The Nuclear Pore Complex, Nuclear Transport, and Apoptosis1

279

MINIREVIEW / MINISYNTHE` SE

The nuclear pore complex, nuclear transport, and apoptosis1

Birthe Fahrenkrog

Abstract: The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eu- karyotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically dis- cussed. Key words: apoptosis, nuclear pore complex, nuclear transport. Re´sume´ : Le complexe du pore nucleáire (CPN) est le seul passage entre le noyau et le cytoplasme des cellules eucaryotes en interphase, et il facilite tous les ećhanges entre ces 2 compartiments cellulaires. Ainsi, le CPN et le transport nucleáire jouent un roˆle important dans la translocation des signaux d’apoptose de la membrane cellulaire vers le noyau ou` ils de´- clenchent les modifications biochimiques et morphologiques observeés durant l’apoptose. De rećents travaux donnent a` penser que la corre´lation entre le CPN, le transport nucleáire et l’apoptose de´passe le simple fait que les CPN facilitent le transport nucleáire de joueurs cle´s implique´s dans le programme de mort cellulaire. Dans ce contexte, l’accessibilite´ des re´gulateurs cle´s de l’apoptose semble eˆtre fortement moduleé par le transport nucleáire; par exemple, un import nucleáire alte´re´ pourrait eˆtre un dećlencheur d’apoptose. Dans cette synthe`se, les rećentes observations sur le lien e´troit, inattendu entre les CPN, le transport nucleáire, et l’apoptose seront pre´senteés et feront l’objet d’une discussion ećlaireé. Mots cle´s:apoptose, complexe du pore nucleáire, transport nucleáire. [Traduit par la Re´daction]

Introduction transport pathway is the import of proteins carrying a so- called classical nuclear localization signal (NLS), which is The bidirectional transport of macromolecules between characterized by a stretch of basic amino acids (Fig. 1). The the nucleus and the cytoplasm is mediated by nuclear pore NLS is recognized by the adapter protein importin a, which, complexes (NPCs), supramolecular assemblies embedded in in turn, interacts with the actual transport receptor importin the double membrane of the nuclear envelope (NE) (Fah- b. The importin–cargo complex translocates through the renkrog et al. 2004; Fahrenkrog and Aebi 2003). Nuclear NPC via interaction of the receptor with a subset of nuclear transport depends on transport receptors known as importins pore proteins (nucleoporins). Once the complex reaches the or exportins (also collectively called karyopherins; Fried and nuclear side of the NPC, binding of RanGTP to importin b Kutay 2003; Pemberton and Paschal 2005). Karyopherins induces a conformational change in the receptor, which leads generally bind the small GTPase Ran in its GTP-bound state, to the dissociation of the cargo from the receptor as well as which controls the directionality of nuclear transport. Most dissociation of the receptor from the NPC, whereby the re- of the karyopherins are able to bind to their cargo directly, ceptor is recycled back to the cytoplasm. Conversely, in the although some use adapter molecules. The best-characterized presence of RanGTP, exportins bind to export cargo that harbor a nuclear export signal (NES) (Fig. 1). The heterotri- Received 6 June 2005. Published on the NRC Research Press meric export complexes are able to interact with the NPC Web site at http://cjpp.nrc.ca on 25 April 2006. via the exportin transport receptor and after NPC transloca- B. Fahrenkrog. M.E. Mu¨ller Institute for Structural Biology, tion they become dissociated upon RanGTP hydrolysis by Biozentrum, University of Basel, Klingelbergstr. 70, 4056 Basel, RanGAP at the cytoplasmic face of the NPC. The exportin Switzerland (e-mail: [email protected]). shuttles back into the nucleus, and RanGDP is re-imported 1This paper is one of a selection of papers published in this into the nucleus by its own transport receptor, termed NTF2 Special Issue, entitled The Nucleus: A Cell Within A Cell. (Fried and Kutay 2003; Pemberton and Paschal 2005). Once

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Fig. 1. Nuclear transport cycles. Importins bind to cargo molecules harboring a nuclear localization signal (NLS) in the cytoplasm and mediate the interaction with the nuclear pore complex (NPC). In the nucleus, after NPC translocation, RanGTP binds to the importin, which induces a conformational change in the importin and leads to the dissociation of the cargo-receptor complex and the release from the NPC. The importin–RanGTP complex then recycles back into the cytoplasm, where RanGTP is displaced upon GTP hydrolysis mediated by a concerted action of RanGAP, RanBP1, and RanBP2 at the cytoplasmic filaments. RanGDP is imported back into the nucleus by its own import receptor, called NTF2. Exportins bind to cargo harboring a nuclear export signal (NES), which requires the presence of RanGTP. An export complex becomes dissociated upon GTP hydrolysis at the cytoplasmic face of the NPC and the exportin shuttles back to the nucleus.

