Regulation of tumor suppressors by nuclear-cytoplasmic shuttling

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Experimental Cell Research 282 (2003) 59 – 69

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Review

Regulation of tumor suppressors by nuclear-cytoplasmic shuttling Megan Fabbro and Beric R. Henderson* Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead 2145 New South Wales, Australia Received 18 June 2002, revised version received 16 September 2002

Abstract Tumor suppressor proteins control the proliferation and survival of normal cells; consequently, their inactivation by gene mutations can initiate or drive cancer progression. Most tumor suppressors have been identified by genetic screening, and in many cases their function and regulation are poorly understood. Ten such proteins were recently shown to contain nuclear transport signals that facilitate their “shuttling” between the nucleus and cytoplasm. This type of dynamic intracellular movement not only regulates protein localization, but also often impacts on function. Here, we review the pathways by which tumor suppressors such as APC, p53, VHL, and BRCA1 cross the nuclear envelope and the impact of regulated nuclear import/export on protein function. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Nuclear transport; Nuclear export; Tumor suppressors; Cancer

Introduction Over the past few years, many fields of biological research have converged in the study of nucleocytoplasmic transport, providing exciting and highly significant advances in our understanding of protein subcellular localization and the mechanisms by which proteins “shuttle” between the nucleus and cytoplasm [1– 4]. Protein transport in either direction across the nuclear envelope involves sequential steps. These include: (a) recognition of the protein import/export signal by an import or export receptor, (b) docking of the protein/receptor assembly at the nuclear pore complex, (c) translocation across the nuclear pore, (d) release of the transported protein, and (e) recycling of transport factors for continued cycles of transportation [1,2]. Although broadly defined, each of the above steps is complex and involves the intricate interplay of multiple protein components. Nuclear transport is proving to be a fundamental and critical mechanism for regulating not only protein localization, but also protein function [5]. Therefore, it is hardly surprising that deregulation of nuclear transport is implicated in the mislocalization and altered function of a variety of proteins, in-

* Corresponding author. Fax: ⫹61-2-9845-9102. E-mail address: [email protected] (B.R. Henderson).

cluding tumor suppressors. The mistargeting of tumor suppressors can have dire cellular consequences and potentially lead to the initiation and progression of cancer. Cancer is one of the leading causes of mortality in western countries. Many hereditary cancers are initiated by germline mutations that target one allele of a tumor suppressor gene, and tumor development in these cancer patients typically results from either somatic loss or inactivation of the remaining wild-type allele, as proposed originally by Knudson’s “two-hit” hypothesis [6,7]. Over 30 different tumor suppressor genes have been identified and shown to exhibit germline mutations (and often mutational inactivation) in various human cancers. Some tumor suppressor genes encode proteins of such basic importance to normal cell growth and survival that their deficiency contributes to several tumor types; one such gene is p53 [8]. The normal role of most tumor suppressor genes involves regulation of the cell’s response to growth stimuli, DNA damage, and cell cycle checkpoints, as defects in the control of these mechanisms may contribute to the initiation or progression of cancer. Ten different tumor suppressors are regulated by nuclear cytoplasmic shuttling (Tables 1 and 2), and aberrations in the nuclear transport pathways can cause cytoplasmic mislocalization (e.g., for p53 [9] and BRCA1 [10,11]) in some tumors. Here, we discuss the

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Table 1 The nuclear localization sequences and import pathways utilized by shuttling tumor suppressor proteins Tumor suppressor p53

p73 Beclin BRCA1

Steady-state localization Nuclear

Nuclear Cytoplasmic Nuclear

APC

Cytoplasmic

VHL PML Smad4 p130

Cytoplasmic Nuclear Cytoplasmic Nuclear

INI1/hSNF5

Nuclear

Nuclear import

References

Import elements

Sequence

Bipartite NLS1 NLS2 NLS3 Bipartite NLS Not defined NLS1 NLS2 RING domain NLS1 NLS2 ARM repeats Bipartite NLS n.d. NLS NLS1 NLS2 NLS3 Pocket domain n.d.

305

Receptor/pathway

KRALPNNTSSSPQPKKKP LKSKKGQ375 379 RHKKLM384 327 KRAFKQSPPAVPALGAGVKKRR348

Importin-␣/␤ Importin-␣/␤ Importin-␣/␤ Importin-␣/␤

503

KRKRRP508 PKKNRLRRKS615 a.a. 1–109 1787 GKKKKP1772 2048 PKKKKP2053 a.a. 302–625 1 PRR(X)53RPRPV60

Importin-␣/␤ Importin-␣/␤ BARD1 Importin-␣/␤ Importin-␣/␤ B56␣ Importin-␣/␤

37

Importin-␣/␤ Importin-␣/␤ Importin-␣/␤ Importin-␣/␤ Not defined

322

369

606

KRAIESLVKKLKEKKDELD55 PSKRLRE1087 1098 PTKKRGI1104 935 KRKRR939 a.a. 417–1024 1081

[15] [12] [12] [22] [99] [20, 21, 100] [20, 21] [29] [19] [19] [26] [23] [101] [25] [24] [24] [24] [24] [59]

Note. Critical amino acids are shown in boldface.

subtle levels at which shuttling itself is regulated and how altered tumor suppressor localization may impact on cancer.

with recent findings on other tumor suppressors. The C-terminus of the p53 tumor suppressor contains two basic nuclear localization signals (NLSs, see Table 1) [12], similar to those first identified in SV40 large T antigen [13] and nucleoplasmin [14]. They consist of very short amino acid stretches rich in basic positively charged amino acid residues, such as lysine and arginine [13]. p53 also contains another type of NLS at its C-terminus (a bipartite NLS), comprising two basic clusters separated by 10 –12 variable amino acids [15]. All of these nuclear import sequences are recognized by an NLS receptor (a heterodimeric complex composed of importin ␣ and ␤) [16,17] that facilitates the nuclear import of p53 driven by the

Tumor suppressor nuclear transport pathways Nuclear import NLS-mediated nuclear import p53 is arguably the best studied tumor suppressor protein, and thus much of our discussion will at least partly revolve around the regulation of p53 transport and how this compares Table 2 The nuclear export sequences and export pathway utilized by tumor suppressors Tumor suppressor p53 p73 Beclin BRCA1 APC

VHL PML Smad4 p130 INI1/hSNF5

Nuclear export

References

Export elements

Sequence

NES1 NES2 NES NES NES NES1 NES2 NES3 NES4 NES5 Exon 2 NES NES NES NES

11

Receptor/ pathway 27

EPPLSQETFSDLWKLLP EMFRELNEALELKD352 387 ILMKLKESLELM378 180 LQMELKELALE190 81 QLVEELLKIICAFQLDTGL99 63 SGQIDLLERLKELNLDSSN81 161 QLQNLTKRIDSLPLTENF178 1494 ESTPDGFSCSSSLSALSLDEP1514 1545 EGTPINFSTATSLSDLTIESP1565 2015 EDTPVCFSRNSSLSSLSIDSE2035 a.a. 114–154 700 FQEAISGFLAALPLIRER717 138 SPGIDLSGLTLQ149 217 MISDDLVNSYHLLLCALDLVYG236 266 LNIMVGNISLV278 339

Note. Critical amino acids are shown in boldface.

