functional partner, MdmX (Mdm4), age by 5 Gy of IR, p53 accumulates in the nuclei of cells and which lacks the enzymatic capacity to ubiquitinate p53, also induces apoptosis in spleen, thymus, bone marrow, intestine and induces embryonic lethality when deleted but at a later stage of ependyma. p53 levels also increase in kidney, osteocytes, myocar- 50 54 development. In consideration of the other E3-ligases of p53, dium and salivary glands but no cell death occurs. In liver, skel- important questions are whether these other E3-ligases of p53 etal muscle and brain tissue, there is no response to IR at the level are expressed during mouse embryonic development, as well as of p53 stability or apoptosis. In an intriguing study, Mdm2 was when and where expression occurs. To address these questions, shown to regulate mutant p53 protein stability as well as normal databases such as EMAGE (www.emouseatlas.org/emage/home. p53 protein levels in a mouse model, but this regulatory control php) offer collected information regarding expression of specific was lacking in liver tissue. genes during mouse embryonic development. Comparison of whole-mount in situ expression analysis allows some assessment Partners of E3-Ligase Function of specific E3-ligases that target p53. Here, we present interpreta - tion of more robust, available data and not all members listed in Tissue-specificity may lie at the level of E3-ligase expression Table 1 are analyzed. or whether the specific E2 needed for function is expressed The primary literature reveals that Mdm2 expression during (see Table 1). Although UbcH5 is a common E2-partner of many mouse embryogenesis is detectable, by in situ analysis, in neural E3-ligases, there are a notable few that are more active or exclu- folds and in migratory neural crest cells, 7.5–9 dpc. After these sively active with other E2 proteins. Even UbcH5 has its a, b and stages of critical neural crest development, Mdm2 is expressed c subtypes and their expression and interactions with E3-ligases more ubiquitously in the embryo. Interestingly, embryonic offer another level of regulation for specificity. Additionally one lethality in Mdm2-null mouse models occurs during peri-im- E2 in particular, Ubc13, can directly ubiquitinate p53 without plantation; thus, curbing p53 from massive cell death occurs intervention of a partner E3. much earlier than stages of detectable Mdm2 expression. This E3-ligase functions are inu fl enced by proteins that interact underscores the possibility that E3-ligases of p53 may acquire with or regulate the E3-ligase, in addition to an E2 protein. specific functions at different stages of development and/or in MdmX is a well-known example of a regulatory partner that 30,31 specific cells. alters Mdm2 activity and even its stability. Whether MdmX In contrast to detectable Mdm2 expression, Trim24 at 10.5 activity is more or less inu fl ential in specific cell types or tissues is dpc is visibly expressed at the tip of the hindlimb bud and in the unknown and other previously noted modifiers of Mdm2 func - 37 38 39 40 41 facial primordia, primarily the nasal eminence, with more mes- tions, p300, Y Y1, Gankyrin, K AP1 and Siva1, offer other enchymal appearance. Cop1, at the same stage of development, is regulatory nodes. As an example of potential tissue-specificity also expressed in the facial primordia but with additional expres- and its effects, deletion of Trim24 in mice led to tumor develop- sion in the forelimb bud and ear. CARP1 at 10.5 dpc is expressed ment in the liver. These tumors are responsive to retinoic acid in the outflow tract of the developing heart, the distal fore - treatment, suggesting that Trim24 primarily functions as a co- limb bud, as well as the facial primordia. Makorin Ring Finger regulator of the retinoic acid receptor rather than an E3-ligase of 668 Cancer Biology & Therapy volume 10 issue 7 7,10,11,14,63-66,68,69 p53 in the liver. Whether E3-ligases interact with p53 in a direct in some depth. Table 2 summarizes and updates or indirect manner (Table 1), protein partners and upstream some of the significant modifications of p53 and the enzymes regulation likely inu fl ence their activities, stability or expression. that create or abolish them. Though these modifications may be crucial for p53 functions in cultured cells, in vivo models sug- Evolution of E3-Ligases that Target p53 gest that PTMs play subtle, modulatory roles in regulating p53 66,70 functions. Therefore, the key regulatory switch in turning on Evolutionary conservation may offer some sense of the most and off p53 response may be direct or indirect regulation of p53 basic of E3-ligase functions and whether a single ancestral stability through ubiquitin modification. E3 was duplicated or multiple E3-ligases arose separately over One possibility is that among the collection of E3-ligases are time. A review by Abrams and colleagues suggests that control specific readers of p53 PTMs, which evolved in order to respond of p53 protein stability may be restricted to vertebrate develop- to particular inductive signals that write a pattern of PTMs. The ment although orthologs and structural conservation of p53 exist type of structural domains known to interact with modified across invertebrate phyla. Mdm2 and MdmX are not conserved lysine residues, such as Bromo with acetylated lysines as well as in flies and worms, but recent work from our laboratory sug - PHD and chromodomain with specific patterns of methylated gest that other regulators of p53, which serve as E3-ligases in lysines, are present in a subset of E3-ligases that target p53. For mammalian cells, are active in invertebrates. We used mosaic example, TRIM24 possesses a tandem PHD-Bromo domain deletion analysis in Drosophila to assess the outcome of bonus unit, but the ability of TRIM24 to recognize p53 that is acety- loss of function. Bonus is the single Drosophila representative lated and/or methylated is untested. Cop1 has a WD40-domain, of the TIF1 sub-family of TRIM (RING/B-box/Coiled-coil or which can mediate protein-protein interactions, not notably Tripartite Motif ) proteins and closest in homology to TRIM24 dependent on PTMs. ARF-BP1 has a WWE domain that acts among the TRIM24/28/33 members. Loss of bonus induces similarly but preferentially in the ubiquitin pathway. Other spe- apoptosis and cleavage of Caspase 3. Cell death is apparently due cific proteins that target cytoplasmic p53 for ubiquitin-mediated to unrestrained D-p53 activity, as RNAi-mediated depletion of degradation, proteins p300 and CREB-binding protein (CBP), D-p53 rescues the phenotype. Drosophila bonus may be an ances- have Bromo domains as well as poly-ubiquitin ligase (E4) activi- tral negative regulator of p53; molecular studies are needed to ties that tag p53 for proteolysis. These proteins are histone ace- show that ubiquitination and E3-ligase activity are the basis of tyl transferases that mediate acetylation of target proteins, e.g., this regulation. Evolutionary comparisons between all E3-ligase p53 and histones H3 and H4, in addition to recognizing the regulators of p53 and conserved proteins are needed to reveal acetylated motif. Acetylation of p53 occurs during activation of 44,72 comparable pathways and conserved functions. p53, in response to a variety of stresses; thus, these E4 pro- teins may also be involved in the termination of a stress-induced Upstream Regulation and Downstream Response p53-response. Protein-protein interactions between E3-ligases and p53 do The most striking aspect of p53-signaling, in response to stress not necessarily rely on independent, structural domain recogni- or inductive signaling, is the tightly regulated process of post- tion, but occur as a result of allosteric recognition of surfaces or transcriptional modification of p53, which guide p53 activi - interfaces created by p53 protein folding or charge. Interestingly, ties primarily by dictating p53-interactions with proteins that viral protein BZLF1 directly functions as an adaptor component control: (1) the levels of p53, (2) the ability of p53 to bind to of the ECS (ElonginB/C-Cul2/5-SOCS-box protein) ubiquitin DNA, (3) the subcellular localization of p53, (4) interaction with ligase complex and targets phosphorylated p53, induced by viral regulatory proteins, and thus determine the physiological out- lytic replication. Signaling to E3-ligases, as well as p53, alters come of p53 activation. A comparison can be made to chroma- interactions between them, e.g., phosphorylation of S166, S188, tin, where PTMs of specific histone residues are interpreted by S395, Y276 or Y394 of Mdm2 disrupts the p53-Mdm2 associa- histone “reader” proteins via specific, specialized domains, while tion. An E3-ligase may be regulated by signaling, as a result of other specialized enzymes “write” the PTM code. A few readers stress or other regulatory pathways, which induce PTMs or the and writers identified for histones are known to interact and/or association of protein partners that modify the feedback loops of 59-62 23 modify p53 as well. regulation that exist between p53 and its E3-ligase partners. Covalent