arriving at the nuclear side of the NPC, RanGDP is tingtin disease, immune deficiency, and cancer (Lawen recharged with GTP by the RanGEF, called RCC1, a 2003; Riedl and Shi 2004). Apoptosis is morphologically chromatin-associated nuclear protein. characterized by a loss of cell volume, plasma membrane The NPC is composed of a set of ~30 different proteins blebbing, chromatin condensation, and nuclear fragmenta- (Rout et al. 2000; Cronshaw et al. 2002), called nucleopor- tion, followed by packaging of the cellular contents into ins. Owing to the highly symmetrical organization of the membrane-enclosed vesicles called apoptotic bodies. Cas- NPC (Fig. 2), nucleoporins exist typically in 8 or multiple pases, a specific group of cysteine proteases, have been of 8 copies per NPC, resulting in a molecular mass of shown to be the key mediators of the cell death program, ~120 MDa (Fahrenkrog et al. 2004; Fahrenkrog and Aebi and their activation leads to cleavage of a selected group of 2003). Immuno-EM studies, both in yeast and vertebrates, substrates (Fischer et al. 2003), ultimately yielding the bio- have unveiled that most of the nucleoporins are symmetri- chemical and morphological changes that characterize apop- cally distributed on both sides of the NPC, whereas only a tosis. few are located asymmetrically either at the nuclear or the Two main apoptotic pathways lead to the activation of cytoplasmic periphery of the NPC (Fahrenkrog and Aebi caspases (Lawen 2003; Riedl and Shi 2004). An extrinsic 2003; Rout et al. 2003; Fahrenkrog et al. 2004). Roughly pathway (Fig. 3) is initiated by ligand binding to cell surface one third of the nucleoporins harbor distinct domains of death receptors such as Fas or the TNFa receptor. This re- phenylalanine-glycine (FG) repeats in their amino acid se- sults in the recruitment of adapter proteins such as FADD quence, and it is believed that these FG-repeat regions medi- (Fas-associated death domain) or TRADD (tumor necrosis ate the main interaction between the NPC and the soluble factor receptor-associated death domain) to the cell mem- transport receptors. brane and to the activation of a so-called initiator caspase, Apoptosis is a physiological form of programmed cell such as caspase-8. Activation of caspase-8, in turn, promotes death and crucial for normal development, tissue homeosta- activation of an effector caspase such as caspase-3 and sub- sis, and the elimination of damaged cells. Defects in apopto- sequent apoptosis. sis have been linked to various diseases including An intrinsic or mitochondrial pathway (Fig. 3) can be ac- neurodegenerative disorders such as Alzheimer’s and Hun- tivated by a variety of intra- or extracellular stresses such as