CRM1 CRM1 CRM1 CRM1 CRM1 CRM1 CRM1 CRM1 CRM1 CRM1 CRM1-independent pathway CRM1 CRM1 CRM1 CRM1

[60] [54] [22] [99] [102] [74, 103] [74, 103] [76] [76] [76] [45] [33] [25, 104] [24] [59]

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cycling of GTP hydrolysis involving the small GTPase, Ran [18]. The two types of NLS described above are also found in many other tumor suppressors, namely APC [19], BRCA1 [20,21], p73 [22], VHL [23], p130 [24], and Smad4 [25] (see Table 1), and are important for targeting these proteins to the nucleus via the importin-␣/␤ receptor pathway. NLS-independent nuclear import Although NLSs are the most common type of nuclear import element, other sequences are often important for targeting certain proteins to the nucleus. For example, APC and BRCA1 were recently shown to enter the nucleus via pathways that are quite independent of their NLSs. In the case of the colon cancer-associated protein, APC, the majority of cancer mutations result in deletion of the centrally located NLSs, and yet these truncated forms of the protein can still enter the nucleus very efficiently. An additional nuclear import sequence was subsequently mapped to the N-terminal ARM domain of APC [26], a sequence comprising seven armadillolike repeats similar to those identified in the APC-binding partner, ␤-catenin [27]. Interestingly, its association with B56␣, a regulatory subunit of protein phosphatase 2A, stimulated ARM-dependent nuclear localization of APC and it was proposed that B56␣ functions as a nuclear chaperone [26]. The ARM repeats of ␤-catenin can mediate nuclear import independent of the importin receptors [28], suggesting a similar possibility for the APC ARM domain. The breast cancer-linked tumor suppressor, BRCA1, which is equipped with two centrally located NLSs, nevertheless can enter the nucleus in an NLS-independent manner as described above for APC. The additional nuclear targeting element of BRCA1 was identified as the N-terminal RING-finger domain, the deletion of which caused nuclear exclusion of BRCA1 [29]. This was the first report of a zinc-finger-like domain functioning as a nuclear targeting signal and is potentially important given that many other nuclear proteins, including p53, also contain RING domains. The BRCA1 RING sequence constitutes the major part of a binding site for the BRCA1 binding partner, BARD1 [30,31]. In transfected breast cancer cells, the coexpression of BARD1 induced the nuclear accumulation of not only wild-type BRCA1, but also NLS-deficient forms of BRCA1, and appears to carry BRCA1 into the nucleus via a “piggy-back” mechanism [29]. This alternative transport route was found to be functionally important in recruiting BRCA1 to specific nuclear foci associated with DNA repair, in particular for those alternatively spliced variants of BRCA1 which lack nuclear localization signals and absolutely require BARD1 for nuclear entry [29]. Nuclear export CRM1-dependent nuclear export The protein sequences responsible for nuclear export are quite different from those used for import, and they bind a different type of transporter molecule. The most common

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type of nuclear export signal (NES) is contained within a short peptide sequence with closely spaced large hydrophobic amino acids (L x(1–3) L x(2– 4) L x L), in particular leucine or isoleucine [32,33], that are recognized and bound by the export receptor, CRM1/exportin [34 –37]. The interaction between an NES and CRM1 is stabilized in a trimeric complex following association with RanGTP [34]. The first protein found to be exported through the CRM1-dependent pathway was the HIV regulatory protein, Rev [38]. Since the identification of the Rev NES, similar export sequences have been identified in dozens of cellular proteins with diverse functions. A critical reagent enabling the identification of NES-containing proteins was the CRM1-specific inhibitor, leptomycin B [39,40]. Nine of the 10 tumor suppressors whose localization is regulated by shuttling between the nucleus and cytoplasm have been reported to contain at least one Rev-like NES (see Table 2). CRM1-independent nuclear export In the nuclear export field, the CRM1 protein remains the best studied and most important exporter molecule yet identified. However, other nuclear export sequences have been mapped in a small group of proteins, including the RNA processing molecules hnRNP A1 [41], hnRNP K [42], and Hur [43]. The sequences identified in these proteins are quite large and poorly characterized, although it is known that they do not respond to leptomycin B and thus do not act through the CRM1 pathway [33]. In addition to CRM1, a second RanGTP-associated nuclear export receptor was recently discovered and identified as the calcium-regulated protein, calreticulin [44]. The role of calreticulin in nuclear export is less well defined, and while it can bind to some CRM1-binding sequences [44], its range of target sequences is not yet known. Only one tumor suppressor, the von Hippel Lindau (VHL) protein, appears able to exit the nucleus independent of CRM1. VHL nuclear export is facilitated by exon 2 sequences; however, it does not contain a functional Rev-type NES [45]. The VHL nuclear export pathway involves an active process that, like the CRM1 pathway, is driven by ATP hydrolysis and requires the Ran-GTPase [46]. It would be extremely interesting to test the possible involvement of calreticulin in VHL nuclear export, or indeed the export of other tumor suppressors.

Regulation of nuclear transport and function Tumor suppressor proteins are regulated by a number of different strategies to avoid abnormal cellular growth. Many of their functions are localized within a specific cellular compartment; therefore, the regulation of tumor suppressor subcellular localization can impact on their activity. Below, we discuss various mechanisms for regulating tumor suppressor localization and activity by altering nuclear transport.