modifications of p53 occur on more than 40 dif - ferent amino acid residues and likely provide fine-tuning and E3-Ligases Targeted to Specific Outcomes specificity of p53 activation. These PTMs range from phos - phorylation, acetylation, methylation, ubiquitination, sumoyla- Mouse knock-in models have been created that express mutant tion, neddylation, glycosylation, ribosylation and more recently forms of p53 (p53-6KR or p53-7KR), where C-terminal lysines 10,63-67 O-GlcNAcylation. Depending on the type of stress, specific are mutated and cannot be ubiquitinated, acetylated or methyl- signaling events are activated that target precise residues on p53 ated. These mice are viable and their mutant p53 has a normal half- 47,48 protein resulting in a stress-to-enzyme-to-residue specific modifi - life and response to stress. Elucidation of alternative pathways, 68 49 cation on p53 protein. Discussing all these modifications and the which act in addition to Mdm2, began with Pirh2, and offered enzymes responsible (writers) in detail is beyond the scope of this some idea of whether specific E3-ligases are involved in response review, however several comprehensive reviews cover this topic to certain stimuli or to different aspects of p53 regulation. The www.landesbioscience.com Cancer Biology & Therapy 669 Table 2. p53 post-translational modifications and modifying enzymes Residue of p53 Modification Modifiers De-modifiers S6 P JNK2, CKiδ/ε - S9 P CK1δ/ε - S15 P ATM, ATr , DNA-PK, AMPK, mTOr , r SK2, CDK5 PP1, PPM1D T18 P TTK, CHK2, vr K1 - S20 P CHK2, PiK3 - S33 P CDK9, CDK5, CAK/CDK7, GSK3β, P38K - S37 P ATr , Pr AK PP1, PP2A S46 P CDK5, AMPKα, HiPK2, P38K, PKCδ, DYr K2 PP2A T55 P er K2, TAF1 PP2A, B56Y T81 P JNK2 - K120 Ac TiP60 - T155 P CSN - K164 Ac P300 (KAT3B) - S215 P STKi5 - e255, e258, e259 r PAr P-1 (poly ADP-ribosylase) - K305 Ac P300 (KAT3B) - S313, S314 P CHK1, CHK2 - S315 P STKi5, CDK9, CDK2 CDC14A L320 Ac PCAF (KAT2B) HDAC1 S366 P CHK2, iκBK2 - 1 2 K370 Me , Me SMYD2 (KMT3C) LSD1 K372 Me SeT7/9 (KMT3) - K373 Ac P300 (KAT3B) HDAC1 S376 P PKC, GSK3β - T377 P CHK1, CHK2 - S378 P PKC, CHK1, CHK2 - K382 Ac P300 (KAT3B) HDAC1, Sir T1 K382 Me SeT8 (KMT5A) - K386 S SUMO-1 - T387 P CHK1 - S392 P CDK9, PKr , FACT (CK2) PP1 K320, K321, K370, K372, K373 N8 FBXO11, MDM2 - K120, K132, K139, K164 Ub - - K291, K292 Ub MKr N1 - K320 Ub e4F1 - K351, K357 Ub MSL2 - K101, K370, K372, K373, K381, Ub MDM2 - K382, K386 1 2 P, Phosphorylation; Ac, Acetylation; Me , Mono-methylation; Me , Di-methylation; r , r ibosylation; S, Sumoylation; N8, Neddylation; Ub, Ubiquitination. primary function of Pirh2 may be terminating the p53 response loops. Mdm2, Pirh2, COP1 and additional E3-ligases, including 58 76 77 78 79 56 to stimuli, because Pirh2 interacts preferentially with formed TRIM24, TOPORS, ARF-BP1, W WP1, ICP0, Ubc13, 80 81 82 83 84 p53-tetramers, an association stimulated during stress activation CUL7, CARP1 and CARP2, Synoviolin, CHIP, E4F1, 74 85 86 of p53. COP1, another E3-ligase with a RING domain, nega- E4orf6 and E1B55K, MSL2, the human kelch domain tively regulates p53 levels by ubiquitination but its targeted resi- containing F-box protein, JFK, and Makorin Ring Finger 75 88 dues are unknown. Similar to Mdm2, the genes encoding Pirh2 Protein-1, MKRN1, mediate K48-linked polyubiquitination of and COP1 are regulated by p53 and, like other E3-ligases listed p53 and degradation. However, mono- or K63-linked ubiquitina- in Table 1, are thought to participate in autoregulatory feedback tion is linked to other functions, e.g., nuclear export and cytosolic 670 Cancer Biology & Therapy volume 10 issue 7 4 localization of p53, as well as alteration of p53 transcriptional and decay, even in normal, unstressed cells, offers further frontiers activities. Limited conjugation of ubiquitin by Mdm2, WWP1 to determinations of relevant PTMs of p53, the signaling pathways and MSL2 induces nuclear export of p53; ICP0 promotes accu- involved and whether any known E3-ligases, or those yet to be mulation of ubiquitinated p53 at nuclear foci; E4F1, an atypical discovered, are involved in this regulatory program. Together these E3-ligase, forms K48-linked mono-, di- or tri-ubiquitinated p53 p53 regulators may alter p53 functions, terminate p53-signaling, that activates cell cycle arrest at G /G ; whereas, CUL7 gener- and/or maintain p53 at low levels bearable by normal, unstressed 0 1 ates mono- or di-ubiquitinated p53 to repress p53 transcrip- cells. Targeting E3-ligases to inhibit ubiquitin-mediated degra- 78-80,84,86 tional activities by unknown mechanisms. Ubc-13 is an dation and restore p53 functions holds considerable therapeutic E2-enzyme, capable of independently ubiquitinating p53, prevent promise. The multiple layers of negative and positive regulation p53 tetramerization. Thus, p53-ubiquitination can come in sev- involved in controlling p53 offer challenges to understanding these eral flavors and the specic E fi 3-ligase involved in modification of critical pathways that regulate p53 stability. p53 can greatly alter the outcome. Acknowledgements So Many E3-ligases that Target p53 We apologize to colleagues for our failure to cite their work due to space limitations. We thank Y. Furuta for his assistance in inter- In this discussion, we offer multiple ways to parse the E3-ligases of pretation of expression analyses. Research in the Barton labora- p53 and suggest specialization among them in their roles as con- tory is supported by the Mitchell Foundation, CellCentric, Ltd., trollers of p53. Further development of animal models, analysis in and grants from the National Institutes of Health (GM081627 multiple model organisms and testing of numerous hypotheses are and DK070824). A.K.J. is an Odyssey Fellow supported by the needed to sort out the roles of the many E3-ligases active in regula- Odyssey Program and The Laura and John Arnold Foundation at tion of p53. Recent evidence that p53 exhibits cycles of induction The University of Texas MD Anderson Cancer Center. 32. Singh RK, Iyappan S, Scheffner M. Hetero- 19. Kubbutat MH, Jones SN, Vousden KH. Regulation of References p53 stability by Mdm2. Nature 1997; 387:299-303. oligomerization with MdmX rescues the ubiquitin/ 1. Soussi T, Beroud C. Assessing TP53 status in human Nedd8 ligase activity of RING finger mutants of 20. Jones SN, Roe AE, Donehower LA, Bradley A. Rescue tumours to evaluate clinical outcome. Nat Rev Cancer Mdm2. J Biol Chem 2007; 282:10901-7. of embryonic lethality in Mdm2-deficient mice by 2001; 1:233-40. 33. Meek DW, Knippschild U. Posttranslational modifica- absence of p53. Nature 1995; 378:206-8. 2. Hainaut P, Hollstein M. p53 and human cancer: The tion of MDM2. Mol Cancer Res 2003; 1:1017-26. 21. Montes de Oca Luna R, Wagner DS, Lozano G. Rescue first ten thousand mutations. Adv Cancer Res 2000; 34. Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai of early embryonic lethality in mdm2-deficient mice by 77:81-137. deletion of p53. Nature 1995; 378:203-6. KK, Yuan ZM. DNA damage-induced MDMX deg- 3. Vousden KH, Prives C. Blinded by the Light: The radation is mediated by MDM2. J Biol Chem 2003; 22. Lu WJ, Abrams JM. Lessons from p53 in non-mamma- Growing Complexity of p53. Cell 2009; 137:413-31. 278:45946-53. lian models. Cell Death Differ 2006; 13:909-12. 4. Loewer A, Batchelor E, Gaglia G, Lahav G. Basal 35. Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. 23. Wu X, Bayle JH, Olson D, Levine AJ. The p53- dynamics of p53 reveal transcriptionally attenuated Mono- versus poly-ubiquitination: differential control mdm-2 autoregulatory feedback loop. Genes Dev pulses in cycling cells. Cell 142:89-100. of p53 fate by Mdm2. Science 2003; 302:1972-5. 1993; 7:1126-32. 5. Hu W, Chan CS, Wu R, Zhang C, Sun Y, Song JS, et 36. Nie L, Sasaki M, Maki CG. Regulation of p53 nuclear 24. Stommel JM, Wahl GM. Accelerated MDM2 auto-deg- al. Negative Regulation of Tumor Suppressor p53 by export through sequential changes in conformation and radation induced by DNA-damage kinases is required MicroRNA miR-504. Mol Cell 38:689-99. ubiquitination. J Biol Chem 2007; 282:14616-25. for p53 activation. EMBO J 2004; 23:1547-56. 6. Vogelstein B, Lane D, Levine AJ. Surfing the p53 net- 37. Grossman SR, Deato ME, Brignone C, Chan HM, 25. Manfredi JJ. The Mdm2-p53 relationship evolves: work. Nature 2000; 408:307-10. Kung AL, Tagami H, et al. Polyubiquitination of p53 Mdm2 swings both ways as an oncogene and a tumor 7. Horn HF, Vousden KH. Coping with stress: multiple by a ubiquitin ligase activity of p300. Science 2003; suppressor. Genes Dev 24:1580-9. ways to activate p53. Oncogene 2007; 26:1306-16. 300:342-4. 26. Momand J, Zambetti GP, Olson DC, George D, 8. Riley T, Sontag E, Chen P, Levine A. Transcriptional 38. Sui G, Affar el B, Shi Y, Brignone C, Wall NR, Yin P, et Levine AJ. The mdm-2 oncogene product forms a com- control of human p53-regulated genes. Nat Rev Mol al. Yin Yang 1 is a negative regulator of p53. Cell 2004; plex with the p53 protein and inhibits p53-mediated Cell Biol 2008; 9:402-12. 117:859-72. transactivation. Cell 1992; 69:1237-45. 