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Fig. 2. Schematic representation of the 3-D architecture of the nu- insights into this important cross-talk between nuclear trans- clear pore complex (NPC). The main structural components of the port and apoptosis will be the focus of this review. NPC include the central framework (i.e., the part of the NPC that is embedded in the double membrane of the nuclear envelope (NE)), Regulation of the subcellular localization of the cytoplasmic ring moiety with the cytoplasmic filaments, and the apoptotic key-players nuclear ring moiety, which is decorated by the nuclear basket. En- closed by the central framework is the central pore of the NPC, Inhibition of active nuclear import by, for example, WGA which mediates the macromolecular exchange between the cyto- or antibodies to importin a (Yasuhara et al. 1997), or by the plasm and the nucleus. This figure was modeled with a visual pro- presence of dominant negative forms of Drosophila RCC1, gramming environment (ViPEr) at the Scripps Research Institute the RanGEF (see above; Shi and Skeath 2004), prevents nu- (http://www.Scripps.edu). clear apoptosis, suggesting that important mediators of apoptosis are not able to enter the nuclear compartment. Caspase- 3 is thought to be involved in these nuclear morphology changes that occur during apoptosis (Fig. 3; Lawen 2003; Riedl and Shi 2004). In fact, many substrates for caspase-3 have been identified in the nucleus (Fischer et al. 2003), whereas the inactive procaspase-3 resides in the cytoplasm. Upon activation, caspase-3 translocates from the cytoplasm into the nuclei of apoptotic cells (Kamada et al. 2005). Nu- clear import of caspase-3 requires proteolytic activation and substrate recognition, and, contrary to earlier assumptions (Faleiro and Lazebnik 2000), it appears to be a signal-dependent, receptor-mediated process (Kamada et al. 2005). The specific nuclear import pathway for caspase-3, however, remains to be elucidated. Nuclear localization has also been shown for the executioner caspase-6 (Schickling et al. 2001) and the initiator caspase-2 (Paroni et al. 2002), although again the specific import pathways remain unclear. Caspases are not the only key regulators of apoptosis that translocate from the cytoplasm to the nucleus, but already cell death regulators upstream of the caspases reveal tempo- rary nuclear localization. For example, FADD is an adaptor protein that bridges the interaction between death receptors and initiator caspases (see Fig. 3), thereby inferring a cytoplasmic localization of FADD. However, a systematic analysis of the subcellular localization of FADD has revealed that oxidative stress or cytotoxic drugs. Activation of the mito- FADD is primarily localized in the nucleus of adherent cells chondrial pathway leads to the release of cytochrome c (Screaton et al. 2003). from the mitochondria into the cytosol, where it recruits the Lacking a putative NLS, the nuclear import of FADD is apoptotic protease activating factor-1 (Apaf-1), which, in regulated by phosphorylation, whereas FADD export from turn, catalyzes the activation of, for example, initiator the nucleus is mediated by exportin-5 (Screaton et al. 2003). caspase-9 with subsequent activation of caspase-3. Another death receptor adaptor protein is TRADD. For the nuclear manifestation of apoptosis, death signals TRADD harbors both a bipartite NLS and an NES, and in- have to be transduced to the nucleus. In fact, it has been hibition of nuclear export by LMB, a specific inhibitor of shown that cross-talk between the nuclear and cytoplasmic the export receptor CRM1 (Nishi et al. 1994; Kudo et al. compartments plays an essential role in propagating the 1998), results in accumulation of TRADD within PML (pro- death program. Many nuclear proteins such as lamins, the myelocytic leukemia protein) nuclear bodies (Morgan et al. lamin B receptor (LBR), the lamina-associated protein 2002). In these PML nuclear bodies TRADD is able to me- LAP2a, emerin, and nucleoporins are substrates for caspases diate caspase-independent nuclear apoptosis, whereas, in (Fischer et al. 2003; Ferrando-May et al. 2001; Kihlmark et contrast, TRADD-induced apoptosis in the cytoplasm is al. 2001). Moreover, the intracellular localization of apopto- caspase-dependent. Hence, TRADD apparently can lead to sis regulators is tightly controlled by nuclear import or ex- the activation of at least 2 distinct apoptosis pathways port signals. For example, inhibitor of apoptosis proteins (Morgan et al. 2002). (IAPs), which are typically able to bind and inhibit caspases Release of cytochrome c from the mitochondria marks a in the cytoplasm, have been shown to be kept inactive in the major event during progression of apoptosis (Desagher and nucleus (Plenchette et al. 2004; Rodriguez et al. 2002; Sa- Martinou 2000) as it leads to the formation of the apopto- muel et al. 2005; Vischioni et al. 2004). In contrast, the some and subsequent caspase activation. In addition, cyto- apoptosis-inducing factor (AIF) is inhibited by cytosolic re- chrome c may also play a role in caspase-independent tention via the heat shock protein 70 (HSP70; Gurbuxani et nuclear apoptosis (Nur-E-Kamal et al. 2004); cytochrome c, al. 2003). Therefore, nuclear transport appears to provide an once released from the mitochondria, gradually accumulates important level of control for regulating apoptosis. Recent in the nucleus of apoptotic cells, an event that coincides

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Fig. 3. The 2 major apoptotic pathways. The extrinsic pathway can be activated by ligand binding to death receptors in the cell membrane such as Fas. The extrinsic pathway leads to the activation of caspase-8, which, in turn, activates caspase-3. The intrinsic pathway can be activated by, for example, cytotoxic drugs or UV light. Activation of the intrinsic pathway leads to release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c binds and activates Apaf-1, which then facilitates activation of caspase-9, followed by activation of caspase-3. The extrinsic and the intrinsic pathway can crosstalk via caspase-8-mediated cleavage of BID. Cleaved BID, in turn, induces cytochrome c release from the mitochondria. Caspases can be inhibited by inhibitor of apoptosis (IAP) proteins such as XIAP, which, in turn, can be antagonized by mitochondrial proteins such as Smac/DIABLO or Omi/HtrA2. Arrows indicate activation, and perpendicular lines indicate inhibition.

