p53 regulation by post-translational modification and nuclear ... - Nature

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Oncogene (1999) 18, 7656 ± 7665 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

p53 regulation by post-translational modi®cation and nuclear retention in response to diverse stresses Gretchen S Jimenez1, Shireen H Khan1,2, Jayne M Stommel1,2 and Georey M Wahl*,1 1 2

Gene Expression Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, California, CA 92037, USA; Department of Biology, University of California at San Diego, La Jolla, California, CA 92037, USA

p53 activation by diverse stresses involves post-translational modi®cations that alter its structure and result in its nuclear accumulation. We will discuss several unresolved topics regarding p53 regulation which are currently under investigation. DNA damage is perhaps the best-studied stress which activates p53, and recent data implicate phosphorylation at N-terminal serine residues as critical in this process. We discuss recent data regarding the potential kinases which modify p53 and the possible role of the resulting phosphorylation events. By contrast, much less is understood about agents which disrupt the mitotic spindle. The cell cycle phase, induction signal, and biochemical mechanism of the reversible arrest induced by microtubule disruption are currently under investigation. Finally, a key event in response to any genotoxic stress is the accumulation of p53 in the nucleus. The factors which determine the steady state level of p53 are starting to be elucidated, but the mechanisms responsible for nuclear accumulation and nuclear export remain controversial. We discuss new studies revealing a mechanism for nuclear retention of p53, and the potential contributions of MDM2 to this process. Keywords: p53; DNA damage; nuclear export signal; microtubule depolymerization; post translational modi®cation

Introduction New revelations continue to emerge concerning the mechanisms that control p53 activation in response to a wide range of input signals. This tumor suppressor maintains genetic stability by responding to multiple environmental stresses including DNA damage, ribonucleotide depletion, microtubule disruption, redox modulation, cell adhesion, and hypoxia, as well as `genetic' stresses created by activated oncogenes (reviewed in Giaccia and Kastan, 1998). These diverse stimuli appear to invoke a similar set of responses to achieve p53 activation: p53 must ®rst accumulate in the nucleus, and then bind to DNA as a tetramer to transcriptionally regulate a growing list of target genes including p21, GADD45, cyclin G, 14-3-3sigma, thrombospondin 1, MDM2, IGF-BP3, and bax (reviewed in El-Deiry, 1998; Gottlieb and Oren, 1996; Ko and Prives, 1996), as well as c-fos (Elkeles et al., 1999) and others. However, depending on the stress and cell type, p53 activation can lead to responses as *Correspondence: GM Wahl

dierent as a reversible cycle arrest, induction of a senescent-like state, or apoptosis. Clearly, the induction of the irreversible states of senescence or apoptosis requires a permanent activation of p53 which commits the cell to exit the cycle forever. On the other hand, some agents, such as nocodazole and colcemid, cause a reversible arrest. This raises the important question of whether stimuli that induce cell cycle exit induce dierent alterations in p53 than those that result in a reversible response, which could result in regulation of dierent downstream genes. We infer that there must also be attenuation mechanisms that reverse p53 function subsequent to removal of certain stresses. Since p53 has the power to either target a cell for death or allow it to survive, it must be under rigorous and complex control. p53 contains an N-terminal transactivation domain, a core DNA binding domain, and a C-terminal multifunctional regulatory domain (reviewed in Ko and Prives, 1996). Highly conserved residues in its N- and C-terminal domains are targets for potential post-translational modi®cation via phosphorylation, dephosphorylation or acetylation (reviewed in Giaccia and Kastan, 1998). We will discuss how post-translational modi®cation may in¯uence p53 function in a variety of ways. The possibilities include eects on binding to other proteins, such as coactivators or negative regulators, and induction of higher order structural changes mediated by highly conserved amino acid motifs encoded within the primary sequence. For example the multifunctional C-terminus contains a newly identi®ed nuclear export signal (NES) within the tetramerization domain (Stommel et al., 1999). Data are discussed which indicate that the NES is occluded in the p53 tetramer, the active DNA binding conformation. N-terminal phosphorylation and p53 activation p53 protein is translocated to the nucleus, stabilized and rendered competent for DNA binding and transactivation in response to DNA damage. Many studies have attempted to correlate the contributions of p53 protein accumulation, post-translational modification, and binding to regulatory proteins with the ability of p53 to bind DNA and transactivate downstream target genes. p53 transactivation appears to require its interaction with components of TFIID, including the TATA box binding protein (TBP) and TBP-associated factors (TAFs), via the p53 N-terminal transactivation domain (Lu and Levine, 1995; Thut et al., 1995; Xiao et al., 1994). More recently, transcriptional coactivators p300 and CREB-binding protein (CBP) have also been shown to enhance p53 transactivation