9. Laptenko O, Prives C. Transcriptional regulation by 39. Higashitsuji H, Itoh K, Sakurai T, Nagao T, Sumitomo 27. Chen J, Marechal V, Levine AJ. Mapping of the p53 p53: one protein, many possibilities. Cell Death Differ Y, Masuda T, et al. The oncoprotein gankyrin binds to and mdm-2 interaction domains. Mol Cell Biol 1993; 2006; 13:951-61. MDM2/HDM2, enhancing ubiquitylation and degra- 13:4107-14. dation of p53. Cancer Cell 2005; 8:75-87. 10. Carter S, Vousden KH. Modifications of p53: competing 28. Yu GW, Rudiger S, Veprintsev D, Freund S, Fernandez- for the lysines. Curr Opin Genet Dev 2009; 19:18-24. 40. Wang C, Ivanov A, Chen L, Fredericks WJ, Seto E, Fernandez MR, Fersht AR. The central region of Rauscher FJ, 3rd, Chen J. MDM2 interaction with 11. Kruse JP, Gu W. Modes of p53 regulation. Cell 2009; HDM2 provides a second binding site for p53. Proc 137:609-22. nuclear corepressor KAP1 contributes to p53 inactiva- Natl Acad Sci USA 2006; 103:1227-32. tion. EMBO J 2005; 24:3279-90. 12. Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin 29. Shimizu H, Burch LR, Smith AJ, Dornan D, Wallace ligases. Annu Rev Biochem 2009; 78:399-434. 41. Du W, Jiang P, Li N, Mei Y, Wang X, Wen L, et al. M, Ball KL, Hupp TR. The conformationally flex- Suppression of p53 activity by Siva1. Cell Death Differ ible S9-S10 linker region in the core domain of p53 13. Chen ZJ, Sun LJ. Nonproteolytic functions of ubiq- 2009; 16:1493-504. uitin in cell signaling. Mol Cell 2009; 33:275-86. contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo. J Biol Chem 42. Xirodimas DP, Saville MK, Bourdon JC, Hay RT, 14. Brooks CL, Gu W. p53 ubiquitination: Mdm2 and 2002; 277:28446-58. Lane DP. Mdm2-mediated NEDD8 conjugation of beyond. Mol Cell 2006; 21:307-15. p53 inhibits its transcriptional activity. Cell 2004; 30. Marine JC, Francoz S, Maetens M, Wahl G, Toledo 15. Mukhopadhyay D, Riezman H. Proteasome- 118:83-97. F, Lozano G. Keeping p53 in check: essential and independent functions of ubiquitin in endocytosis and synergistic functions of Mdm2 and Mdm4. Cell Death 43. Carter S, Bischof O, Dejean A, Vousden KH. C-terminal signaling. Science 2007; 315:201-5. Differ 2006; 13:927-34. modifications regulate MDM2 dissociation and nuclear 16. Jain AK, Barton MC. Regulation of p53: TRIM24 export of p53. Nat Cell Biol 2007; 9:428-35. 31. Uldrijan S, Pannekoek WJ, Vousden KH. An essential enters the RING. Cell Cycle 2009; 8:3668-74. function of the extreme C-terminus of MDM2 can be 44. Tang Y, Zhao W, Chen Y, Zhao Y, Gu W. Acetylation 17. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes provided by MDMX. EMBO J 2007; 26:102-12. is indispensable for p53 activation. Cell 2008; the rapid degradation of p53. Nature 1997; 387:296-9. 133:612-26. 18. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 1997; 420:25-7. www.landesbioscience.com Cancer Biology & Therapy 671 45. Itahana K, Mao H, Jin A, Itahana Y, Clegg HV, 59. Liu L, Scolnick DM, Trievel RC, Zhang HB, 75. Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, Lindstrom MS, et al. Targeted inactivation of Mdm2 Marmorstein R, Halazonetis TD, Berger SL. p53 sites Dowd P, et al. The ubiquitin ligase COP1 is a critical RING finger E3 ubiquitin ligase activity in the mouse acetylated in vitro by PCAF and p300 are acetylated in negative regulator of p53. Nature 2004; 429:86-92. reveals mechanistic insights into p53 regulation. Cancer vivo in response to DNA damage. Mol Cell Biol 1999; 76. Rajendra R, Malegaonkar D, Pungaliya P, Marshall H, Cell 2007; 12:355-66. 19:1202-9. Rasheed Z, Brownell J, et al. Topors functions as an E3 46. Ringshausen I, O’Shea CC, Finch AJ, Swigart LB, Evan 60. Huang J, Perez-Burgos L, Placek BJ, Sengupta R, ubiquitin ligase with specific E2 enzymes and ubiquiti- GI. Mdm2 is critically and continuously required to Richter M, Dorsey JA, et al. Repression of p53 activ- nates p53. J Biol Chem 2004; 279:36440-4. suppress lethal p53 activity in vivo. Cancer Cell 2006; ity by Smyd2-mediated methylation. Nature 2006; 77. Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF- 10:501-14. 444:629-32. BP1/Mule is a critical mediator of the ARF tumor 47. Feng L, Lin T, Uranishi H, Gu W, Xu Y. Functional 61. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, suppressor. Cell 2005; 121:1071-83. analysis of the roles of posttranslational modifications Richter M, et al. p53 is regulated by the lysine demeth- 78. Laine A, Ronai Z. Regulation of p53 localization and at the p53 C terminus in regulating p53 stability and ylase LSD1. Nature 2007; 449:105-8. transcription by the HECT domain E3 ligase WWP1. activity. Mol Cell Biol 2005; 25:5389-95. 62. Huang J, Dorsey J, Chuikov S, Perez-Burgos L, Zhang Oncogene 2007; 26:1477-83. 48. Krummel KA, Lee CJ, Toledo F, Wahl GM. The X, Jenuwein T, et al. G9a and Glp methylate lysine 373 79. Boutell C, Everett RD. The herpes simplex virus type C-terminal lysines fine-tune p53 stress responses in a in the tumor suppressor p53. J Biol Chem 285:9636-41. 1 (HSV-1) regulatory protein ICP0 interacts with and mouse model but are not required for stability control 63. Murray-Zmijewski F, Slee EA, Lu X. A complex bar- Ubiquitinates p53. J Biol Chem 2003; 278:36596-602. or transactivation. Proc Natl Acad Sci USA 2005; code underlies the heterogeneous response of p53 to 80. Andrews P, He YJ, Xiong Y. Cytoplasmic localized 102:10188-93. stress. Nat Rev Mol Cell Biol 2008; 9:702-12. ubiquitin ligase cullin 7 binds to p53 and promotes cell 49. Leng RP, Lin Y, Ma W, Wu H, Lemmers B, Chung S, 64. Bode AM, Dong Z. Post-translational modification of growth by antagonizing p53 function. Oncogene 2006; et al. Pirh2, a p53-induced ubiquitin-protein ligase, p53 in tumorigenesis. Nat Rev Cancer 2004; 4:793-805. 25:4534-48. promotes p53 degradation. Cell 2003; 112:779-91. 65. Olsson A, Manzl C, Strasser A, Villunger A. How 81. Yang W, Rozan LM, McDonald ER, 3rd, Navaraj A, 50. Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke important are post-translational modifications in p53 Liu JJ, Matthew EM, et al. CARPs are ubiquitin ligases V, Jochemsen AG, Lozano G. Rescue of embryonic for selectivity in target-gene transcription and tumour that promote MDM2-independent p53 and phospho- lethality in Mdm4-null mice by loss of Trp53 suggests a suppression? Cell Death Differ 2007; 14:1561-75. p53ser20 degradation. J Biol Chem 2007; 282:3273-81. nonoverlapping pathway with MDM2 to regulate p53. 66. Toledo F, Wahl GM. Regulating the p53 pathway: in 82. Yamasaki S, Yagishita N, Sasaki T, Nakazawa M, Kato Nat Genet 2001; 29:92-5. vitro hypotheses, in vivo veritas. Nat Rev Cancer 2006; Y, Yamadera T, et al. Cytoplasmic destruction of p53 51. Daujat S, Neel H, Piette J. Preferential expression of 6:909-23. by the endoplasmic reticulum-resident ubiquitin ligase Mdm2 oncogene during the development of neural ‘Synoviolin’. EMBO J 2007; 26:113-22. 67. Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim crest and its derivatives in mouse early embryogenesis. YS, Cho JW. Modification of p53 with O-linked 83. Esser C, Scheffner M, Hohfeld J. The chaperone- Mech Dev 2001; 103:163-5. N-acetylglucosamine regulates p53 activity and stabil- associated ubiquitin ligase CHIP is able to target 52. Chavez-Reyes A, Parant JM, Amelse LL, de Oca Luna ity. Nat Cell Biol 2006; 8:1074-83. p53 for proteasomal degradation. J Biol Chem 2005; RM, Korsmeyer SJ, Lozano G. Switching mechanisms 280:27443-8. 68. Meek DW, Anderson CW. Posttranslational of cell death in mdm2- and mdm4-null mice by dele- Modification of p53: cooperative integrators of func- 84. Le Cam L, Linares LK, Paul C, Julien E, Lacroix M, tion of p53 downstream targets. Cancer Res 2003; tion. Cold Spring Harb Perspect Biol 2009; 1:000950. Hatchi E, et al. E4F1 is an atypical ubiquitin ligase 63:8664-9. 69. Harris SL, Levine AJ. The p53 pathway: positive and that modulates p53 effector functions independently of 53. Bourdon JC. p53 and its isoforms in cancer. Br J degradation. Cell 2006; 127:775-88. negative feedback loops. Oncogene 2005; 24:2899-908. Cancer 2007; 97:277-82. 70. Donehower LA, Lozano G. 20 years studying p53 func- 85. Querido E, Blanchette P, Yan Q, Kamura T, Morrison 54. Midgley CA, Owens B, Briscoe CV, Thomas DB, Lane M, Boivin D, et al. Degradation of p53 by adenovirus tions in genetically engineered mice. Nat Rev Cancer DP, Hall PA. Coupling between gamma irradiation, 2009; 9:831-41. E4orf6 and E1B55K proteins occurs via a novel mecha- p53 induction and the apoptotic response depends nism involving a Cullin-containing complex. Genes 71. Shi D, Pop MS, Kulikov R, Love IM, Kung AL, upon cell type in vivo. J Cell Sci 1995; 108:1843-8. Dev 2001; 15:3104-17. Grossman SR. CBP and p300 are cytoplasmic E4 55. Terzian T, Suh YA, Iwakuma T, Post SM, Neumann 86. Kruse JP, Gu W. MSL2 promotes Mdm2-independent polyubiquitin ligases for p53. Proc Natl Acad Sci USA M, Lang GA, et al. The inherent instability of mutant 2009; 106:16275-80. cytoplasmic localization of p53. J Biol Chem 2009; INK4a p53 is alleviated by Mdm2 or p16 loss. Genes Dev 284:3250-63. 72. Gu W, Roeder RG. Activation of p53 sequence-specific 2008; 22:1337-44. DNA binding by acetylation of the p53 C-terminal 87. Sun L, Shi L, Li W, Yu W, Liang J, Zhang H, et al. 56. Laine A, Topisirovic I, Zhai D, Reed JC, Borden KL, JFK, a Kelch domain-containing F-box protein, links domain. Cell 1997; 90:595-606. Ronai Z. Regulation of p53 localization and activity by the SCF complex to p53 regulation. Proc Natl Acad Sci 73. Sato Y, Kamura T, Shirata N, Murata T, Kudoh A, Ubc13. Mol Cell Biol 2006; 26:8901-13. USA 2009; 106:10195-200. Iwahori S, et al. Degradation of phosphorylated p53 57. Khetchoumian K, Teletin M, Tisserand J, Mark M, by viral protein-ECS E3 ligase complex. PLoS Pathog 88. Lee EW, Lee MS, Camus S, Ghim J, Yang MR, Oh Herquel B, Ignat M, et al. Loss of Trim24 (Tif1alpha) W, et al. Differential regulation of p53 and p21 by 2009; 5:1000530. gene function confers oncogenic activity to retinoic MKRN1 E3 ligase controls cell cycle arrest and apop- 74. Sheng Y, Laister RC, Lemak A, Wu B, Tai E, Duan S, acid receptor alpha. Nat Genet 2007; 39:1500-6. tosis. EMBO J 2009; 28:2100-13. et al. Molecular basis of Pirh2-mediated p53 ubiquity- 58. Allton K, Jain AK, Herz HM, Tsai WW, Jung SY, Qin lation. Nat Struct Mol Biol 2008; 15:1334-42. J, et al. Trim24 targets endogenous p53 for degrada- tion. Proc Natl Acad Sci USA 2009; 106:11612-6. 672 Cancer Biology & Therapy volume 10 issue 7 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Making sense of ubiquitin ligases that regulate p53