with the release of acetylated histone H2A from the nu- nucleus thereby inducing DNA fragmentation (Henkart cleus to the cytoplasm. The translocation of cytochrome c 1994; Trapani 1998). GraB nuclear import is mediated by from the cytoplasm to the nucleus, in turn, occurs inde- importin a in a Ran-independent manner (Blink et al. pendently of caspase-3 activation. Therefore, cytochrome c 2005). Strikingly, graB also directly interacts with importin that is able to directly bind to chromatin may be involved b, but this interaction inhibits the nuclear import of graB in caspase-independent chromatin remodeling during apop- rather than enhancing it (Blink et al. 2005). tosis (Nur-E-Kamal et al. 2004). Proteinase inhibitor 9 (PI-9) is a human serpin and a very Just like cytochrome c, the flavoprotein AIF is released efficient inhibitor of graB. Like graB, PI-9 predominantly from the mitochondria into the cytosol upon stimulation of localizes to the nucleus, but it has the ability to shuttle be- cell death. Once in the cytosol, AIF is translocated to the tween the nucleus and the cytoplasm (Bird et al. 2001). nucleus, where it triggers caspase-independent nuclear Binding of PI-9 to graB reduces binding of importina/b to changes (Susin et al. 1999; Daugas et al. 2000). Binding of graB, which most likely prevents graB nuclear import by HSP70 to AIF leads to cytosolic retention of AIF, thereby masking the NLS of graB. Moreover, PI-9 contains an NES, keeping it away from its nuclear targets in an event that ar- and complex formation of PI-9 and graB in the nucleus may rests progression of apoptosis (Gurbuxani et al. 2003). therefore primarily result in the nuclear export of graB, Cytotoxic lymphocytes (CTLs) and natural killer (NK) thereby preventing graB-mediated nuclear apoptosis (Blink cells kill intracellular parasites and tumor cells by using ei- et al. 2005). ther the Fas pathway or the granula exocytosis pathway Modulating the nuclear import and export of pro-apoptotic (Henkart 1994; Trapani 1998). In the latter, perforin medi- proteins does not appear to be the only control level to regu- ates the entry of the serine proteinase granzyme B (graB) late apoptosis by nuclear transport. Nuclear retention of into the target cell, which then activates caspases, cleaves a apoptosis inhibitors appears to be a regulatory element of variety of other proteins, and rapidly translocates into the apoptosis as well. IAPs are a family of negative regulators