Regulation of p53 by diverse stresses GS Jimenez et al

(Gu et al., 1997; Lill et al., 1997; Scolnick et al., 1997). On the other hand, binding of MDM2, which is a downstream p53 target, downregulates p53 transactivation (Wu et al., 1993). This may be explained by both steric interference between MDM2 and the coactivators, as MDM2 binds p53 at amino acids 17 ± 27 (Chen et al., 1993; Kussie et al., 1996). As MDM2 also targets p53 for degradation (Haupt et al., 1997; Kubbutat et al., 1997, 1998), this region is crucial to the concurrent regulation of p53 transactivation and stability. Phosphorylation of N-terminal amino acids could contribute to p53 regulation by aecting the binding of co-activators and the negative regulator MDM2. p53 is a good in vitro kinase substrate for casein kinase I (CKI) at S6 and S9 (Milne et al., 1992), DNAdependent protein kinase (DNA-PK) at S15 and S37 (Lees-Miller et al., 1992; Shieh et al., 1997), c-Jun kinase (JNK) at S37 (Milne et al., 1995), and CDK activating kinase (CAK) at S33 (Ko et al., 1997). The importance of these potential phosphorylation sites has been assessed by mutating them individually or in combination and (over)expressing the respective mutants in various cell lines (Ashcroft et al., 1999; Fiscella et al., 1993; Fuchs et al., 1995; Mayr et al., 1995). These studies have yielded con¯icting results, but indicated an importance for phosphorylation at S15 and S37 in maintaining protein stability and transactivation properties. The importance of Nterminal phosphorylation was assessed in vivo in recent experiments using two dimensional peptide mapping to show that in response to ionizing radiation (IR), three sites in the N-terminus of p53 are phosphorylated, two of which reside in the ®rst 24 amino acids (Siliciano et al., 1997). One interpretation consistent with these data is that N-terminal phosphorylation interferes with MDM2 binding, which would be expected to allow association of p53 with co-activators and prevent MDM2 from targeting p53 for cytoplasmic degradation (Pise-Masison et al., 1998; Shieh et al., 1997). Phosphorylation of p53 at S15 and S37 in vitro with puri®ed DNA dependent protein kinase (DNAPK) impaired not only the ability of MDM2 to interact with p53, but also its ability to block p53-dependent transcription (Shieh et al., 1997). This model was further supported in vivo by a damage-induced decrease in the binding of MDM2 to p53. In addition, immunoprecipitation studies using an antibody speci®c for phosphoserine 15 showed that this site is phosphorylated in response to DNA damage (Siliciano et al., 1997). The contribution of N-terminal phosphorylation to the damage response is becoming increasingly clear, but the identi®cation of the upstream kinases which respond to various stresses is still being debated. Three members of the PIK-related kinase family, DNA-PK, ATM (the kinase mutated in patients with ataxia telangiectasia), and ATR (the ataxia telangiectasia related kinase), have been proposed as upstream activators of p53 (Banin et al., 1998; Canman et al., 1998; Kastan et al., 1992; Shieh et al., 1997; Siliciano et al., 1997; Tibbetts et al., 1999; Woo et al., 1998). DNA-PK could easily be activated by DNA damage as it is recruited to diverse aberrant DNA structures by the Ku70/80 heterodimer which binds the DNA-PK catalytic subunit (cs) (reviewed in Jeggo, 1997; Smith

and Jackson, 1999). DNA-PK subunits are suciently abundant to locate, bind to, and become activated by a solitary double strand break, which is the minimum amount of damage needed to trigger a p53-dependent arrest response (Di Leonardo et al., 1994; Huang et al., 1996). Even though DNA-PK phosphorylates p53 in vitro at S15 and S37, a signi®cant controversy has emerged over whether it plays a physiological role in p53 activation (Lees-Miller et al., 1992). Until recently, studies involving DNA-PK utilized cells derived from mice with a form of severe combined immunode®ciency (SCID) (Blunt et al., 1995; Kirchgessner et al., 1995). Primary embryonic ®broblasts from SCID mice arrested like wildtype cells in response to DNA damage (Fried et al., 1996; Guidos et al., 1996; Gurley and Kemp, 1996; Huang et al., 1996; Nacht et al., 1996; Rathmell et al., 1997), which is not expected if DNA-PK is required to generate the Nterminal phosphorylations that prevent MDM2 binding. However, the SCID MEFs may have sucient residual kinase activity for p53 to be activated with near normal kinetics (Danska et al., 1996; Woo et al., 1998). Data suggesting the importance of DNA-PK in p53 activation emerged from a recent study utilizing SCGR11, a transformed cell line derived from SCID mouse ®broblasts. p53-dependent DNA binding and transactivation were shown to be aberrant in these cells. The largely in vitro studies led the authors to conclude that DNA-PK is necessary, but not sucient, for induction of p53 DNA binding and transcriptional activity (Woo et al., 1998). We used primary mouse embryonic ®broblasts (MEFs) derived from mice in which the DNA-PK gene was inactivated via homologous recombination in order to compare the cell cycle responses and p53 activation kinetics to those in the SCGR11 cell line. We showed that SCGR11 contains a mutation in the p53 core DNA binding domain (Jimenez et al., 1999). This mutation is similar to those found in many cancers (Hainaut and Milner, 1993), which explains the inability of p53 target genes to be induced in SCGR11 cells and the lack of DNA binding exhibited by SCGR11 derived p53. During our rigorous analysis of each step involved in a p53 dependent response utilizing DNA-PKcs knockout MEFs, we showed that in response to DNA damage, p53 accumulates in the nucleus, is phosphorylated on the mouse equivalent of S15, binds DNA, transactivates target genes, and mediates a robust G1 phase cell cycle arrest with kinetics which are similar to those of normal cells (Jimenez et al., 1999). Therefore, DNA-PK activity is not required to activate p53 in response to DNA damage. The ATM and ATR kinases are much stronger candidates for upstream regulators of p53. AT de®cient cells exhibit a delayed p53 accumulation after ionizing radiation (IR) (Canman et al., 1994; Kastan et al., 1992; Lavin et al., 1994; Zu and Baltimore, 1996) with an accompanying delay in S15 phosphorylation (Siliciano et al., 1997). In vivo and in vitro evidence shows that ATM and ATR will phosphorylate p53 on S15 (Banin et al., 1998; Canman et al., 1998; Khanna et al., 1998; Tibbetts et al., 1999). Although the response is delayed, p53 is eventually induced and phosphorylated in IR-treated AT de®cient cells