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review review Cancer Biology & Therapy 10:7, 665-672; October 1, 2010; © 2010 Landes Bioscience Making sense of ubiquitin ligases that regulate p53 Abhinav K. Jain and Michelle Craig Barton* Department of Biochemistry and Molecular Biology; Center for Stem Cell and Development Biology; Graduate Program in Genes and Development; UT-Houston Graduate School of Biomedical Sciences; UT MD Anderson Cancer Center; Houston, TX USA Key words: tumor suppressor, proteasome, protein degradation, tissue-specificity, developmental expression, cancer, E3-ligase threatens. Single-cell analyses reveal that p53 is post-translation- The functions of p53 most highly associated with the well- ally regulated not only in response to stress that threatens genomic studied tumor suppressor are its abilities to induce cell cycle stability, but also during normal DNA replication. Although the arrest and apoptosis in response to cellular stresses. r ecent majority of control is exercised at a post-translational level, p53 progress underscores that p53 is a multi-functional protein mRNA levels are also controlled at post-transcriptional levels by with activities that range beyond tumor suppression to normal micro-RNAs (miRNAs), e.g., miRNA-504, which targets p53 homeostasis, metabolism, fertility and differentiation. A message for degradation. unifying theme of these studies is that p53 is first and foremost a transcription factor; and control of p53 protein stability The major outcome of p53 regulation is alteration of its nuclear determines its ability to carry out this task. There are an concentration and, thus, its ability to interact with chromatin 6-9 expanding number of e3-ubiquitin ligase proteins that target and function as a transcription factor (Fig. 1). This is primarily p53 for ubiquitin tagging and protein degradation. This review achieved by a highly regulated balancing act between p53 protein discusses these many effectors of p53 protein degradation, degradation and protein synthesis. At the fulcrum of this balance and our task is to provide some level of understanding as to is a collection of proteins that ligate ubiquitin to lysine (K) resi- their differences and their similarities. Further, we propose dues, as a PTM of p53 and regulate protein stability. Ubiquitin how some degree of specialization may be assigned to the e3- is added at a specific amino acid residue as a growing chain or ligases, in their navigation toward a common goal of regulating monomeric unit, which flags p53 for elimination by the protea - p53 protein levels, and emphasize that better understanding 10,11 some complex or modifies activity of p53, respectively. The of the mechanisms involved in e3-ligase functions is needed to E3-ubiquitin ligase determines target protein selection and inter- further their potential as therapeutic targets. action, bringing a complex of E1/E2/ubiquitin together in asso- ciation with the target protein to complete transfer and linkage of a ubiquitin moiety. Ubiquitin polymerization versus monomer The tumor suppressor p53 receives considerable attention within addition is determined by the lysine amino acid (K48 versus K63) 13,14 and without the cell, as a key player in tumor prevention and the of ubiquitin that is covalently linked to p53. Conjugation of 1,2 most frequently mutated gene in human cancers. A majority of a single ubiquitin molecule to one or more lysines of a target human tumors harbor mutations in the TP53 gene and, in other protein is involved in a variety of cellular processes, including 1,2 15 cases, p53 is inactivated by other mechanisms. The functions protein trafficking, DNA repair and transcriptional regulation. of p53 in preventing propagation of DNA damage and genome Recent, comprehensive reviews that discuss the collection of pro- instability are of major import and multiple signaling pathways teins, which regulate p53 protein levels by ubiquitination and are 11,16 converge on p53 to control its protein stability and activities. On themselves regulated by p53, are available. Here, we update and off signals in the form of post-translational modifications this growing number of E3-ligases and further discuss a potential (PTMs), when enzymes covalently link chemical groups to the biological rationale for the large group of proteins intent on nega- amino acid structure of p53, are read into actions that dictate sta- tive regulation of p53 via ubiquitin addition. bility, protein partner and chromatin interactions, as well as sub- cellular localization, of p53. Recent studies offer an expanding The Principle Player: Mdm2 list of p53 regulatory functions that additionally impact devel- opment, differentiation and metabolism. The power of p53, to The paradigm of p53 modification by the addition of ubiquitin shut down the cell cycle or induce cellular death, is held at bay and subsequent regulation of protein stability seemed straight- during normal cellular homeostasis, but is activated when trouble forward with the discovery of Mdm2 and its RING-domain- 14,17-19 dependent ubiquitination of p53. In fact, deletion of Mdm2 from the mouse genome illustrates the consequences of unleash- *Correspondence to: Michelle Craig Barton; Email: [email protected] ing p53 from regulatory control by Mdm2. Knockout of Mdm2 Submitted: 08/13/10; Accepted: 08/29/10 Previously published online: is early embryonic lethal, a phenotype that is rescued by loss 20,21 www.landesbioscience.com/journals/cbt/article/13445 of Trp53 expression. Mdm2 has its homologue in humans, DOI: 10.4161/cbt.10.7.13445 where the protein is referred to as HDM2 while the gene remains www.landesbioscience.com Cancer Biology & Therapy 665 Figure 1. Complex control of p53 protein levels. Mdm2 as an example: An intricate regulatory mechanism controls ubiquitination of p53 and dictates its activity. MDM2 in partnership with MDMX poly-ubiquitinates p53, which is targeted for proteasomal degradation. MDM2 also directs self- destruction and MDMX degradation, mediated by its ri NG-domain. Mono-ubiquitination of p53 by MDM2 leads to its nuclear export. Direct binding with MDMX may also render p53 transcriptionally inactive. p53 upregulates transcriptional activation of MDM2 to complete a negative feedback loop. MDM2, but no Mdm2 homologue exists in invertebrates even contain single point mutations within the DBD site of Mdm2 where p53 is conserved. Efforts of a number of labs define an interaction, have increased levels of Mdm2-dependent ubiquit- autoregulatory circuit between the E3-ligase Mdm2 and p53; ination but are not degraded. Mdm2 has a highly similar part- where p53 activates expression of MDM2, Mdm2/HDM2 in ner called MdmX or Mdm4, which is a RING-domain protein turn associates with p53, p53 is ubiquitinated and degraded by defective in ubiquitin ligase activity. MdmX has considerable the proteasome complex and MDM2 expression declines; the inu fl ence on Mdm2 and p53: MdmX inhibits p53 transcrip- loop continues to maintain both Mdm2/HDM2 and p53 at low tional functions by direct binding and interacts with Mdm2 via 23 31,32 levels in normal cells (Fig. 1). Further studies revealed how their respective RING domains (Fig. 1). Multiple upstream Mdm2-directed stability of p53 is regulated in response to stress modifying enzymes regulate both p53-Mdm2 interactions and signaling, which temporarily breaks the Mdm2-p53 circuitry and protein levels during the activation of p53. During this process, 24 24,34 allows p53 protein levels to increase. Mdm2 levels are them- Mdm2 may ubiquitinate itself and its partner, MdmX. selves highly regulated and finely tuned; aberrant Mdm2 may Further investigations show that Mdm2-mediated regulation either contribute to oncogenesis or tumor