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Table 3 Regulation of nuclear transport of tumor suppressor proteins Tumor suppressor

Binding partners

Posttranslational modification

Cancer mutations

Retention

Cell adhesion

p53

HDM2 mediates nuclear export by ubiquitinating p53 [63, 64]; tetramerization of p53 masks NES2 [54]

DNA damage induced phosphorylation inhibits NES1 activity [60]; ubiquitination enhances export [65,66]

Cancer mutations cause defects in p53 nuclearcytoplasmic shuttling [105]

Not defined

BRCA1

BARD1 mediates nuclear import and enhances foci formation [29]

Not defined

Exon 11 splice variants reduce import [21]

APC

B56␣ stimulates APC nuclear import [26]

Phosphorylation at either CK2 site or PKA site [19]

VHL

Not defined

Not defined

INI/hSNP5

Not defined

Not defined

Truncating colon cancer mutations do not impair nuclear shuttling [26] Renal cancer mutations target exon 2 sequences implicated in export [45,91] C-terminal truncations unmask the NES [59]

c-Abl1, Hdm2 cancer mutants and p14ARF sequesters HDM2 preventing nuclear export of p53 [63, 64]; cytoplasmic sequestration by the glucocortoid receptor [81] BARD1 masks the BRCA1 NES retaining BRCA1 in the nucleus [29] Cytoplasmic retention when associating with microtubules [78, 80]

Masking of transport signals The masking of nuclear import or export signals, either by changes in protein folding/conformation or by binding of another protein, provides a quick and efficient mechanism for preventing signal recognition by the nuclear transport machinery. The masking of nuclear export signals by protein cofactors was previously shown to regulate localization of the transcription factors NF-AT [47] and PBX1/EXD [48], whose export signals are inaccessible to CRM1 following association with calcineurin and PREP1/HTH, respectively. Recently, the nuclear export of p53, BRCA1, and INI1 has been shown (or proposed) to be regulated by NES masking (Table 3). p53 NES masking The p53 protein is regulated by cytoplasmic degradation and is most often detectable within the nucleus, where it functions as a transcription factor involved in maintaining genomic integrity by controling cell cycle progression and cell survival [8]. In response to cellular stresses such as DNA damage, p53 becomes transiently stabilized and translocates to the nucleus. Nuclear entry of p53 is generally NLS-dependent, but is also stimulated by its association with microtubules, mediated by the minus-end-directed microtubule motor, dynein [49] (see Fig. 1). Genotoxic stress induces p53 to activate a suite of genes that facilitate cell growth arrest or apoptosis, including the cyclin-dependent kinase inhibitor, p21 [50,51], the pro-apoptotic factor, bax [52], and the oncogene Hdm2 [53]. Within the nucleus, p53 forms a tetrameric complex, which is transcriptionally ac-

Not defined

Cell density [77]

Not defined

Cell density [23]; RNA Pol II activity stimulates VHL export [45]

Not defined

Not defined

tive. It was hypothesized that tetramer formation of p53 masks the C-terminal NES, thus trapping p53 in the nucleus and increasing its overall transcriptional activity [54]. More recent findings question this notion (see below). BRCA1 NES masking We described above how the RING domain of BRCA1 acts as a nuclear localization element by mediating association with the binding partner, BARD1 [29]. The recently solved structure of the BRCA1/BARD1 heterodimer predicts that the BRCA1 NES lies within the binding interface between these two RING-domain proteins [31]. This raised the possibility that BARD1 binding masks the BRCA1 NES, leading to inhibition of CRM1-mediated nuclear export of BRCA1. Indeed, this idea was confirmed by showing that BARD1 anchors BRCA1 in the nucleus only when the NES is positioned adjacent to the RING domain and not when it is relocated to the C-terminus [29]. These findings have implications for the role of BRCA1 in DNA repair, as the coexpression of BARD1 enhances BRCA1 nuclear localization and nuclear foci formation, particularly in response to DNA damage [29]. Such BRCA1-positive nuclear foci are associated with DNA repair [55]. More than 75% of BRCA1 is thought to be complexed with BARD1 in living cells [56]. The finding that BARD1 can import and trap BRCA1 in the nucleus explains why BRCA1 is so frequently detected in the nucleus, even though it contains an export signal. It further implies that in order to escape from the nucleus, BRCA1 must disengage from BARD1, and that signaling mechanisms likely exist for this purpose. The negative regulation of BRCA1 shut-

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Fig. 1. Factors involved in the regulation of nuclear-cytoplasmic shuttling of the tumor suppressors p53, BRCA1, and APC. p53 (dark gray ellipse), BRCA1 (light gray ellipse) and APC (black ellipse) can shuttle between the nucleus and cytoplasm of cells and are regulated by various factors as shown. NLS, white triangle. NES, gray triangle. RING domain, white oval. ARM domain, white square. C.S.D., cytoplasmic sequestration domain. ⫹, positive regulators. ⫺, negative regulators.

tling has functional implications. For example, it may explain why BRCA1 and BARD1, when bound together, are less prone to cytoplasmic degradation [57], and it further implies that protein substrates targeted by the BRCA1/ BARD1 E3 ubiquitin ligase activity may be predominantly nuclear. The impact of BARD1-mediated nuclear retention on other BRCA1 cellular activities remains to be defined. Furthermore, it is not known whether other RING domain binding proteins such as BAP1 also inhibit the nuclear export of BRCA1. These examples highlight the importance of NES masking in regulating protein function. Certain other tumor suppressors, such as the INI1 protein (see below), may also be regulated by NES masking. Effect of cancer-associated mutations on nuclear import or export Since tumor suppressors are, by definition, proteins whose function is altered by genetic mutation in cancer cells, we will briefly outline the known effects of clinicallyrelevant mutations on tumor suppressor transport and localization. First, the BRCA1 protein was reported some years ago to mislocalize to the cytoplasm in breast cancer cells, but not in normal cells [10,11]. Did these controversial observations

reflect a possible mutation-induced shift in the balance of nuclear import/export rates for BRCA1? Although most laboratories now agree that BRCA1 is predominantly detected in the nucleus, a systematic analysis of the nuclear transport and localization of various BRCA1 mutants has revealed that a subset of C-terminal truncations actually impair BRCA1 nuclear import in breast cancer cells, but do not affect nuclear export (Rodriguez and Henderson, unpublished data). The ability of cancer mutations to deregulate both correct nuclear targeting and nuclear functioning seems highly effective and may apply to other types of tumor suppressors. On the other hand, even the most deleterious cancer-linked truncations of APC that remove more than 80% of the peptide sequence do not seem to disrupt its ability to move in or out of the nucleus, as shown recently for both transiently expressed and endogenous forms of APC in colon cancer cells [26]. It is possible, therefore, that nuclear-cytoplasmic shuttling of APC is integral to cell survival and that impairment of its trafficking is toxic to cells. INI1 (integrase interactor 1)/hSNF5 is a tumor suppressor and forms a key component of the SWI/SNF chromatin remodeling complex [58]. Like BRCA1 and p53, INI1 is most frequently detected in the nucleus of cells [59], but contains a nuclear export signal that mediates CRM1-de-