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(Siliciano et al., 1997). This suggests that another kinase, such as ATR, may take over for some of the ATM function in cells obtained from AT patients or atm7/7 mice. In fact, overexpression of an inactive form of ATR in normal ®broblasts mimics several phenotypes of AT defective cells (Cliby et al., 1998). Interestingly, p53 protein accumulation and S15 phosphorylation occur with normal kinetics in UVirradiated AT de®cient cells (Canman et al., 1994; Khanna and Lavin, 1993; Siliciano et al., 1997). Since the UV response is normal in AT de®cient cells, it appears that dierent upstream signal transducers activate p53 in response to dierent forms of DNA damage. While many studies have focused on the N-terminal modi®cation events in response to ionizing or gamma radiation, the phosphorylation of S15 and S37 are likely to represent only a fraction of the modi®cations. For example, recent data suggest that phosphorylation of S20, not S15, disrupts the association of MDM2 with p53 (Chabib et al., submitted; Shieh et al., 1999; Unger et al., 1999). Therefore, S20 could be the second damage-responsive phosphorylation site in the ®rst 24 amino acids indicated by 2 D peptide mapping (Siliciano et al., 1997). The identity of the kinase responsible for this modi®cation has not been determined. The question remains as to the importance of phosphorylation in maintaining protein stability versus activation of p53 as a transcription factor. For example, unmodi®ed p53, which has been stabilized by treatment with a proteasome inhibitor, shows the same kinetics of binding to and dissociation from DNA as p53 from IR-treated cells, as monitored by electrophoretic mobility shift assay (Siliciano et al., 1997). However, p53 in cells treated with proteasome inhibitor does not have the same ability to transactivate downstream targets as the p53 in IR-treated cells (Siliciano et al., 1997). Although these data indicate that post-translational modi®cation is important, p53 retained transciptional transactivation function after mutation of all known phosphorylation sites in the Nand C-termini to alanine (Ashcroft et al., 1999). Only mutation of S15 and S37 to aspartic acid disrupted MDM2-mediated degradation, but only slightly (Ashcroft et al., 1999). The C-terminus contains several sites of potential modi®cation, and interactions of the C-terminus with residues in the middle of the protein have long been known to prevent p53 from binding DNA (Hupp et al., 1992; Ko and Prives, 1996). p53 is phosphorylated in vitro on S315 by cdc2 and cdk2, on S378 by protein kinase C, and on S392 by casein kinase II (Milczarek et al., 1997). There is also growing evidence for activation of p53 through dephosphorylation. For instance, S376 appears phosphorylated in unirradiated cells but becomes dephosphorylated rapidly subsequent to ionizing irradiation (Waterman et al., 1998). This dephosphorylation event leads to association with a 14-3-3 protein, which correlates temporally with p53 activation (Waterman et al., 1998). Similarly, acetylation of the C-terminus on K382 by p300 or on K320 by PCAF enhances sequence speci®c DNA binding in vitro (Liu et al., 1999; Sakaguchi et al., 1998). Acetylationspeci®c and phosphospeci®c antibodies reveal that S33 and S37 are phosphorylated after UV or IR, along

with acetylation at K382, while K320 is acetylated only after UV (Sakaguchi et al., 1998). In vitro studies indicate that the N-terminal phosphorylation events may facilitate C-terminal acetylation (Sakaguchi et al., 1998). More recently, it has been reported that the tetramerization domain is necessary to facilitate Nterminal p53 phosphorylation (Shieh et al., 1999). Therefore, multiple modi®cations on both ends of p53, coupled with the structural changes they elicit, are likely to occur in response to DNA damage. These modi®cations appear to in¯uence both the association of p53 with regulatory proteins and its ability to regulate the transcription of target genes. Despite the correlations between damage and p53 modi®cation, there is still much confusion over the role of post-translational modi®cation in p53 function. We discussed above the potential delicate interplay of several modi®cations of p53 in mediating a damage response, raising the expectation that their mutation would lead to gross alterations in p53 function. Con¯icting data regarding the contribution of individual phosphorylation events to p53 function suggest that either the contribution of post-translational modi®cation is subtle, or that experimental systems which cannot accurately recapitulate p53 expression may fail to reveal the eects of such mutations on cell cycle progression, apoptosis, genetic instability and other important biological manifestations of p53 function. With the growing number of specialized antibodies recognizing the acetylated or phosphorylated forms of p53, study of the events occurring in vivo during expression of the endogenous gene should become practical. Another important target for future study is the modi®cation of p53-associated proteins. For instance, it was shown that DNA-PK could phosphorylate MDM2 in vitro (Mayo et al., 1997). It may be that analysis of both p53 and its binding partners will be required to understand which modi®cations on each protein play important biological roles. Finally, new elements intrinsic to the p53 protein itself, such as the newly identi®ed nuclear export signal (NES) within the tetramerization domain, point us toward new levels of regulation in p53 function which are only now being elucidated (see below). Disruption of microtubule organization induces an arrest in a 4N G1-like state via the components of the p53 G1 arrest pathway Faithful genome replication and maintenance of diploidy are commonly compromised in human neoplasms. The components of the machinery that ensure that duplicated chromosomes are segregated evenly between two daughter cells remain to be elucidated. However, recent data implicate p53 as part of a checkpoint that senses microtubule disruption, and that prevents the outgrowth of aneuploid variants that emerge after exposure of cells to microtubule inhibitors. A key link was established when Brian Reid and colleagues demonstrated that in rodent cells microtubule disruption in combination with p53 loss allows downstream events (DNA replication) to occur prior to the successful completion of upstream events (mitosis)