suppression, as recently of p53 is more complex than initially thought, as Mdm2 also reviewed in ref. 25. inu fl ences subcellular localization and transcriptional potential Repression of p53 activity by Mdm2 may be achieved by two of p53 (Fig. 1). By conjugating a single ubiquitin onto one or major mechanisms: (1) by promoting p53 degradation and (2) more lysines within the carboyx (C)-terminus and DBD of p53, 35,36 by disrupting the ability of p53 to function as a transcription Mdm2 enhances the nuclear export of p53. How Mdm2 17,19,26 factor. Mdm2 binds p53 at its amino (N)-terminus to block activity is regulated to effect mono- versus poly-ubiquitination the transactivation domain of p53; additionally a second bind- of p53 is not well understood. Several reports suggest that other 37 38 39 40 ing site for Mdm2 within the DNA-binding domain (DBD) of proteins, including p300, YY1, Gankyrin, KAP1 and 28,29 41 p53 is known. Interestingly, some mutant forms of p53, which Siva1, may modulate Mdm2-mediated poly-ubiquitination of 666 Cancer Biology & Therapy volume 10 issue 7 Table 1. Ubiquitin e3-ligases that regulate p53 activity p53- Ligase Type E2 Ub p53-Lysine residue p53 status Mode responsive K370, K372, K373, Mdm2 r iNG Yes UbcH5b Mono/Poly Degradation Direct K381, K382, K386 Pirh2 r iNG Yes UbcH5b Poly - Degradation Direct Cop1 r iNG Yes UbcH5b Poly - Degradation Direct Tr iM24 r iNG - UbcH8 Poly - Degradation Direct Ar F-BP1 HeCT No UbcH5c Poly - Degradation Direct CAr P1/2 r iNG - - Poly - Degradation Direct TOPOr S r iNG - UbcH5a/c, UbcH6 Poly - Degradation Direct Synoviolin r iNG - UbcH5c Poly - Degradation Direct CHiP - - UbcH5b Poly - Degradation Direct Skp1-Cul1- JFK r iNG - - Poly - Degradation r bx1-F-box MKr N1 r iNG No - - K291, K292 Degradation Direct e4orf6 and elonginB-Cul5- r iNG - UbcH5a Poly - Degradation e1B55K r bx1 Translocation to iCP0 r iNG - UbcH5a, UbcH6 Poly - Direct Nuclear Foci MSL2 r iNG - - Poly K351, K357 Nuclear export Direct Transcriptional CUL7 - - - Mono/Di - Direct inactivation w w P1 HeCT Yes UbcH5c Mono/Poly - Nuclear export Direct Prevent Ubc13 - r epressed Ubc13 Poly - Direct Tetramerization Translocation to e4F1 - - - Poly K320 Direct Chromatin elonginB/c- BZLF1 - - UbcH5a/c Poly - Degradation Cul2/5-SOCS p53, while others support a model where the levels of Mdm2 endogenous p53 mouse model that allows rapid and reversible determine the type of ubiquitin conjugated to p53. Mdm2 also toggling of endogenous p53 between nucleus and cytoplasm, inhibits p53 by promoting its conjugation to the ubiquitin-like Evan and colleagues showed that, although p53 is simultaneously molecule NEDD8. Modifications of C-terminal lysines of p53 active in all tested tissue of Mdm2-deficient mice, its levels fall by ubiquitination, neddylation, sumoylation or acetylation affect rapidly after functional restoration post DNA-damage, suggest- interactions between p53 and Mdm2 to compete for Mdm2- ing that a negative feedback loop remains operable even in absence 43,44 mediated ubiquitination. of Mdm2. Other reports of in vivo, mouse knock-in models that express mutant forms of p53 (p53-6KR or p53-7KR), lacking the Nothing is Simple about Regulation of p53 majority of ubiquitination sites for Mdm2, show that mutant p53 47,48 has a normal half-life and is stabilized and activated by stress. With further studies of p53 regulation, the model of Mdm2 as Thus, alternative pathways for p53-degradation likely exist inde- sole arbiter of p53 protein levels began to erode. Genetic knock-in pendent of Mdm2. methodology was used to create mice that express E3-ligase defi - Benchimol and colleagues were the first to show that Mdm2 cient Mdm2 (a C462A mutant), which retains the ability to bind is not alone as an E3-ligase capable of controlling p53 protein to p53 and theoretically inhibit p53 transcriptional activity with- levels by ubitiquin-tagging p53 for degradation. The identifi - out ubiquitination. Surprisingly, these mice exhibit p53-depen- cation of Pirh2, another RING-domain E3-ligase that mediates dent embryonic lethality, similar to Mdm2-null mice, and also degradation of p53 by direct interaction, was soon followed by retain ubiquitination and degradation of Mdm2. These results discoveries of several other proteins that regulate p53 at the level suggest that protein-protein interactions between p53 and Mdm2 of protein stability. Table 1 presents a summary of numerous are insufficient to block p53-mediated apoptosis during embry - E3-ligases that target p53 and underscores the major premise of onic development and, more importantly, Mdm2-independent this review: Why are there so many E3-ligases that negatively reg- pathways must exist that regulate protein stability of both p53 ulate p53? We will address this question in a teleological manner and Mdm2 by ubiquitin modification. Using a switchable, by assuming there is specificity among these negative regulators www.landesbioscience.com Cancer Biology & Therapy 667 of p53. We offer the multi-component What, When and Where Protein-1 (MKRN1) displays expression in developing neural tis- Hypothesis of specificity: (1) E3-ligase proteins are expressed in sues, including the forebrain and the ear at 10.5 dpc. ARF-BP1 distinct tissues or cell types at specific times of development. A appears to be ubiquitously expressed but available data are com- corollary to this is that tissue- or stage-specific expression of the piled at a later stage of 14.5 dpc. At this same stage, Synoviolin1 required E2 or co-regulators dictates function of an E3-ligase. is primarily expressed in selected bone and cartilaginous sites of (2) Interactions between p53 and an E3-ligase are determined by the embryo. These examples of embryonic expression support readout of a p53 “PTM code”, e.g., situational signaling to p53 the hypothesis that the many E3-ligases of p53 display distinct leads to modification of specific residues, which likely determine expression patterns during embryonic development. protein-protein interactions, including those with E3-ligases. (3) Further analyses are needed to encompass adult tissues and An E3-ligase mediates ubiquitination of a defined residue of p53 expression levels, in order to address a major question of the that determines a specific outcome or timing of delayed versus roles that E3-ligases may play in specific tumor development immediate degradation. (4) An E3-ligase itself acquires specific and potential treatments based on these functions. An intrigu- PTMs under a specific condition that determines its interaction ing aspect of p53 regulation, which is less studied than most, with p53 and ultimately the status of p53 activity. (5) Targeting is that the p53 gene encodes other expressed p53 protein iso- p53 for ubiquitination by an E3-ligase is specific to the isoform of forms, which have been associated with specific cancers. These p53 under different situations. isoforms are expressed as a result of secondary promoter use and alternative splicing, which create a form of p53 truncated at the Embryonic Expression of E3-Ligases that Target p53 N-terminus. As the N-terminus is the site of interaction with Mdm2, additional E3-ligases might provide a “failsafe” mecha- None of the regulators, listed in Table 1, has been scrutinized nism to keep these truncated p53 proteins in check and regulate at the level of Mdm2, especially in development of mouse mod- their protein stability. Additionally, work with mouse models els of function. Lack of Mdm2 causes embryonic lethality and, suggests that specificity in regulation of full-length p53 occurs in the category of embryonic regulation of p53, Mdm2 is the at a tissue-specific level. When normal mice are exposed to dam - 20,21 principle player. Its functional partner, MdmX (Mdm4), age by 5 Gy of IR, p53 accumulates in the nuclei of cells and which lacks the enzymatic capacity to ubiquitinate p53, also induces apoptosis in spleen, thymus, bone marrow, intestine and induces embryonic lethality when deleted but at a later stage of ependyma. p53 levels also increase in kidney, osteocytes, myocar- 50 54 development. In consideration of the other E3-ligases of p53, dium and salivary glands but no cell death occurs. In liver, skel- important questions are whether these other E3-ligases of p53 etal muscle and brain tissue, there is no response to IR at the level are expressed during mouse embryonic development, as well as of p53 stability or apoptosis. In an intriguing study, Mdm2 was when and where expression occurs. To address these questions, shown to regulate mutant p53 protein stability as well as normal databases such as EMAGE (www.emouseatlas.org/emage/home. p53 protein levels in a mouse model, but this regulatory control php) offer collected information regarding expression of specific was lacking in liver tissue. genes during mouse embryonic development. Comparison of whole-mount in situ expression analysis allows some assessment Partners of E3-Ligase Function of specific E3-ligases that target p53. Here, we present interpreta - tion of more robust, available data and not all members listed in Tissue-specificity