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pendent export [59]. There is evidence to suggest that INI1 nuclear transport is tightly regulated by NES masking, ensuring that INI1 localizes to the nucleus where it is associated with cell cycle arrest, an important aspect of its tumor suppressor function. Interestingly, an INI1 cancer mutant (found in malignant rhabdoid tumors) is predominantly localized to the cytoplasm due to unmasking of the export signal, which apparently results from deletion of the Cterminal 66 amino acids of INI1 [59]. These results suggest that a conformational change caused by deletion of the C-terminus, exposes the INI1 export signal, and stimulates INI1 movement to the cytoplasm. This INI1 cancer mutant is no longer able to induce cell cycle arrest [59]. These examples, which represent only those few proteins so far analyzed, reveal the profound functional effects mediated by cancer mutations through alterations in protein localization. Modification of protein transport signals The nuclear translocation of proteins can also be altered following the posttranslational modification of specific sequences within or proximal to nuclear import/export signals. The changes, which potentially include phosphorylation, ubiquitinylation, glycosylation, and sumoylation, can result from a variety of signaling pathways [5]. In cancer, the nuclear shuttling ability of two tumor suppressors, p53 and APC, is regulated by phosphorylation and/or ubiquitinylation. As mentioned previously, p53 is anchored in the nucleus following DNA damage, and this block to export was thought to partly reflect masking of a C-terminal NES following tetramer formation [54]. This mechanism, however, does not account for the action of a recently identified N-terminal NES in p53 [60]. The new findings suggest that following DNA damage, two serine residues (S15 and S20) located within the N-terminal NES become phosphorylated and that this phosphorylation event blocks p53 nuclear export [60]. The nuclear-cytoplasmic shuttling of p53 is also regulated by a different type of amino acid modification: ubiquitinylation. Hdm2 is a critical mediator of p53 nuclear export and stability. Hdm2 promotes degradation of p53 by causing its ubiquitinylation in unstressed cells [8], thereby down-regulating its growth inhibitory activity. The Hdm2dependent degradation of p53 is stimulated by nuclear export, however, while this requires an association between p53 and Hdm2, Hdm2 does not physically carry p53 out of the nucleus as was previously thought [61,62]. The Hdm2dependent export of p53 requires an intact p53 NES, but not the Hdm2 NES [63,64]. Moreover, a RING domain mutant of Hdm2 that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that Hdm2 ubiquitin ligase activity is required for the Hdm2-mediated p53 export and degradation. Indeed, other studies have since revealed that Hdm2 ubiquitinylation of the C-terminus of p53 enhances nuclear export [65,66]. Furthermore, the ubiquitinylation of

several lysine residues in the p53 C-terminus, as well as lysine 305, was required to expose the NES even when p53 was bundled as a tetramer [65]. These findings indicate that p53 ubiquitinylation induces conformational changes that affect p53 nuclear export. Several other posttranslational modifications of p53 have also been described, such as sumoylation [67], acetylation [68], glycosylation [69], and ribosylation by PARP [70,71], which may also participate in regulating p53 nuclear transport [72]. APC is most frequently detected within the cytoplasm of cells; however, APC is capable of entering the nucleus [73,74] where it is known to associate with, and export, the oncogenic transcriptional activator, ␤-catenin [74 –76]. APC nuclear import is mediated by two NLSs found within the central portion of the protein and by an N-terminal ARM domain. The APC NLSs appear to be regulated by phosphorylation at potential casein kinase 2 and protein kinase A sites which flank the NLS sequences [19,77]. Phosphorylation of the casein kinase 2 site increased nuclear import of APC NLS-␤-galactosidase constructs, whereas phosphorylation of the protein kinase A site decreased the import of such constructs [77]. The impact of phosphorylation on nuclear transport of full-length APC remains to be determined. Nuclear or cytoplasmic anchorage The subcellular positioning of proteins can also be affected by nuclear or cytoplasmic sequestration. This form of regulation, leading to immobilization of a protein within a specific cellular compartment, influences the nuclear-cytoplasmic shuttling of APC and p53 (Fig. 1). The C-terminus of APC (which is deleted by most APC cancer mutations) exhibits a tight association with cytoskeletal microtubules [78,79]. This association may be important for anchoring APC in the cytoplasm and in turn is linked to the proposed role of APC in stabilizing the growing plus-ends of microtubule fibers [80]. Similarly, p53 association with the glucocortoid receptor results in its cytoplasmic anchorage [81]. It is possible that other shuttling tumor suppressors are also sequestered in the cytoplasm by association with binding partners. For example, the BRCA1 nuclear localization signals are in some instances bound by BRAP2, a cytoplasmic protein that may contribute to the cytoplasmic sequestration of BRCA1 [82]. Furthermore, reports suggest the presence of a cytoplasmic sequestration domain (CSD) in the Cterminus of p53 that inhibits importin-␣ association with the dominant C-terminal NLS1 [83,84], preventing p53 nuclear import. A number of factors also control nuclear retention of p53, most often through regulation of its association with Hdm2. Following cellular stress, the ability of Hdm2 to bind p53 is impaired, preventing p53 export and degradation and increasing its stability and activity. Similar effects are elicited after DNA damage by c-Abl, and even by Hdm2 splice variants (lack p53 binding domain) that are commonly

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Fig. 2. Regulating nuclear function or cytoplasmic turnover by nuclear export. The transcription factors or activators, HIF␣, ␤-catenin, and p53, are regulated by nuclear export and proteasomal degradation by their shuttling partners, VHL, APC, and Hdm2, respectively. Whereas current evidence suggests that Hdm2 and VHL stimulate export/degradation of p53 and HIF␣ by promoting their ubiquitinylation, it is thought that APC directly binds and exports ␤-catenin from nucleus to cytoplasm, where it is then phosphorylated prior to ubiquitinylation and degradation. It remains unclear whether the Hdm2/p53 or VHL/HIF␣ protein pairs can exit the nucleus as a complex.

found in cancers [85,86]. The nucleolar tumor suppressor p19ARF increases p53 stability and nuclear activity in two ways; by sequestering Hdm2 to the nucleolus [87] and by inhibiting Hdm2-mediated ubiquitination of p53 [88]. Alternatively, binding to the retinoblastoma protein (Rb) results in formation of a trimeric Rb/Hdm2/p53 complex in which p53 transcriptional activity is decreased, but p53induced apoptosis is increased [89]. Cell signaling Various cellular signals can influence protein targeting and localization (see Table 3). For instance, APC localization is apparently influenced by cell density. APC was reported to be nuclear in subconfluent epithelial cells, but less nuclear in superconfluent cultures due to impaired nuclear import or enhanced cytoplasmic retention [77]. Putative casein kinase 2 and protein kinase A phosphorylation sites were proposed to control the density-dependent localization of APC [77]. Changes in cell density were reported to have a similar impact on VHL localization. Lee et al. observed that VHL is predominantly nuclear in sparse cell

cultures, whereas in confluent cultures it shifts to the cytoplasm [23]. However, it is unclear what mechanisms are required to relocalize VHL in response to cell density signals. Recently it was shown that in confluent cells, expression of VHL results in a reduction in cell growth [90], indicating that cytoplasmic localization of VHL could be important for VHL-mediated cell growth inhibition. VHL nuclear transport is also regulated by RNA polymerase II activity. In this regard, inhibition of RNA polymerase II decreases VHL nuclear export [45], whereas active RNA polymerase II causes VHL to be exported to the cytoplasm by exon2 sequences [45]. Interestingly, these sequences are frequently targeted by mutations in human renal clear cell carcinoma [91], raising the possibility that VHL mutations may modulate its localization and activity.