(Cross et al., 1995). The consequence of this loss of coordination is an increase in the number of cells with greater than 4N DNA content. Thus, loss of p53 leads to DNA reduplication after exposure to agents that inhibit mitotic division, such as nocodazole and colcemid. These data led to the proposal that p53 functions in a mitotic spindle checkpoint in which it monitors the proper assembly of the mitotic spindle and prevents exit from mitosis under conditions that hinder cell division (Cross et al., 1995). Subsequent studies showed that loss of pRb function in rodent ®broblasts led to an increase in polyploidy after exposure to nocodazole or colcemid for extended time periods (Di Leonardo et al., 1997a). Hence, loss of p53 and pRb allow DNA replication to occur prior to completion of the upstream mitotic events. Rereplication was also observed in human ®broblasts lacking functional p53 or pRb due to inactivation by HPV16 E6 or E7, suggesting that the controls coordinating the proper progression of the cycle are conserved between species (Di Leonardo et al., 1997b; Thomas et al., 1996). However, others report that DNA reduplication only occurs in human ®broblasts expressing p53 missense mutations which confer a dominant gain-of-function phenotype (Gualberto et al., 1998). The authors argue that loss of p53 is not sucient to induce polyploidization in human cells, but rather, a speci®c class of p53 missense mutations imparts an oncogenic phenotype that promotes polyploidy. The con¯icting reports may re¯ect dierences in cell types, drug concentrations and length of exposure to the microtubule destabilizing agents. In fact, high concentrations of nocodazole were shown to prevent rereplication in baby hamster kidney cells (Andreassen and Margolis, 1994). Moreover, several dierent cell lines varied in their propensity to rereplicate after colcemid treatment, suggesting that cell types dier in their response to microtubule inhibitors (Kung et al., 1990). The available data allow two possible models to be proposed, one of which implicates p53 and pRb in novel roles during G2/M to prevent cell cycle advancement in the presence of spindle poisons. This would be analogous to the Saccharomyces cerevisiae bub mutants that become polyploid under microtubule depolymerizing conditions (Hoyt et al., 1991). BUB1 encodes a protein kinase that is required to arrest cells prior to anaphase following spindle disruption (Farr and Hoyt, 1998; Roberts et al., 1994). A second model proposes that extended exposure to microtubule destabilizing agents does not result in a long term arrest in mitosis; rather, the cells escape the mitotic block and enter a stage similar to G1 during which p53 and pRB mediate the arrest response. Persuasive data from several dierent experimental approaches demonstrate that extended exposure to microtubule destabilizing agents does not lead to a sustained mitotic arrest in mammalian cells. Instead, the cells undergo `mitotic slippage' whereby cells with a 4N DNA content bypass the mitotic block and maintain a long term arrest in a stage biochemically similar to G1 (Andreassen and Margolis, 1994; Kung et al., 1990; Rieder and Palazzo, 1992). This raises the possibility that p53 and pRb prevent reduplication after the cells have undergone mitotic slippage and enter a 4N `G1-like' state. We will refer to this stage as a `G1-like' stage

since the classical de®nition of G1 requires that the cells have 2N DNA content. There is now compelling evidence that, independent of the p53 or pRb status, extended exposure of mammalian ®broblasts to nocodazole leads to a transient mitotic arrest during which the cells have elevated levels of cyclin B1, MPM-2 reactivity (a marker of M phase), condensed chromosomes, and a rounded mitotic morphology (Bunz et al., 1998; Khan and Wahl, 1998; Lanni and Jacks, 1998; Minn et al., 1996; Notterman et al., 1998). The cells may spend up to 6 h struggling through mitosis before adapting to the presence of nocodazole and escaping the mitotic block (Lanni and Jacks, 1998). Once the ®broblasts bypass this block, they enter a phase exhibiting many characteristics of G1: cyclin B1 levels are low, cyclins D and E are high, p53 activation occurs, and pRb is hypophosphorylated (Khan and Wahl, 1998; Lanni and Jacks, 1998; Minn et al., 1996; Notterman et al., 1998). The nocodazole induced rereplication observed in p53 and pRb-de®cient cells occurs only after a sign®cant delay, which corresponds to the length of time required for the fastest progressing cells to escape the mitotic block. Taken together, the data support a role for p53 and pRb in preventing entry into the ensuing S phase by mediating an arrest during a G1like state. p53 mediates a G1 arrest in response to DNA damage and ribonucleotide depletion, in part, by activating the cyclin dependent kinase inhibitor p21 (El-Deiry et al., 1993; Harper et al., 1993). Mouse embryonic ®broblasts lacking p21 were originally reported to arrest after microtubule disruption (Deng et al., 1995); however, subsequent reports showed that p217/7 MEFs rereplicate after exposure to nocodazole or colcemid and induction of the p21 protein occurs within 24 h in wildtype, but not p53 null MEFs (Khan and Wahl, 1998; Lanni and Jacks, 1998). This suggests that p53 mediates the nocodazole induced G1 arrest, in part, through activation of p21. Curiously, overexpression of a transactivation-de®cient form of p53 in immortal murine ®broblasts apparently can also prevent rereplication upon exposure to microtubule inhibitors, suggesting that the p53-mediated activation of p21 is not absolutely required to induce the arrest response (Notterman et al., 1998). Indeed, a loss of p53 leads to a greater proportion of polyploid cells than does p21 loss (Khan and Wahl, 1998), it is possible that the role of p53 in this response transcends p21 activation. However, it is also possible that overexpression of certain mutant forms of p53 may produce the observed microtubule inhibitor-induced cell cycle arrest by interfering with gene expression in ways that would not occur with normal levels of p53 expression. It remains to be determined if endogenous levels of transcriptionally defective mutant p53 also retain the ability to prevent rereplication in the presence of nocodazole. Mouse embryonic ®broblasts lacking both p16Ink4a and p19ARF also showed increased polyploidy after exposure to nocodazole or colcemid, suggesting that the loss of pRb and/or p53 regulation in these MEFs promotes DNA reduplication (Khan and Wahl, 1998). This raises questions about the individual roles of the tumor suppressors p16Ink4a and p19ARF in preventing rereplication. p16Ink4a may prevent S phase entry