may lie at the level of E3-ligase expression Table 1 are analyzed. or whether the specific E2 needed for function is expressed The primary literature reveals that Mdm2 expression during (see Table 1). Although UbcH5 is a common E2-partner of many mouse embryogenesis is detectable, by in situ analysis, in neural E3-ligases, there are a notable few that are more active or exclu- folds and in migratory neural crest cells, 7.5–9 dpc. After these sively active with other E2 proteins. Even UbcH5 has its a, b and stages of critical neural crest development, Mdm2 is expressed c subtypes and their expression and interactions with E3-ligases more ubiquitously in the embryo. Interestingly, embryonic offer another level of regulation for specificity. Additionally one lethality in Mdm2-null mouse models occurs during peri-im- E2 in particular, Ubc13, can directly ubiquitinate p53 without plantation; thus, curbing p53 from massive cell death occurs intervention of a partner E3. much earlier than stages of detectable Mdm2 expression. This E3-ligase functions are inu fl enced by proteins that interact underscores the possibility that E3-ligases of p53 may acquire with or regulate the E3-ligase, in addition to an E2 protein. specific functions at different stages of development and/or in MdmX is a well-known example of a regulatory partner that 30,31 specific cells. alters Mdm2 activity and even its stability. Whether MdmX In contrast to detectable Mdm2 expression, Trim24 at 10.5 activity is more or less inu fl ential in specific cell types or tissues is dpc is visibly expressed at the tip of the hindlimb bud and in the unknown and other previously noted modifiers of Mdm2 func - 37 38 39 40 41 facial primordia, primarily the nasal eminence, with more mes- tions, p300, Y Y1, Gankyrin, K AP1 and Siva1, offer other enchymal appearance. Cop1, at the same stage of development, is regulatory nodes. As an example of potential tissue-specificity also expressed in the facial primordia but with additional expres- and its effects, deletion of Trim24 in mice led to tumor develop- sion in the forelimb bud and ear. CARP1 at 10.5 dpc is expressed ment in the liver. These tumors are responsive to retinoic acid in the outflow tract of the developing heart, the distal fore - treatment, suggesting that Trim24 primarily functions as a co- limb bud, as well as the facial primordia. Makorin Ring Finger regulator of the retinoic acid receptor rather than an E3-ligase of 668 Cancer Biology & Therapy volume 10 issue 7 7,10,11,14,63-66,68,69 p53 in the liver. Whether E3-ligases interact with p53 in a direct in some depth. Table 2 summarizes and updates or indirect manner (Table 1), protein partners and upstream some of the significant modifications of p53 and the enzymes regulation likely inu fl ence their activities, stability or expression. that create or abolish them. Though these modifications may be crucial for p53 functions in cultured cells, in vivo models sug- Evolution of E3-Ligases that Target p53 gest that PTMs play subtle, modulatory roles in regulating p53 66,70 functions. Therefore, the key regulatory switch in turning on Evolutionary conservation may offer some sense of the most and off p53 response may be direct or indirect regulation of p53 basic of E3-ligase functions and whether a single ancestral stability through ubiquitin modification. E3 was duplicated or multiple E3-ligases arose separately over One possibility is that among the collection of E3-ligases are time. A review by Abrams and colleagues suggests that control specific readers of p53 PTMs, which evolved in order to respond of p53 protein stability may be restricted to vertebrate develop- to particular inductive signals that write a pattern of PTMs. The ment although orthologs and structural conservation of p53 exist type of structural domains known to interact with modified across invertebrate phyla. Mdm2 and MdmX are not conserved lysine residues, such as Bromo with acetylated lysines as well as in flies and worms, but recent work from our laboratory sug - PHD and chromodomain with specific patterns of methylated gest that other regulators of p53, which serve as E3-ligases in lysines, are present in a subset of E3-ligases that target p53. For mammalian cells, are active in invertebrates. We used mosaic example, TRIM24 possesses a tandem PHD-Bromo domain deletion analysis in Drosophila to assess the outcome of bonus unit, but the ability of TRIM24 to recognize p53 that is acety- loss of function. Bonus is the single Drosophila representative lated and/or methylated is untested. Cop1 has a WD40-domain, of the TIF1 sub-family of TRIM (RING/B-box/Coiled-coil or which can mediate protein-protein interactions, not notably Tripartite Motif ) proteins and closest in homology to TRIM24 dependent on PTMs. ARF-BP1 has a WWE domain that acts among the TRIM24/28/33 members. Loss of bonus induces similarly but preferentially in the ubiquitin pathway. Other spe- apoptosis and cleavage of Caspase 3. Cell death is apparently due cific proteins that target cytoplasmic p53 for ubiquitin-mediated to unrestrained D-p53 activity, as RNAi-mediated depletion of degradation, proteins p300 and CREB-binding protein (CBP), D-p53 rescues the phenotype. Drosophila bonus may be an ances- have Bromo domains as well as poly-ubiquitin ligase (E4) activi- tral negative regulator of p53; molecular studies are needed to ties that tag p53 for proteolysis. These proteins are histone ace- show that ubiquitination and E3-ligase activity are the basis of tyl transferases that mediate acetylation of target proteins, e.g., this regulation. Evolutionary comparisons between all E3-ligase p53 and histones H3 and H4, in addition to recognizing the regulators of p53 and conserved proteins are needed to reveal acetylated motif. Acetylation of p53 occurs during activation of 44,72 comparable pathways and conserved functions. p53, in response to a variety of stresses; thus, these E4 pro- teins may also be involved in the termination of a stress-induced Upstream Regulation and Downstream Response p53-response. Protein-protein interactions between E3-ligases and p53 do The most striking aspect of p53-signaling, in response to stress not necessarily rely on independent, structural domain recogni- or inductive signaling, is the tightly regulated process of post- tion, but occur as a result of allosteric recognition of surfaces or transcriptional modification of p53, which guide p53 activi - interfaces created by p53 protein folding or charge. Interestingly, ties primarily by dictating p53-interactions with proteins that viral protein BZLF1 directly functions as an adaptor component control: (1) the levels of p53, (2) the ability of p53 to bind to of the ECS (ElonginB/C-Cul2/5-SOCS-box protein) ubiquitin DNA, (3) the subcellular localization of p53, (4) interaction with ligase complex and targets phosphorylated p53, induced by viral regulatory proteins, and thus determine the physiological out- lytic replication. Signaling to E3-ligases, as well as p53, alters come of p53 activation. A comparison can be made to chroma- interactions between them, e.g., phosphorylation of S166, S188, tin, where PTMs of specific histone residues are interpreted by S395, Y276 or Y394 of Mdm2 disrupts the p53-Mdm2 associa- histone “reader” proteins via specific, specialized domains, while tion. An E3-ligase may be regulated by signaling, as a result of other specialized enzymes “write” the PTM code. A few readers stress or other regulatory pathways, which induce PTMs or the and writers identified for histones are known to interact and/or association of protein partners that modify the feedback loops of 59-62 23 modify p53 as well. regulation that exist between p53 and its E3-ligase partners. Covalent modifications of p53 occur on more than 40 dif - ferent amino acid residues and likely provide fine-tuning and E3-Ligases Targeted to Specific Outcomes specificity of p53 activation. These PTMs range from phos - phorylation, acetylation, methylation, ubiquitination, sumoyla- Mouse knock-in models have been created that express mutant tion, neddylation, glycosylation, ribosylation and more recently forms of p53 (p53-6KR or p53-7KR), where C-terminal lysines 10,63-67 O-GlcNAcylation. Depending on the type of stress, specific are mutated and cannot be ubiquitinated, acetylated or methyl- signaling events are activated that target precise residues on p53 ated. These mice are viable and their mutant p53 has a normal half- 47,48 protein resulting in a stress-to-enzyme-to-residue specific modifi - life and response to stress. Elucidation of alternative pathways, 68 49 cation on p53 protein. Discussing all these modifications and the which act in addition to Mdm2, began with Pirh2, and offered enzymes responsible (writers) in detail is beyond the scope of this some idea of whether specific E3-ligases are involved in response review, however several comprehensive reviews cover this topic to certain stimuli or to different aspects of p53 regulation. The www.landesbioscience.com Cancer Biology & Therapy 669 Table 2. p53 post-translational modifications and modifying enzymes Residue