Link between nuclear export and protein degradation p53 and Hdm2 provide the best characterized examples of how regulated nuclear shuttling can impact on protein activity. p53 functions as a transcriptional regulator in the

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nucleus, affecting multiple pathways that collectively influence the maintenance of genomic integrity [8]. This activity is controled in normal cells by Hdm2 (outlined above), which tightly regulates nuclear p53 levels by promoting nuclear export and cytoplasmic turnover (see Fig. 2). There are some striking similarities between this aspect of p53/ Hdm2 regulation and the regulation of other tumor suppressors/oncogene pairs, in particular APC and ␤-catenin. In this latter case, oncogenic ␤-catenin is the transcriptional activator (up-regulating genes such as c-myc and cyclin D1) whose activity is down-regulated by the tumor suppressor, APC [92]. Like Hdm2, APC stimulates the nuclear export and degradation of ␤-catenin [74,75] (see Fig. 2). However unlike Hdm2, it is thought that APC can bind to and ferry ␤-catenin from nucleus to cytoplasm for degradation, since mutagenesis of the APC NES reduces the rate of degradation of ␤-catenin [74]. Other shuttling tumor suppressors act in a similar manner. The tumor suppressor, VHL, when complexed with elongin B, elongin C, and cullin-2, exhibits E3 ubiquitin ligase activity and is capable of mediating the ubiquitinylation and subsequent degradation of the transcription factor, hypoxia inducible factor ␣ (HIF␣) under “normoxia,” or normal oxygen conditions [93], whereas during “hypoxia,” HIF␣ becomes stabilized and activates gene transcription in the nucleus. The VHL-mediated degradation of HIF␣ requires a direct association between the two proteins, as indicated by the finding that a VHL cancer mutant unable to bind HIF␣ consequently does not affect its ubiquitinylation and degradation [93]. Moreover, as shown for the Hdm2/ p53 and APC/␤-catenin pairings, it does appear that upon reoxygenation, VHL shuttling activity stimulates HIF␣ degradation by promoting its export from nucleus to cytoplasm [94] (see Fig. 2). There is a striking analogy here between p53 and HIF-␣, in that both proteins must be ubiquitinylated in order to gain passage from the nucleus. It is therefore possible that ubiquitin modification tags proteins for both nuclear export and proteasome-mediated degradation. It is not yet known whether ubiquitination of ␤-catenin influences its egress from the nuclear compartment. BRCA1 also exhibits E3 ubiquitin ligase activity, which is substantially enhanced following its heterodimerization with BARD1 [95–97]. Although no target substrates of BRCA1/BARD1 ligase activity have been identified, it does appear that upon association, BRCA1 and BARD1 themselves become ubiquitinylated [98]. Therefore, we speculate that upon dissociation from BARD1, the ubiquitinylated form of BRCA1 is then exported to the cytoplasm and degraded by the proteasome complex. This is consistent with reports that BRCA1/BARD1 complexes are restricted to the nucleus [29] and are more stable than free BRCA1 or BARD1 [57] and may prove an important mechanism for regulating the transcriptional and DNA repair activities of BRCA1 [57], because many tumor suppressors function in the nucleus but are regulated at the level of degradation, it

should prove profitable to test other tumor suppressors for links between ubiquitinylation and nuclear export.

References [1] E.A. Nigg, Nucleocytoplasmic transport: Signals, mechanisms and regulation, Nature 386 (1997) 779 –787. [2] D. Gorlich, Transport into and out of the cell nucleus, EMBO J. 17 (1998) 2721–2727. [3] J. Moroianu, Nuclear import and export: Transport factors, mechanisms and regulation, Crit. Rev. Euk. Gene Exp. 9 (1999) 89 –106. [4] D.A. Jans, C.-Y. Xiao, M.H.C. Lam, Nuclear targeting signal recognition: A key control point in nuclear transport? BioEssays 22 (2000) 532–544. [5] J.K. Hood, P.A. Silver, Diverse nuclear transport pathways regulate cell proliferation and oncogenesis, Biochim. Biophysi. Acta 1471 (2000) M31–M41. [6] A.G. Knudson, Antioncogenes and human cancer, Proc. Natl. Acad. Sci. USA 90 (1993) 10914 –10921. [7] B. Vogelstein, K.W. Kinzler, The multistep nature of cancer, Trends Genet. 9 (1993) 138 –141. [8] D.B. Woods, K.H. Vousden, Regulation of p53 function, Exp. Cell Res. 264 (2001) 56 – 66. [9] U.M. Moll, G. Riou, A.J. Levine, Two distinct mechanisms alter p53 in breast cancer: Mutation and nuclear exclusion, Proc. Natl. Acad. Sci. USA 89 (1992) 7262–7266. [10] Y. Chen, C.-F. Chen, D.J. Riley, D.C. Allred, P.-L. Chen, D.V. Hoff, C.K. Osbome, W.-H. Lee, Aberrant subcellular localization of BRCA1 in breast cancer, Science 270 (1995) 789 –791. [11] J. Taylor, M. Lymboura, P.E. Pace, R.P.A. Hem, A.J. Desai, S. Shousha, R.C. Coombes, S. Ali, An important role for BRCA1 in breast cancer progression is indicated by its loss in a large proportion of non-familial breast cancers, Int. J. Cancer 79 (1998) 334 – 342. [12] G. Shaulsky, N. Goldfinger, A. Benzeev, V. Rotter, Nuclear accumulation of p53 protein is mediated by several nuclear-localization signals and plays a role in tumorigenesis, Mol. Cell. Biol. 10 (1990) 6565– 6577. [13] D. Kalderon, B.L. Roberts, W.D. Richardson, A.E. Smith, A short amino acid sequence able to specify nuclear location, Cell 39 (1984) 499 –509. [14] C. Dingwall, R.A. Laskey, Nuclear targeting sequences—A consensus? Trends Biol. Sci. 16 (1991) 178 –181. [15] S.-H. Liang, M.F. Clarke, A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain, J. Biol. Chem. 274 (1999) 32699 –32703. [16] A.H. Corbett, P.A. Silver, Nucleocytoplasmic transport of macromolecules, Microbiol. Mol. Biol. Rev. 61 (1997) 193–211. [17] L.F. Pemberton, G. Blobel, J.S. Rosenblum, Transport routes through the nuclear pore complex, Curr. Opin. Cell Biol. 10 (1998) 392–399. [18] M.S. Moore, G. Blobel, The GTP-binding protein Ran/TC4 is required for protein import into the nucleus, Nature 365 (1993) 661– 663. [19] F. Zhang, R.L. White, K.L. Neufeld, Phosphorylation near nuclear localization signals regulates nuclear import of adenomatous polyposis coli protein, Proc. Natl. Acad. Sci. USA 97 (2000) 12577– 12582. [20] C.-F. Chen, S. Li, Y. Chen, P.-L. Chen, Z.D. Sharp, W.-H. Lee, The nuclear localization sequences of the BRCA1 protein interact with the importin-␣ subunit of the nuclear transport signal, J. Biol. Chem. 271 (1996) 32863–32868. [21] S. Thakur, H.B. Zhang, Y. Peng, H. Le, B. Carroll, T. Ward, J. Yao, L.M. Farid, F.J. Couch, R.B. Wilson, B.L. Weber, Localization of