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by inhibiting the phosphorylation of pRb which promotes the sequestration of the E2F family of transcription factors (Chellappan et al., 1991; Serrano et al., 1993). p19ARF may also promote G1 arrest in the presence of microtubule antagonists by interfering with the MDM2-mediated degradation of p53 and thereby promoting the stability of p53 (Pomerantz et al., 1998; Zhang et al., 1998). The percentage of polyploid cells is signi®cantly higher in p53 null and pRb null MEFs compared to the p16Ink4a/p19ARF double knockout MEFs (Di Leonardo et al., 1997a; Khan and Wahl, 1998) which suggests that loss of p16Ink4a and p19ARF is not functionally equivalent to loss of either p53 or pRb. A majority of normal primary human or mouse ®broblasts treated with nocodazole arrest in the ®rst G1 with 2N DNA content, suggesting that the signal for arrest is generated prior to entry into mitosis (Di Leonardo et al., 1997a; Khan and Wahl, 1998). The signal for arrest generated by microtubule disruption, therefore, appears to be independent of either aberrant mitotic spindle assembly or chromosome segregation. This raises the intriguing question of the nature of the signal generated during G1 by microtubule disruption that activates p53. The signal may be generated by aberrant centrosome duplication, disruption of organelle and protein tracking along the microtubules, changes in the equilibrium of monomeric/dimeric tubulin to polymerized tubulin, or disruption of the cell polarity in interphase cells. It remains to be determined if p53 directly senses microtubule disarray during G1 or if there are proteins upstream of p53 that sense the disorganization and relay the signal for arrest to p53. It is evident that the tumor suppressors p53 and pRb serve as a key link between cell cycle progression and the integrity of the microtubule network. Hence, loss of the ability to sense changes in the cytoskeleton may be an additional factor that contributes to the production of aneuploid variants which are so frequently detected in cells undergoing tumor progression. Linking p53 activation with its subcellular localization Although p53 gene mutation occurs in a substantial fraction of human cancers, many tumors contain p53 that is functionally inactivated through other mechanisms. One such mechanism involves aberrant subcellular localization. Constitutive cytoplasmic localization of wildtype p53 has been reported in in¯ammatory breast carcinoma (Moll et al., 1992), colorectal adenocarninoma (Bosari et al., 1995; Sun et al., 1992), undierentiated neuroblastoma (Moll et al., 1995), hepatocellular carcinoma (Ueda et al., 1995), and retinoblastoma (Schlamp et al., 1997), and is associated with tumor metastasis and poor long-term patient survival (Stenmark-Askmalm et al., 1994; Sun et al., 1992). In addition, some viruses induce uncontrolled cell proliferation through a strategy involving p53 cytoplasmic sequestration. For example, the adenovirus E1B-55-kDa protein anchors p53 in cytoplasmic structures, reducing p53 transcriptional activity (Blair Zajdel and Blair, 1988; Konig et al., 1999; Sarnow et al., 1982; Yew and Berk, 1992; Zantema et al., 1985). The hepatitis B virus HBx