of p53 Modification Modifiers De-modifiers S6 P JNK2, CKiδ/ε - S9 P CK1δ/ε - S15 P ATM, ATr , DNA-PK, AMPK, mTOr , r SK2, CDK5 PP1, PPM1D T18 P TTK, CHK2, vr K1 - S20 P CHK2, PiK3 - S33 P CDK9, CDK5, CAK/CDK7, GSK3β, P38K - S37 P ATr , Pr AK PP1, PP2A S46 P CDK5, AMPKα, HiPK2, P38K, PKCδ, DYr K2 PP2A T55 P er K2, TAF1 PP2A, B56Y T81 P JNK2 - K120 Ac TiP60 - T155 P CSN - K164 Ac P300 (KAT3B) - S215 P STKi5 - e255, e258, e259 r PAr P-1 (poly ADP-ribosylase) - K305 Ac P300 (KAT3B) - S313, S314 P CHK1, CHK2 - S315 P STKi5, CDK9, CDK2 CDC14A L320 Ac PCAF (KAT2B) HDAC1 S366 P CHK2, iκBK2 - 1 2 K370 Me , Me SMYD2 (KMT3C) LSD1 K372 Me SeT7/9 (KMT3) - K373 Ac P300 (KAT3B) HDAC1 S376 P PKC, GSK3β - T377 P CHK1, CHK2 - S378 P PKC, CHK1, CHK2 - K382 Ac P300 (KAT3B) HDAC1, Sir T1 K382 Me SeT8 (KMT5A) - K386 S SUMO-1 - T387 P CHK1 - S392 P CDK9, PKr , FACT (CK2) PP1 K320, K321, K370, K372, K373 N8 FBXO11, MDM2 - K120, K132, K139, K164 Ub - - K291, K292 Ub MKr N1 - K320 Ub e4F1 - K351, K357 Ub MSL2 - K101, K370, K372, K373, K381, Ub MDM2 - K382, K386 1 2 P, Phosphorylation; Ac, Acetylation; Me , Mono-methylation; Me , Di-methylation; r , r ibosylation; S, Sumoylation; N8, Neddylation; Ub, Ubiquitination. primary function of Pirh2 may be terminating the p53 response loops. Mdm2, Pirh2, COP1 and additional E3-ligases, including 58 76 77 78 79 56 to stimuli, because Pirh2 interacts preferentially with formed TRIM24, TOPORS, ARF-BP1, W WP1, ICP0, Ubc13, 80 81 82 83 84 p53-tetramers, an association stimulated during stress activation CUL7, CARP1 and CARP2, Synoviolin, CHIP, E4F1, 74 85 86 of p53. COP1, another E3-ligase with a RING domain, nega- E4orf6 and E1B55K, MSL2, the human kelch domain tively regulates p53 levels by ubiquitination but its targeted resi- containing F-box protein, JFK, and Makorin Ring Finger 75 88 dues are unknown. Similar to Mdm2, the genes encoding Pirh2 Protein-1, MKRN1, mediate K48-linked polyubiquitination of and COP1 are regulated by p53 and, like other E3-ligases listed p53 and degradation. However, mono- or K63-linked ubiquitina- in Table 1, are thought to participate in autoregulatory feedback tion is linked to other functions, e.g., nuclear export and cytosolic 670 Cancer Biology & Therapy volume 10 issue 7 4 localization of p53, as well as alteration of p53 transcriptional and decay, even in normal, unstressed cells, offers further frontiers activities. Limited conjugation of ubiquitin by Mdm2, WWP1 to determinations of relevant PTMs of p53, the signaling pathways and MSL2 induces nuclear export of p53; ICP0 promotes accu- involved and whether any known E3-ligases, or those yet to be mulation of ubiquitinated p53 at nuclear foci; E4F1, an atypical discovered, are involved in this regulatory program. Together these E3-ligase, forms K48-linked mono-, di- or tri-ubiquitinated p53 p53 regulators may alter p53 functions, terminate p53-signaling, that activates cell cycle arrest at G /G ; whereas, CUL7 gener- and/or maintain p53 at low levels bearable by normal, unstressed 0 1 ates mono- or di-ubiquitinated p53 to repress p53 transcrip- cells. Targeting E3-ligases to inhibit ubiquitin-mediated degra- 78-80,84,86 tional activities by unknown mechanisms. Ubc-13 is an dation and restore p53 functions holds considerable therapeutic E2-enzyme, capable of independently ubiquitinating p53, prevent promise. The multiple layers of negative and positive regulation p53 tetramerization. Thus, p53-ubiquitination can come in sev- involved in controlling p53 offer challenges to understanding these eral flavors and the specic E fi 3-ligase involved in modification of critical pathways that regulate p53 stability. p53 can greatly alter the outcome. Acknowledgements So Many E3-ligases that Target p53 We apologize to colleagues for our failure to cite their work due to space limitations. We thank Y. Furuta for his assistance in inter- In this discussion, we offer multiple ways to parse the E3-ligases of pretation of expression analyses. Research in the Barton labora- p53 and suggest specialization among them in their roles as con- tory is supported by the Mitchell Foundation, CellCentric, Ltd., trollers of p53. Further development of animal models, analysis in and grants from the National Institutes of Health (GM081627 multiple model organisms and testing of numerous hypotheses are and DK070824). A.K.J. is an Odyssey Fellow supported by the needed to sort out the roles of the many E3-ligases active in regula- Odyssey Program and The Laura and John Arnold Foundation at tion of p53. Recent evidence that p53 exhibits cycles of induction The University of Texas MD Anderson Cancer Center. 32. Singh RK, Iyappan S, Scheffner M. Hetero- 19. Kubbutat MH, Jones SN, Vousden KH. Regulation of References p53 stability by Mdm2. Nature 1997; 387:299-303. oligomerization with MdmX rescues the ubiquitin/ 1. Soussi T, Beroud C. Assessing TP53 status in human Nedd8 ligase activity of RING finger mutants of 20. Jones SN, Roe AE, Donehower LA, Bradley A. Rescue tumours to evaluate clinical outcome. Nat Rev Cancer Mdm2. J Biol Chem 2007; 282:10901-7. of embryonic lethality in Mdm2-deficient mice by 2001; 1:233-40. 33. Meek DW, Knippschild U. Posttranslational modifica- absence of p53. Nature 1995; 378:206-8. 2. Hainaut P, Hollstein M. p53 and human cancer: The tion of MDM2. Mol Cancer Res 2003; 1:1017-26. 21. Montes de Oca Luna R, Wagner DS, Lozano G. Rescue first ten thousand mutations. Adv Cancer Res 2000; 34. Kawai H, Wiederschain D, Kitao H, Stuart J, Tsai of early embryonic lethality in mdm2-deficient mice by 77:81-137. deletion of p53. Nature 1995; 378:203-6. KK, Yuan ZM. DNA damage-induced MDMX deg- 3. Vousden KH, Prives C. Blinded by the Light: The radation is mediated by MDM2. J Biol Chem 2003; 22. Lu WJ, Abrams JM. Lessons from p53 in non-mamma- Growing Complexity of p53. Cell 2009; 137:413-31. 278:45946-53. lian models. Cell Death Differ 2006; 13:909-12. 4. Loewer A, Batchelor E, Gaglia G, Lahav G. Basal 35. Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. 23. Wu X, Bayle JH, Olson D, Levine AJ. The p53- dynamics of p53 reveal transcriptionally attenuated Mono- versus poly-ubiquitination: differential control mdm-2 autoregulatory feedback loop. Genes Dev pulses in cycling cells. Cell 142:89-100. of p53 fate by Mdm2. Science 2003; 302:1972-5. 1993; 7:1126-32. 5. Hu W, Chan CS, Wu R, Zhang C, Sun Y, Song JS, et 36. Nie L, Sasaki M, Maki CG. Regulation of p53 nuclear 24. Stommel JM, Wahl GM. Accelerated MDM2 auto-deg- al. Negative Regulation of Tumor Suppressor p53 by export through sequential changes in conformation and radation induced by DNA-damage kinases is required MicroRNA miR-504. Mol Cell 38:689-99. ubiquitination. J Biol Chem 2007; 282:14616-25. for p53 activation. EMBO J 2004; 23:1547-56. 6. Vogelstein B, Lane D, Levine AJ. Surfing the p53 net- 37. Grossman SR, Deato ME, Brignone C, Chan HM, 25. Manfredi JJ. The Mdm2-p53 relationship evolves: work. Nature 2000; 408:307-10. Kung AL, Tagami H, et al. Polyubiquitination of p53 Mdm2 swings both ways as an oncogene and a tumor 7. Horn HF, Vousden KH. Coping with stress: multiple by a ubiquitin ligase activity of p300. Science 2003; suppressor. Genes Dev 24:1580-9. ways to activate p53. Oncogene 2007; 26:1306-16. 300:342-4. 26. Momand J, Zambetti GP, Olson DC, George D, 8. Riley T, Sontag E, Chen P, Levine A. Transcriptional 38. Sui G, Affar el B, Shi Y, Brignone C, Wall NR, Yin P, et Levine AJ. The mdm-2 oncogene product forms a com- control of human p53-regulated genes. Nat Rev Mol al. Yin Yang 1 is a negative regulator of p53. Cell 2004; plex with the p53 protein and inhibits p53-mediated Cell Biol 2008; 9:402-12. 117:859-72. transactivation. Cell 1992; 69:1237-45. 9. Laptenko O, Prives C. Transcriptional regulation by 39. Higashitsuji H, Itoh K, Sakurai T, Nagao T, Sumitomo 27. Chen J, Marechal V, Levine AJ. Mapping of the p53 p53: one protein, many possibilities. Cell Death Differ Y, Masuda T, et al. The oncoprotein gankyrin binds to and mdm-2 interaction domains. Mol Cell Biol 1993; 2006; 13:951-61. MDM2/HDM2, enhancing ubiquitylation and degra- 13:4107-14. dation of p53. Cancer Cell 2005; 8:75-87. 10. Carter S, Vousden KH. Modifications of p53: competing 28. Yu GW, Rudiger S, Veprintsev D, Freund S, Fernandez- for the lysines. Curr Opin Genet Dev 2009; 19:18-24. 40. Wang C, Ivanov A, Chen L, Fredericks WJ, Seto E, Fernandez MR, Fersht AR. The central region of Rauscher FJ, 3rd, Chen J. MDM2 interaction with 11. Kruse JP, Gu W. Modes of p53 regulation. Cell 2009; HDM2 provides a second binding site for p53. Proc 137:609-22. nuclear corepressor KAP1 contributes to p53 inactiva- Natl Acad Sci USA 2006; 103:1227-32. tion. EMBO J 2005; 24:3279-90. 12. Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin 29. Shimizu H, Burch LR, Smith AJ, Dornan D, Wallace ligases. Annu Rev Biochem 2009; 78:399-434. 