M. Fabbro, B.R. Henderson / Experimental Cell Research 282 (2003) 59 – 69

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

BRCA1 and a splice variant identifies the nuclear localization signal, Mol. Cell. Biol. 17 (1997) 444 – 452. Inoue, T., Stuart, J., Leno, R. and Maki, C.G. (2002). Nuclear import and export signals in control of the p53-related protein p73. J. Biol. Chem.277, 15053–15060. S. Lee, D.Y.T. Chen, J.S. Humphrey, J.R. Gnarra, W.M. Linehan, R.D. Klausner, Nuclear/cytoplasmic localization of the von HippelLindau tumor suppressor gene product is determined by cell density, Proc. Natl. Acad. Sci. USA 93 (1996) 1770 –1775. A. Chestukhin, L. Litovchick, K. Rudich, J.A. DeCaprio, Nucleocytoplasmic shuttling of p130/RBL2: Novel regulatory mechanism, EMBO J. 22 (2002) 453– 468. C.E. Pierreux, F.J. Nicolas, C.S. Hill, Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus, Mol. Cell Biol. 20 (2000) 9041–9054. M.A. Galea, A. Eleftheriou, B.R. Henderson, ARM domain-dependent nuclear import of adenomatous polyposis coli protein is stimulated by the B56␣ subunit of protein phosphatase 2A, J. Biol. Chem. 276 (2001) 45833– 45839. M. Peifer, S. Berg, A.B. Reynolds, A repeating amino acid motif shared by proteins with diverse cellular roles, Cell 76 (1994) 789 – 791. F. Fagotto, U. Gluck, B.M. Gumbiner, Nuclear localization signalindependent and importin/karyopherin-independent nuclear import of beta-catenin, Curr. Biol. 8 (1998) 181–190. M. Fabbro, J.A. Rodriguez, R. Baer, B.R. Henderson, BARD1 induces BRCA1 intranuclear foci formation by increasing RINGdependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export, J. Biol. Chem. 277 (2002) 21315–21324. L.C. Wu, Z.W. Wang, J.T. Tsan, M.A. Spillman, A. Phung, X.L. Xu, M.C. Yang, L.Y. Hwang, A.M. Bowcock, R. Baer, Identification of a RING protein that can interact in vivo with the BRCA1 gene product, Nat. Genet. 14 (1996) 430 – 440. P.S. Brzovic, P. Rajagopal, D.W. Hoyt, M.C. King, R.E. Klevit, Structure of a BRCA1–BARD1 heterodimeric RING–RING complex, Nat. Struct. Biol. 8 (2001) 833– 837. A.M. Bogerd, R.A. Fridell, R.E. Benson, J. Hua, B.R. Cullen, Protein sequence requirements for function of the human T-cell leukemia cirus type 1 Rex nuclear export signal delineated by a novel in vivo randomization selection assay, Mol. Cell. Biol. 16 (1996) 4207– 4214. B.R. Henderson, A. Eleftheriou, A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals, Exp. Cell Res. 256 (2000) 213–224. M. Fornerod, M. Ohno, M. Yoshida, I.W. Mattaj, CRM1 is an export receptor for leucine-rich nuclear export signals, Cell 90 (1997) 1051–1060. B. Ossareh-Nazari, F. Bachelerie, C. Dargemont, Evidence for a role of CRM1 in signal-mediated nuclear protein export, Science 278 (1997) 141–144. K. Stade, C.S. Ford, C. Guthrie, K. Weis, Exportin (Crm1p) is an essential nuclear export factor, Cell 90 (1997) 1041–1050. M. Fukuda, S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, E. Nishida, CRM1 is responsible for intracellular transport mediated by the nuclear export signal, Nature 390 (1997) 308 –311. U. Fischer, J. Huber, W.C. Boelens, L.W. Mattaj, R. Luhrmann, The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs, Cell 82 (1995) 475– 483. B. Wolff, J.J. Sanglier, Y. Wang, Leptomycin B is an inhibitor of nuclear export: Inhibition of nucleo-cytoplasmic translocation of the human imunodeficiency virus type-1 (HIV-1) Rev protein and Revdependent mRNA, Chem. Biol. 4 (1997) 139 –147. N. Kudo, B. Wolff, T. Sekimoto, E.P. Schreiner, Y. Yoneda, M. Yanagida, S. Horinouchi, M. Yoshida, Leptomycin B inhibition of

[41]

[42]

[43] [44]

[45]

[46]

[47] [48]

[49]

[50]

[51]

[52] [53] [54]

[55] [56]

[57]

[58] [59]

[60]

[61]