protein localizes p53 to the cytoplasm and inhibits both its transcriptional activity and its ability to mediate apoptosis (Elmore et al., 1997; Feitelson et al., 1993; Ueda et al., 1995; Wang et al., 1994, 1995). Hepatocellular carcinoma is among the most common malignancies world wide, with over 80% of patients positive for Hepatitis B virus infection. As p53 mutations are found in only one-quarter to one-third of these tumors (Ueda et al., 1995 and references therein), cytoplasmic sequestration is clearly an important mechanism of inactivating p53. p53 cytoplasmic sequestration could result from its anchorage to a cytoplasmic tether or by an imbalance in nuclear-cytoplasmic shuttling. p53 has been shown to bind multiple cytoplasmic proteins (such as, ribosomes (Fontoura et al., 1997), hsc70 (Gannon and Lane, 1991), hsp84 (Sepehrnia et al., 1996) and various cytoskeletal elements (e.g. F-actin (Metcalfe et al., 1999) and vimentin (Klotzsche et al., 1998)) which may hold it in the cytoplasm and/or prevent its import into the nucleus. However, the biological relevance of the formation of these complexes remains to be determined. Alternatively, it was shown recently that in neuroblastoma cells, the rapid, energy-dependent shuttling of p53 between the nucleus and cytoplasm is altered so that hyperactive nuclear export results in net cytoplasmic accumulation (Middeler et al., 1997; Moll et al., 1995; Stommel et al., 1999). This result implies that either an upstream signaling event which results in nuclear export is constitutively active in these cells, or that signals required for nuclear retention are missing. Regardless of the mechanism, these data demonstrate that the appearance of `cytoplasmically-sequestered' p53 in these cells re¯ects the inadequacy of static microscopic observation of ®xed cells for the analysis of a dynamic process. It remains to be determined if the `cytoplasmically-sequestered' p53 in other cancers is also a result of uncontrolled, hyperactive nuclear export, or whether bona ®de cytoplasmic anchoring mechanisms exist. In normal, unstressed cells, p53 subcellular localization is variable and depends on the phase of the cell cycle. p53 is predominantly nuclear from approximately mid G1 to the G1/S transition, then becomes increasingly cytoplasmic as the cell progresses through the cell cycle (David-Pfeuty et al., 1996; Shaulsky et al., 1990a; Takahashi and Suzuki, 1994). This is consistent with the observation that p53 is predominantly cytoplasmic in mouse ES cells, which alternate between S and M phases (Aladjem et al., 1998), In response to various cytotoxic and genotoxic stresses, post-translational modi®cations increase p53 stability and lead to nuclear accumulation (Fritsche et al., 1993; Kastan et al., 1991). p53 nuclear accumulation is cell cycle-dependent for some stresses, such as g-irradiation (Komarova et al., 1997), while other stresses, such as hydrogen peroxide and heat shock, induce p53 nuclear translocation and accumulation independently of cell cycle phase (Sugano et al., 1995). The observation that the normal cell cycle controls of p53 subcellular localization are overridden by some activators implies the existence of stress-activated mechanisms for regulating p53 nuclear accumulation. Analysis of girradiated cell fusions shows that p53 nuclear accumulation is determined by a property of the nucleus, indicating that this stress may produce


modi®cations that inhibit p53 nuclear export rather than accelerate its nuclear import (Komarova et al., 1997). Therefore, tight regulation of p53 nuclear export may be crucial for determining the kinetics, duration, and magnitude of a stress response. As p53 exerts many of its eects by transcriptional regulation, its translocation from cytoplasmic sites of synthesis to nuclear target genes is critical for the elicitation of biological responses. Consequently, the failure of p53 to be retained in the nucleus should result in a defective p53 stress response. For example, neuroblastoma and mouse embryonic stem cells have cytoplasmic wildtype p53 and an impaired G1 arrest in response to genotoxic stresses (Aladjem et al., 1998; Moll et al., 1996). A temperature sensitive mutant p53 cannot suppress growth of transformed cells grown at the 37.58C, the temperature at which it is predominantly cytoplasmic, but does so eectively at 32.58C when it is nuclear (Martinez et al., 1991; Michalovitz et al., 1990). In addition, a constitutively cytoplasmic mutant derived from a Burkitt's lymphoma B-cell line cannot suppress the transformation of primary rodent ®broblasts by HPV16-E7 and H-ras, yet is fully capable of transactivating p53-responsive genes and inducing apoptosis when it is forced into the nucleus through transient overexpression (Crook et al., 1998). These results demonstrate the importance of nuclear localization for p53 activity. Some stresses, such as ribonucleotide depletion or microtubule disruption, induce a reversible p53mediated cell cycle arrest (Khan and Wahl, 1998; Linke et al., 1996). To enable renewed progression through the cell cycle, the high levels of nuclear p53 which accumulate in a stress response must be reduced. The proteasome-mediated degradation of p53 probably occurs in the cytoplasm, since the export-inhibiting drug leptomycin B stabilizes p53 (Freedman and Levine, 1998). Therefore, the nuclear export of p53 prior to its degradation in the cytoplasm may be an important component of the mechanism that regulates p53 stability. Because an MDM2 mutant defective for nuclear export is incapable of mediating the degradation of p53 (Freedman and Levine, 1998; Roth et al., 1998), p53 degradation must occur via a mechanism which depends on the export of MDM2. However, the mere co-localization of MDM2 and p53 in the cytoplasm may be insucient to induce p53 degradation, as cytoplasmic wildtype p53 in neuroblastoma cells is immune to degradation by MDM2 (Moll et al., 1996), and an MDM2 mutant defective for nuclear import cannot facilitate the degradation of p53 (Tao and Levine, 1999). It is tempting to speculate that either or both proteins must acquire a modi®cation in the nucleus that allows p53 degradation. This is consistent with the observation that constitutively cytoplasmic p53 in neuroblastoma cells is not only immune to proteasome-mediated degradation by MDM2, but also by HPV E6 (Isaacs et al., 1999). Compartmentalizing the steps that regulate p53 degradation could ensure that newly-synthesized p53 is not degraded prior to reaching the nucleus, and that the half-life of activated, nuclear p53 is prolonged when needed in a stress response. Our understanding of p53 transcriptional activation and stability has grown through years of analysis, but investigation of the factors that regulate p53 sub-