41. Du W, Jiang P, Li N, Mei Y, Wang X, Wen L, et al. M, Ball KL, Hupp TR. The conformationally flex- Suppression of p53 activity by Siva1. Cell Death Differ ible S9-S10 linker region in the core domain of p53 13. Chen ZJ, Sun LJ. Nonproteolytic functions of ubiq- 2009; 16:1493-504. uitin in cell signaling. Mol Cell 2009; 33:275-86. contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo. J Biol Chem 42. Xirodimas DP, Saville MK, Bourdon JC, Hay RT, 14. Brooks CL, Gu W. p53 ubiquitination: Mdm2 and 2002; 277:28446-58. Lane DP. Mdm2-mediated NEDD8 conjugation of beyond. Mol Cell 2006; 21:307-15. p53 inhibits its transcriptional activity. Cell 2004; 30. Marine JC, Francoz S, Maetens M, Wahl G, Toledo 15. Mukhopadhyay D, Riezman H. Proteasome- 118:83-97. F, Lozano G. Keeping p53 in check: essential and independent functions of ubiquitin in endocytosis and synergistic functions of Mdm2 and Mdm4. Cell Death 43. Carter S, Bischof O, Dejean A, Vousden KH. C-terminal signaling. Science 2007; 315:201-5. Differ 2006; 13:927-34. modifications regulate MDM2 dissociation and nuclear 16. Jain AK, Barton MC. Regulation of p53: TRIM24 export of p53. Nat Cell Biol 2007; 9:428-35. 31. Uldrijan S, Pannekoek WJ, Vousden KH. An essential enters the RING. Cell Cycle 2009; 8:3668-74. function of the extreme C-terminus of MDM2 can be 44. Tang Y, Zhao W, Chen Y, Zhao Y, Gu W. Acetylation 17. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes provided by MDMX. EMBO J 2007; 26:102-12. is indispensable for p53 activation. Cell 2008; the rapid degradation of p53. Nature 1997; 387:296-9. 133:612-26. 18. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 1997; 420:25-7. www.landesbioscience.com Cancer Biology & Therapy 671 45. Itahana K, Mao H, Jin A, Itahana Y, Clegg HV, 59. Liu L, Scolnick DM, Trievel RC, Zhang HB, 75. Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, Lindstrom MS, et al. Targeted inactivation of Mdm2 Marmorstein R, Halazonetis TD, Berger SL. p53 sites Dowd P, et al. The ubiquitin ligase COP1 is a critical RING finger E3 ubiquitin ligase activity in the mouse acetylated in vitro by PCAF and p300 are acetylated in negative regulator of p53. Nature 2004; 429:86-92. reveals mechanistic insights into p53 regulation. Cancer vivo in response to DNA damage. Mol Cell Biol 1999; 76. Rajendra R, Malegaonkar D, Pungaliya P, Marshall H, Cell 2007; 12:355-66. 19:1202-9. Rasheed Z, Brownell J, et al. Topors functions as an E3 46. Ringshausen I, O’Shea CC, Finch AJ, Swigart LB, Evan 60. Huang J, Perez-Burgos L, Placek BJ, Sengupta R, ubiquitin ligase with specific E2 enzymes and ubiquiti- GI. Mdm2 is critically and continuously required to Richter M, Dorsey JA, et al. Repression of p53 activ- nates p53. J Biol Chem 2004; 279:36440-4. suppress lethal p53 activity in vivo. Cancer Cell 2006; ity by Smyd2-mediated methylation. Nature 2006; 77. Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF- 10:501-14. 444:629-32. BP1/Mule is a critical mediator of the ARF tumor 47. Feng L, Lin T, Uranishi H, Gu W, Xu Y. Functional 61. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, suppressor. Cell 2005; 121:1071-83. analysis of the roles of posttranslational modifications Richter M, et al. p53 is regulated by the lysine demeth- 78. Laine A, Ronai Z. Regulation of p53 localization and at the p53 C terminus in regulating p53 stability and ylase LSD1. Nature 2007; 449:105-8. transcription by the HECT domain E3 ligase WWP1. activity. Mol Cell Biol 2005; 25:5389-95. 62. Huang J, Dorsey J, Chuikov S, Perez-Burgos L, Zhang Oncogene 2007; 26:1477-83. 48. Krummel KA, Lee CJ, Toledo F, Wahl GM. The X, Jenuwein T, et al. G9a and Glp methylate lysine 373 79. Boutell C, Everett RD. The herpes simplex virus type C-terminal lysines fine-tune p53 stress responses in a in the tumor suppressor p53. J Biol Chem 285:9636-41. 1 (HSV-1) regulatory protein ICP0 interacts with and mouse model but are not required for stability control 63. Murray-Zmijewski F, Slee EA, Lu X. A complex bar- Ubiquitinates p53. J Biol Chem 2003; 278:36596-602. or transactivation. Proc Natl Acad Sci USA 2005; code underlies the heterogeneous response of p53 to 80. Andrews P, He YJ, Xiong Y. Cytoplasmic localized 102:10188-93. stress. Nat Rev Mol Cell Biol 2008; 9:702-12. ubiquitin ligase cullin 7 binds to p53 and promotes cell 49. Leng RP, Lin Y, Ma W, Wu H, Lemmers B, Chung S, 64. Bode AM, Dong Z. Post-translational modification of growth by antagonizing p53 function. Oncogene 2006; et al. Pirh2, a p53-induced ubiquitin-protein ligase, p53 in tumorigenesis. Nat Rev Cancer 2004; 4:793-805. 25:4534-48. promotes p53 degradation. Cell 2003; 112:779-91. 65. Olsson A, Manzl C, Strasser A, Villunger A. How 81. Yang W, Rozan LM, McDonald ER, 3rd, Navaraj A, 50. Parant J, Chavez-Reyes A, Little NA, Yan W, Reinke important are post-translational modifications in p53 Liu JJ, Matthew EM, et al. CARPs are ubiquitin ligases V, Jochemsen AG, Lozano G. Rescue of embryonic for selectivity in target-gene transcription and tumour that promote MDM2-independent p53 and phospho- lethality in Mdm4-null mice by loss of Trp53 suggests a suppression? Cell Death Differ 2007; 14:1561-75. p53ser20 degradation. J Biol Chem 2007; 282:3273-81. nonoverlapping pathway with MDM2 to regulate p53. 66. Toledo F, Wahl GM. Regulating the p53 pathway: in 82. Yamasaki S, Yagishita N, Sasaki T, Nakazawa M, Kato Nat Genet 2001; 29:92-5. vitro hypotheses, in vivo veritas. Nat Rev Cancer 2006; Y, Yamadera T, et al. Cytoplasmic destruction of p53 51. Daujat S, Neel H, Piette J. Preferential expression of 6:909-23. by the endoplasmic reticulum-resident ubiquitin ligase Mdm2 oncogene during the development of neural ‘Synoviolin’. EMBO J 2007; 26:113-22. 67. Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim crest and its derivatives in mouse early embryogenesis. YS, Cho JW. Modification of p53 with O-linked 83. Esser C, Scheffner M, Hohfeld J. The chaperone- Mech Dev 2001; 103:163-5. N-acetylglucosamine regulates p53 activity and stabil- associated ubiquitin ligase CHIP is able to target 52. Chavez-Reyes A, Parant JM, Amelse LL, de Oca Luna ity. Nat Cell Biol 2006; 8:1074-83. p53 for proteasomal degradation. J Biol Chem 2005; RM, Korsmeyer SJ, Lozano G. Switching mechanisms 280:27443-8. 68. Meek DW, Anderson CW. Posttranslational of cell death in mdm2- and mdm4-null mice by dele- Modification of p53: cooperative integrators of func- 84. Le Cam L, Linares LK, Paul C, Julien E, Lacroix M, tion of p53 downstream targets. Cancer Res 2003; tion. Cold Spring Harb Perspect Biol 2009; 1:000950. Hatchi E, et al. E4F1 is an atypical ubiquitin ligase 63:8664-9. 69. Harris SL, Levine AJ. The p53 pathway: positive and that modulates p53 effector functions independently of 53. Bourdon JC. p53 and its isoforms in cancer. Br J degradation. Cell 2006; 127:775-88. negative feedback loops. Oncogene 2005; 24:2899-908. Cancer 2007; 97:277-82. 70. Donehower LA, Lozano G. 20 years studying p53 func- 85. Querido E, Blanchette P, Yan Q, Kamura T, Morrison 54. Midgley CA, Owens B, Briscoe CV, Thomas DB, Lane M, Boivin D, et al. Degradation of p53 by adenovirus tions in genetically engineered mice. Nat Rev Cancer DP, Hall PA. Coupling between gamma irradiation, 2009; 9:831-41. E4orf6 and E1B55K proteins occurs via a novel mecha- p53 induction and the apoptotic response depends nism involving a Cullin-containing complex. Genes 71. Shi D, Pop MS, Kulikov R, Love IM, Kung AL, upon cell type in vivo. J Cell Sci 1995; 108:1843-8. Dev 2001; 15:3104-17. Grossman SR. CBP and p300 are cytoplasmic E4 55. Terzian T, Suh YA, Iwakuma T, Post SM, Neumann 86. Kruse JP, Gu W. MSL2 promotes Mdm2-independent polyubiquitin ligases for p53. Proc Natl Acad Sci USA M, Lang GA, et al. The inherent instability of mutant 2009; 106:16275-80. cytoplasmic localization of p53. J Biol Chem 2009; INK4a p53 is alleviated by Mdm2 or p16 loss. Genes Dev 284:3250-63. 72. Gu W, Roeder RG. Activation of p53 sequence-specific 2008; 22:1337-44. DNA binding by acetylation of the p53 C-terminal 87. Sun L, Shi L, Li W, Yu W, Liang J, Zhang H, et al. 56. Laine A, Topisirovic I, Zhai D, Reed JC, Borden KL, JFK, a Kelch domain-containing F-box protein, links domain. Cell 1997; 90:595-606. Ronai Z. Regulation of p53 localization and activity by the SCF complex to p53 regulation. Proc Natl Acad Sci 73. Sato Y, Kamura T, Shirata N, Murata T, Kudoh A, Ubc13. Mol Cell Biol 2006; 26:8901-13. USA 2009; 106:10195-200. Iwahori S, et al. Degradation of phosphorylated p53 57. Khetchoumian K, Teletin M, Tisserand J, Mark M, by viral protein-ECS E3 ligase complex. PLoS Pathog 88. Lee EW, Lee MS, Camus S, Ghim J, Yang MR, Oh Herquel B, Ignat M, et al. Loss of Trim24 (Tif1alpha) W, et al. Differential regulation of p53 and p21 by 2009; 5:1000530. gene function confers oncogenic activity to retinoic MKRN1 E3 ligase controls cell cycle arrest and apop- 74. Sheng Y, Laister RC, Lemak A, Wu B, Tai E, Duan S, acid receptor alpha. Nat Genet 2007; 39:1500-6. tosis. EMBO J 2009; 28:2100-13. et al. Molecular basis of Pirh2-mediated p53 ubiquity- 58. Allton K, Jain AK, Herz HM, Tsai WW, Jung SY, Qin lation. Nat Struct Mol Biol 2008; 15:1334-42. J, et al. Trim24 targets endogenous p53 for degrada- tion. Proc Natl Acad Sci USA 2009; 106:11612-6. 672 Cancer Biology & Therapy volume 10 issue 7

Journal

Cancer Biology & Therapy – Taylor & Francis

Published: Oct 1, 2010

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