67

signal-mediated nuclear export by direct binding to CRM1, Exp. Cell Res. 242 (1998) 540 –547. W.M. Michael, M. Choi, G. Dreyfuss, A nuclear export signal in hnRNPA1: A signal-mediated, temperature-dependent nuclear protein export pathway, Cell 83 (1995) 415– 422. W.M. Michael, P.S. Eder, G. Dreyfuss, The K nuclear shuttling domain: A novel signal for nuclear import and nuclear export in the hnRNP K protein, EMBO J. 16 (1995) 3587–3598. X.C. Fan, J.A. Steitz, HNS, a nuclear-cytoplasmic shuttling sequence in HuR, Proc. Natl. Acad. Sci. USA 95 (1998) 15293–15298. J.M. Holaska, B.E. Black, D.C. Love, J.A. Hanover, J. Leszyk, B.M. Paschal, Calreticulin is a receptor for nuclear export, J. Cell Biol. 152 (2001) 127–140. S. Lee, M. Neumann, R. Stearman, R. Stauber, A. Pause, G.N. Pavlakis, R.D. Klausner, Transcription-dependent nuclear-cytoplasmic trafficking is required for the function of the von Hippel-Lindau tumour suppressor protein, Mol. Cell. Biol. 19 (1999) 1486 –1497. I. Groulx, M.-E. Bonicalzi, S. Lee, Ran-mediated nuclear export of the von Hippel Lindau tumor suppressor protein occurs independently of its assembly with cullin-2, J. Biol. Chem. 275 (2000) 8991–9000. J. Zhu, F. McKeon, NF-AT activation requires suppression of Crm1dependent export by calcineurin, Nature 398 (1999) 256 –260. J. Berthelsen, C. Kilstrup-Nielsen, F. Blasi, F. Mavilio, V. Zappavigna, The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH, Genes Dev. 13 (1999) 946 –953. S. Karki, E.L.F. Holzbaur, Cytoplasmic dynein and dynactin in cell division and intracellular transport, Curr. Opin. Cell Biol. 11 (1999) 45–53. W.S. El-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, a potential mediator of p53 tumour suppression, Cell 75 (1993) 817– 825. J.W. Harper, G.R. Adami, N. Wei, K. Keyomarsi, S.J. Elledge, The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclindependent kinases, Cell 75 (1993) 805– 816. T. Miyashita, J.C. Reed, Tumor suppressor p53 is a direct transcriptional activator of the human bax gene, Cell 80 (1995) 293–299. Y. Barak, T. Juven, R. Haffner, M. Oren, mdm2 expression is induced by wild type p53 activity, EMBO J. 12 (1993) 461– 468. J.M. Stommel, N.D. Marchenko, G.S. Jimenez, U.M. Moll, T.J. Hope, G.M. Wahl, A leucine-rich nuclear export signal in the p53 tetramerization domain: Regulation of subcellular localization and p53 activity by NES masking, EMBO J. 18 (1999) 1160 –1172. R. Scully, D.M. Livingston, In search of the tumor-suppressor functions of BRCA1 and BRCA2, Nature 408 (2000) 429 – 432. R. Baer, T. Ludwig, The BRCA1/BARD1 heterodimer, a tumor suppressor complex with ubiquitin E3 ligase activity, Curr. Opin. Gen. Dev. 12 (2002) 86 –91. V. Joukov, J. Chen, E.A. Fox, J.B.A. Green, D.M. Livingston, Functional communication between endogenous BRCA1 and its partner, BARD1, during Xenopus laevis development, Proc. Natl. Acad. Sci. USA 90 (2001) 12078 –12083. W. Wang, Purification and biochemical heterogeneity of the mammalian SWI-SNF complex, EMBO J. 15 (1996) 5370 –5382. E. Craig, Z.K. Zhang, K.P. Davies, G.V. Kalpana, A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: Implications for tumorigenesis, EMBO J. 21 (2002) 31– 42. Y. Zhang, Y. Xiong, A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation, Science 292 (2001) 1910 –1915. J. Roth, M. Dobbelstein, D.A. Freedman, T. Shenk, A.J. Levine, Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein, EMBO J. 17 (1998) 554 –564.

68

M. Fabbro, B.R. Henderson / Experimental Cell Research 282 (2003) 59 – 69

[62] W. Tao, A.J. Levine, Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53, Proc. Natl. Acad. Sci. USA 96 (1999) 3077–3080. [63] S.D. Boyd, K.Y. Tsai, T. Jacks, An intact HDM2 RING-finger domain is required for nuclear exclusion of p53, Nat. Cell Biol. 2 (2000) 563–568. [64] R.K. Geyer, Z.K. Yu, C.G. Maki, The MDM2 RING-finger domain is required to promote p53 nuclear export, Nat. Cell Biol. 2 (2000) 569 –573. [65] J. Gu, L. Nei, D. Weiderschain, Z.-M. Yuan, Identification of p53 sequence elements that are required for MDM2-mediated nuclear export, Mol. Cell Biol. 21 (2001) 8533– 8546. [66] M.A.E. Lohrum, D.B. Woods, R.L. Ludwig, E. Balint, K.H. Vousden, C-terminal ubiquination of p53 contributes to nuclear export, Mol. Cell Biol. 21 (2001) 8521– 8532. [67] S.S. Kwek, J. Derry, A.L. Tyner, Z. Shen, A.V. Gudkov, Functional analysis and intracellular localization of p53 modified by SUMO-1, Oncogene 20 (2001) 2587–2599. [68] W. Gu, R.G. Roeder, Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain, Cell 90 (1997) 595– 606. [69] P. Shaw, J. Freeman, R. Bovey, R. Iggo, Regulation of specific DNA binding by p53: Evidence for a role for O-glycosylation and charged residues at the carboxy-terminus, Oncogene 12 (1996) 921–930. [70] J. Wesierska-Gadek, A. Bugajska-Schretter, C. Cemi, ADP-ribosylation of p53 tumor suppressor protein: Mutant but not wild-type p53 is modified, J. Cell. Biochem. 62 (1996) 90 –101. [71] J. Wesierska-Gadek, G. Schmid, C. Cemi, ADP-ribosylation of wild-type p53 in vitro: Binding of p53 protein to specific p53 consensus sequence prevents its modification, Biochem. Biophys. Res. Commun. 224 (1996) 96 –102. [72] E. Appella, C.W. Anderson, Post-translational modifications and activation of p53 by genotoxic stresses, Eur. J. Biochem. 268 (2001) 2764 –2772. [73] K.L. Neufeld, R.L. White, Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein, Proc. Natl. Acad. Sci. USA 94 (1997) 3034 –3039. [74] B.R. Henderson, Nuclear-cytoplasmic shuttling of APC regulates ␤-catenin subcellular localization and turnover, Nat. Cell Biol. 2 (2000) 653– 660. [75] Z.L. Neufeld, F. Zhang, B.R. Cullen, R.L. White, APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export, EMBO Rep. 1 (2000) 519 –523. [76] R. Rosin-Arbesfeld, F. Townsley, M. Bienz, A nuclear export function of the APC tumour suppressor, Nature 406 (2000) 1009 –1012. [77] F. Zhang, R.L. White, K.L. Neufeld, Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein, Mol. Cell Biol. 21 (2001) 8143– 8156. [78] S. Munemitsu, B. Souza, O. Muller, L. Albert, B. Rubinfeld, P. Polakis, The APC gene product associates with microtubules in vivo and promotes their assembly in vitro, J. Cell Biol. 148 (1994) 505–517. [79] K.J. Smith, D.B. Levy, P. Maupin, T.D. Pollard, B. Vogelstein, K.W. Kinzler, Wild-type but not mutant APC associates with the microtubule cytoskeleton, Cancer Res. 54 (1994) 3672–3675. [80] Y. Mimori-Kiyosue, N. Shiina, S. Tsukita, Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells, J. Cell Biol. 148 (2000) 505–517. [81] S. Sengupta, J.L. Vonesch, C. Waltzinger, H. Zheng, B. Wasylyk, Negative cross-talk between p53 and the glucocorticoid receptor and its role in neuroblastoma cells, EMBO J. 19 (2000) 6051– 6064. [82] S. Li, C.-Y. Ku, A.A. Farmer, Y.-S. Cong, C.-F. Chen, W.-H. Lee, Identification of a novel cytoplasmic protein that specifically binds to nuclear localization signal motifs, J. Biol. Chem. 273 (1998) 6183– 6189.