cellular localization is embryonic by comparison. As p53 is too large to passively diuse across the nuclear pore, its subcellular distribution must be mediated by the cell's protein import and export machinery. Facilitated nuclear import of p53 occurs via three lysine-rich nuclear localization signals (NLSs) in the Cterminus at amino acids 305±322 (NLS I), 369-375 (NLS II), and 379±384 (NLS III) (Liang et al., 1998; Shaulsky et al., 1990b). NLS I appears to be the primary NLS as mutation of this sequence alone abrogates p53 nuclear import. In addition, the mutation of the other two NLSs enhance the cytoplasmic sequestration engendered by the NLS I mutation, but by themselves have little eect on p53 localization (Shaulsky et al., 1990b). Little is known about how p53 nuclear import may be regulated. A protein of unknown function, spot-1, interacts with NLS I, but the signi®cance of this binding remains to be determined (Elkind et al., 1995). The cdc2/cdk2 phosphorylation site at S315 and the PCAF acetylation site at K320 both lie within NLS I, and it is not dicult to imagine that these modi®cations could aect association with a nuclear import receptor, especially since the phosphorylation of NLSs has been reported to regulate the import of other proteins such as SWI5 (Jans et al., 1995; Moll et al., 1991), Lamin B2 (Hennekes et al., 1993), v-Jun (Tagawa et al., 1995), and CaM kinase II (Heist et al., 1998). However, since an S315A mutation seems to have no eect on p53 nuclear localization (Liang et al., 1998), it remains to be determined what role these modi®cations may play. The study of p53 nuclear export has become an interesting topic of late, in part because of recent leaps in our understanding of the mechanisms of nuclear export, but also because of the discoveries of nuclear export signals (NESs) in both p53 and MDM2 (Gorlich, 1998; Roth et al., 1998; Stommel et al., 1999). p53 has a leucine-rich, rev-like NES in the Cterminus, which was shown to be functional through multiple `gold standard' assays for export activity (Stommel et al., 1999). For example, the p53 NES can mediate the movement of p53 between nuclei in a heterokaryon, it can facilitate the nuclear export of a non-shuttling heterologous protein, and its activity is sensitive to the export-inhibiting drug, leptomycin B (Stommel et al., 1999). MDM2 has a similar rev-like NES that mediates its nuclear export (Roth et al., 1998). Because MDM2 binding to p53 is necessary for p53 degradation, it was proposed that MDM2 functions as an NES-containing chaperone that mediates p53 export to cytoplasmic proteasomes (Freedman and Levine, 1998; Roth et al., 1998). However, there is currently no direct evidence that MDM2 is required for p53 nuclear export. On the contrary, p53 has been shown to be fully capable of nuclear export independently of MDM2 (Stommel et al., 1999). On the other hand, it is clear that MDM2 nuclear import and export are required for p53 degradation (Freedman and Levine, 1998; Roth et al., 1998; Tao and Levine, 1999). This is consistent with the idea proposed above that MDM2 must receive a modi®cation in the nucleus that makes it competent for p53 degradation. However, the role of MDM2 in p53 subcellular localization is still unclear. A potentially important mechanism by which both p53 subcellular localization and activation can be

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coordinately regulated involves tetramerization. The p53 NES lies within, and shares key residues with, the tetramerization domain, such that the mutation of the conserved leucines of the NES results in the abrogation of both p53 export and tetramerization (Stommel et al., 1999). Examination of three-dimensional structures of the tetramerization domain reveals that the NES is buried at the interface where two dimers combine to make the active tetramer, and that it should be solventaccessible only in the monomeric or dimeric forms (Clore et al., 1995; Jerey et al., 1995; Lee et al., 1994; Mateu and Fersht, 1998; Waterman et al., 1995). Consequently, a nuclear export receptor may be incapable of binding tetrameric p53 due to NES occlusion. Consistent with this hypothesis, a Cterminal peptide consisting of the p53 tetramerization domain inhibits the hyperactive nuclear export observed in neuroblastoma cells (Ostermeyer et al., 1996; Stommel et al., 1999). This appears to be due to the binding of the peptides to the p53 NES such that when these p53/peptide heterocomplexes enter the nucleus, they may be incapable of nuclear export because the binding surface recognized by the export receptor is hidden by the peptide. Tetrameric p53 is most eective at binding and transactivating p53 response elements, and mutations which prevent tetramerization compromise DNA binding, diminish transactivation, and lead to tumorigenesis (Friedman et al., 1993; Hainaut et al., 1994; Halazonetis and Kandil, 1993; Hupp and Lane, 1994; Ishioka et al., 1995; Lomax et al., 1998; McLure and Lee, 1998; Pietenpol et al., 1994; Tarunina et al., 1996; Varley et al., 1996). Therefore, stress-induced modi®cations which allow p53 tetramerization to occur may concurrently contribute to both the formation of DNA-binding tetramers and to their retention in the nucleus. The modi®cations which may mediate simultaneous tetramer formation and NES inhibition are unknown, but are likely to involve regulatory sites in the Cterminus. The C-terminus contains an inhibitory basic domain that may be involved in intramolecular interactions that prevent DNA binding, as peptide binding to this region, or its deletion, enhances this ability (Bayle et al., 1995; Hupp et al., 1995). Because some C-terminal modi®cations are induced by genotoxic agents (including K320 and K382 acetylation (Liu et al., 1999; Sakaguchi et al., 1998), S376 dephosphorylation (Waterman et al., 1998), and S392 phosphorylation (Kapoor and Lozano, 1998; Lu et al., 1998)), it is conceivable that they serve to neutralize the inhibitory basic domain, thus enabling p53 to bind DNA. These modi®cations may induce structural alterations which allow tetramer formation (Sakaguchi et al., 1997) and may consequently aect nuclearcytoplasmic shuttling. In addition, S376, S378, and S392 are phosphorylation sites for PKC, PKA, and CK2 respectively. Each of these kinases has been implicated in regulating nuclear-cytoplasmic shuttling of other proteins, such as diacylglycerol kinase (Topham et al., 1998), dorsal (Briggs et al., 1998; Drier et al., 1999), and SV40 T-antigen (Hubner et al., 1997). In fact, PKC inhibitors induce both p53 DNA binding and nuclear accumulation (Chernov et al., 1998), both of which correlate with tetramer formation. A number of the p53 C-terminal modi®cations mentioned above have been examined quite exten-