[83] S.H. Liang, D. Hong, M.F. Clarke, Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein, J. Biol. Chem. 273 (1998) 19817–19821. [84] S.H. Liang, M.F. Clarke, The nuclear import of p53 is determined by the presence of a basic domain and its relative position to the nuclear localization signal, Oncogene 18 (1999) 2163–2166. [85] M.E. Perry, S.M. Mendrysa, L.J. Saucedo, P. Tannous, M. Holubar, p76 (MDM2) inhibits the ability of p90 (MDM2) to destabilize p53, J. Biol. Chem. 275 (2000) 5733–5738. [86] R.V. Sionov, S. Coen, Z. Goldberg, M. Berger, B. Bercovich, Y. Ben-Neriah, A. Ciechanover, Y. Haupt, c-Abl regulates p53 levels under normal and stress conditions by preventing its nuclear export and ubiquitination, Mol. Cell Biol. 21 (2001) 5869 –5878. [87] J.D. Weber, L.J. Taylor, M.F. Roussel, C.J. Sherr, D. Bar-Sagi, Nucleolar Arf sequesters Mdm2 and activates p53, Nat. Cell Biol. 1 (1999) 20 –26. [88] C.A. Midgley, J.M. Desterro, M.K. Saville, S. Howard, A. Sparks, R.T. Hay, D.P. Lane, An N-terminal p14ARF peptide blocks Mdm2dependent ubiquitination in vitro and can activate p53 in vivo, Oncogene 19 (2000) 2312–2323. [89] J.K. Hsieh, F.S. Chan, D.J. O’Connor, S. Modjtahedi, S. Zhong, X. Lu, RB regulatesc the stability and the apoptotic function of p53 via MDM2, Mol. Cell 3 (1999) 181–193. [90] M. Baba, S. Hirai, S. Kazwakami, T. Kishida, N. Sakai, S. Kaneko, M. Yao, T. Shuin, Y. Kubota, M. Hosaka, S. Ohno, Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth, Oncogene 20 (2001) 2727–2736. [91] Z. Zhuang, J.R. Gnarra, C.F. Dudley, B. Zbar, W.M. Linehan, I.A. Lubensky, Detection of von Hippel-Lindau disease gene mutations in paraffin-embedded sporadic renal cell carcinoma specimens, Mod. Pathol. 9 (1996) 838 – 842. [92] R. Fodde, R. Smits, H. Clevers, APC, signal transduction and genetic instability in colorectal cancer, Nat. Rev. Cancer 1 (2001) 55– 67. [93] K. Tanimoto, Y. Makino, T. Pereira, L. Poellinger, Mechanism of regulation of the hypoxia-inducible factor-1␣ by the von HippelLindau tumor suppressor protein, EMBO J. 19 (2000) 4298 – 4309. [94] I. Groutx, S. Lee, Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein, Mol. Cell. Biol. 22 (2002) 5319 –5336. [95] K.L. Lorick, J.P. Jensen, S. Fang, A.M. Ong, S. Hatakeyama, RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination, Proc. Natl. Acad. Sci. USA 96 (1999) 11354 –11369. [96] R. Hashizume, M. Fukuda, I. Maeda, H. Nishikawa, D. Oyake, Y. Yabuki, H. Ogata, T. Ohta, The RING heterodimer BRCA1– BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation, J. Biol. Chem. 276 (2001) 14537–14540. [97] H. Ruffner, C.A.P. Joazeiro, D. Hemmati, T. Hunter, I.M. Verma, Cancer-predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity, Proc. Nat. Acad. Sci. USA 98 (2001) 5134 – 5139. [98] A. Chen, F.E. Kleiman, J.L. Manley, T. Ouchi, Z.-Q. Pan, Autoubiquitination of the BRCA1/BARD1 RING ubiquitin ligase, J. Biol. Chem. 277 (2002) 22085–22092. [99] X.H. Liang, J. Yu, K. Brown, B. Levine, Beclin 1 contains a leucine-rich nuclear export signal that is required for its autophagy and tumor suppressor function, Cancer Res. 61 (2001) 3443– 3449. [100] C.A. Wilson, L. Ramos, M.R. Villasenor, K.H. Anders, M.F. Press, K. Clarke, B. Karlan, J.-J. Chen, R. Scully, D. Livingston, R.H. Zuch, M.H. Kanter, S. Cohen, F.J. Calzone, D.J. Slamon, Localiza-

M. Fabbro, B.R. Henderson / Experimental Cell Research 282 (2003) 59 – 69 tion of human BRCA1 and its loss in high-grade, non-inherited breast carcinomas, Nat. Genet. 21 (1999) 236 –240. [101] M.T. Daniel, M. Koken, O. Romagne, S. Barbey, A. Bazarbachi, M. Stadler, M.C. Guillemin, L. Degos, C. Chomienne, H.D. The, PML protein expression in hematopoietic and acute promyelocytic leukemia cells, Blood 82 (1993) 1858 –1867. [102] J.A. Rodriguez, B.R. Henderson, Identification of a functional nuclear export signal in BRCA1, J. Biol. Chem. 275 (2000) 38589–38596. [103] K.L. Neufeld, D.A. Nix, H. Bogerd, Y. Kang, M.C. Beckerle, B.R. Cullen, R.L. White, Adenomatous polyposis coli protein

69

contains two nuclear export signals and shuttles between the nucleus and cytoplasm, Proc. Natl. Acad. Sci. USA 97 (2000) 12085–12090. [104] M. Watanabe, N. Masuyama, M. Fukuda, E. Nishida, Regulation of intracellular dynamics of Smad4 by its leucine-rich nuclear export signal, EMBO Rep. 1 (2000) 176 –182. [105] T. Crook, G.A. Parker, M. Rozycka, S. Crossland, M.J. Allday, A transforming p53 mutant, which binds DNA transactivates and induces apoptosis reveals a nuclear: Cytoplasmic shuttling detect, Oncogene 16 (1998) 1429 –1441.