sively, yet much of the published data on their relevance to p53 function is con¯icting. This is in part due to the use of 32P in the assays, which could potentially damage DNA and induce modi®cations which activate p53 and complicate interpretation of the experiments. In addition, the EMSA or transactivation assays typically used to assess p53 activity may be inappropriate for determining the function of these modi®cations, as mutations at sites involved in subcellular localization may be only indirectly relevant to DNA binding. A more careful analysis of the role of these and other sites in p53 nuclear-cytoplasmic shuttling and tetramer formation may reveal how temporal and spatial regulation of p53 activity can be eectively coordinated in response to physiologically and medically relevant stresses. Additional unresolved problems p53 activates both irreversible and reversible cell cycle arrests, but the precise mechanisms leading to the appropriate response remain to be elucidated. It is possible that any stress that produces irreparable DNA damage induces cell cycle exit, while conditions such as ribonucleotide depletion or microtubule perturbation produce a reversible arrest. It is reasonable to propose that p53 activates dierent sets of downstream genes to achieve the appropriate response, but the precise spectrum of dierentially expressed genes is not yet fully known. A more immediate challenge is to identify whether dierent stresses activate p53 by producing dierent modi®cations in its primary structure. The most intensively studied stress response involving p53 is that initiated by ionizing radiation. Initially, an attractive model was that the signal transduction system involved the Ku proteins and their catalytic partner DNA-PK which phosphorylates the relevant regions of the p53 N-terminus (Lees-Miller et al., 1992; Shieh et al., 1997; Woo et al., 1998), but we now know that this model is incorrect (Jimenez et al., 1999). Rather, it seems more likely that the ATM and ATR kinases are responsible for N-terminal p53 phosphorylation following ionizing radiation. However, these proteins have no known Ku-like binding partners, and so it remains to be determined how they `sense' the presence of the relevant lesions. Furthermore, the relevance of S15 phosphorylation has now been brought into question by new evidence showing that S20 plays a more important role in controlling p53 stability (Shieh et al., 1999; Unger et al., 1999). Whether ATM and ATR act in a signal ampli®cation pathway to ensure that p53 is activated when as little as only one lesion is present is also a matter for future investigation. The mechanism by which oncogenes activate p53 is of substantial biological importance, as this stress may provide the most profound selection for loss of p53 function during cancer progression. Furthermore, as activated oncogenes can induce DNA breakage and consequent genomic instability (Denko et al., 1994; Felsher and Bishop, 1999; Fukasawa and Vande Woude, 1997), loss of p53 function in the context of an activated oncogene could signi®cantly accelerate the rate of mutation accumulation and tumor progression. It has been suggested that oncogenes activate p53 by a


mechanism that does not involve DNA damage but does involve activation of p19ARF via hyperproliferative signals (Sherr, 1998). In light of the data indicating that oncogenes can induce DNA damage, it is prudent to revisit this proposal. If no damage is involved, it will be important to ascertain whether stresses which do not induce DNA damage generally work though p19ARF to activate p53. Activation of p53 via p19ARF requires interaction of p19ARF with MDM2 (Sherr, 1998). While p53 has been the focus of studies to analyse stress induced covalent modi®cations, it is clear that these other binding partners will need to be studied with equal scrutiny for the dynamics of the activating mechanisms to be fully understood. Finally, the mechanisms that control the subcellular, or even subnuclear, localization of each of these proteins are only starting to be studied. Indeed, there are already competing hypotheses on the role and relative importance of the nuclear export signals in p53 and MDM2. While structural modifications in latent p53 are presumed to be necessary for tetramer formation and consequent occlusion of the

intrinsic p53 NES, these remain to be identi®ed, as do the modifying enzymes. How the p53 tetramer is destabilized to enable p53 nuclear exit and reversal of a stress response, and whether MDM2 collaborates in reversing p53 nuclear accumulation, are hotly debated issues. As is true in any rapidly moving ®eld, the data generated today only serve to pose more questions to be answered tomorrow. Fortunately, the emergence of many powerful analytical reagents such as sequence and conformation speci®c antibodies, and strategies such as expression arrays and high resolution microscopes, should continue to provide answers at a fast pace for the foreseeable future.

Acknowledgements This work is supported by a postdoctoral fellowship from the NIH (to GS Jimenez), a National Science Foundation Graduate Fellowship (to JM Stommel), and grants from the NIH and the G Harold and Leila Y Mathers Charitable Foundation.

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