# 2006 NRC Canada Fahrenkrog 283 of caspases characterized by the presence of one to three ~70 matin to the inner nuclear membrane. Both are substrates for amino acid zinc-binding baculovirus IAP repeat (BIR) do- caspases (Gotzmann et al. 2000; Buendia et al. 2001), im- mains that are essential for the IAP activity (Salvesen and plying that their cleavage leads to the detachment of the NE Duckett 2002; Shiozaki and Shi 2004). Often IAPs contain a from chromatin during apoptosis. second zinc-binding domain called the RING domain, which In additional to proteins of the inner nuclear membrane, typically has E3 ubiquitin ligase activity, thereby allowing the nucleoporins Nup153, RanBP2/Nup358, Nup214/CAN, self-degradation of the IAPs and degradation of IAP targets POM121, and Tpr were found to be cleaved during apopto- such as caspases, by the 26S proteosome (Salvesen and sis, whereas p62 and gp210 are either unaffected or only late Duckett 2002). substrates for cleavage (Buendia et al. 1999; Ferrando-May IAP activity is required in the cytoplasm, where IAPs can et al. 2001; Kihlmark et al. 2001, 2004). Nup153, POM121, bind and inhibit caspases. However, a number of IAPs ap- and Tpr are degraded in a caspase-3-dependent manner pear to be retained in the nucleus to prevent IAP-mediated (Buendia et al. 1999; Ferrando-May et al. 2001; Kihlmark caspase inhibition. For example, c-IAP1 and c-IAP2 are et al. 2004). Nup153 is thought to connect the NPC with mostly localized to the nucleus (Plenchette et al. 2004; Vi- the nuclear lamina, indicating that Nup153 cleavage in com- schoni et al. 2004; Samuel et al. 2005). Apoptotic stimuli that bination with lamina cleavage leads to diffusion of NPCs trigger either the extrinsic or the intrinsic pathway, in turn, in- within the plane of the NE thereby yielding the typical NPC duce the nuclear export of c-IAP1 in a CRM1-dependent man- clustering observed during apoptosis. ner (Plenchette et al. 2004; Vischoni et al. 2004; Samuel et al. Degradation of NE proteins and nucleoporins takes place 2005). Indeed, nuclear export signals have been identified in in a particular sequential order and is independent of the ac- c-IAP1’s linker regions between its BIR2 and BIR3 domain tual apoptotic stimulus, but requires activation of caspase-3 (Vischioni et al. 2004) as well as in its caspase-recruitment and (or) caspase-6 (Buendia et al. 1999; Ferrando-May et domain (Plenchette et al. 2004). In contrast, no functional al. 2001; Kihlmark et al. 2004). In this context, POM121 NES could be identified in c-IAP2 (Vischioni et al. 2004), and Nup358 appear as early targets for destruction prior to whose nuclear export is also CRM1-dependent (Vischioni et nucleosomal DNA degradation and NPC clustering, whereas al. 2004). Nup153 and lamin B cleavage coincide with the onset of The IAP protein Survivin is overexpressed in many hu- DNA fragmentation and clustering of NPCs (Kihlmark et al. man tumors (Altieri 2004), and it appears to be a nuclear- 2001, 2004). However, the hierarchical sequence and the shuttling protein. Like cIAP1, survivin is actively exported kinetics of degradation may be influenced by the apoptosis- out of the nucleus by CRM1 despite the absence of a func- inducing stimulus (Dynlacht et al. 2000). tional NES (Rodriguez et al. 2002). In contrast to the proteins of the inner nuclear membrane The X-linked IAP protein XIAP is retained in the nucleus and the nucleoporins, the nuclear transport receptors do not upon binding to its antagonist XAF1 (XIAP-associated fac- appear to be targets for caspases (Ferrando-May et al. 2001). tor 1; Liston et al. 2001). In the absence of XAF1, XIAP, However, Ran, importin a, and importin b redistribute across which harbors neither a NLS nor a NES, mainly localizes the NE during staurosporine- or actinomycin D-induced to the cytoplasm. Overexpression of XAF, however, triggers apoptosis. Strikingly, this occurs prior to and independent of XIAP redistribution from the cytoplasm to the nucleus caspase activation (Ferrando-May et al. 2001). In contrast, in (Liston et al. 2001). hydrogen peroxide-treated apoptotic cells Ran and importin b Taken together, active transport between the nucleus and are degraded in a caspase- and proteosome-dependent man- cytoplasm of caspases, IAPs, and other apoptosis regulators ner (Kodiha et al. 2004). In addition, staurosporine- and acti- may constitute an important level of control to modulate the nomycin D-treated cells exhibited mRNA accumulation in cellular death program. the nucleus and an increase in the permeability barrier of the NPC, again, prior to and independent of caspase activation The fate of NE proteins, nucleoporins, and (Ferrando-May et al. 2001; Faleiro and Lazebnik 2000). Therefore, caspase-independent as well as caspase-dependent nuclear transport during apoptosis events lead to alterations in the nuclear transport machinery, The NE is a highly dynamic structure that irreversibly which may give rise to several phenotypes of nuclear apopto- breaks down during apoptosis. Changes in NE morphology, sis. Both the molecular mechanisms and the key players in including clustering of NPCs and the budding and detach- caspase-independent changes in the nuclear transport machi- ment of the nuclear membrane from chromatin, led to the nery remain to be elucidated and represent a great challenge investigation of the fate of NE proteins during apoptosis. In for future research. fact, several NE proteins are cleaved by caspases during apoptosis. The nuclear lamina proteins lamin A/C, lamin B, Impaired nuclear import may trigger and emerin are cleaved by caspase-6 (Buendia et al. 2001). apoptosis More specifically, lamin B is cleaved at a conserved aspar- tate residue located in the hinge region upstream of coil 2 Nuclear transport and NPCs play important roles in the (Gruenbaum et al. 2005). Coil 2 is necessary for the forma- regulation of apoptosis, not only in terms of controlling the tion of stable lamin B homodimers and their lateral interac- subcellular localization of apoptosis mediators, but nuclear tions in lamin filaments, suggesting that cleavage of coil 2 transport is necessary for apoptosis progression (Yasuhara leads to the observed persistent association of lamina et al. 1997; Shi and Skeath 2004). Moreover, recent studies remnants with the NE until late stages of apoptosis. Lamin- support the notion that impaired nuclear import may in fact associated proteins such as LAP2a and LAP2b attach chro- act as a trigger for cells to induce their death program.

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Initially, the cellular protein CC3 was identified as least, ongoing nuclear import is a prerequisite for apoptosis, metastasis suppressor in variant small cell lung carcinoma but, somewhat contradictory, impaired nuclear import can be (Shtivelman 1997). Consistent with this observation, CC3 also a trigger for initiating the cellular death program. overexpression predisposes tumor cells to apoptosis Therefore, the crosstalk between NPC, nuclear transport, (Whitman et al. 2000). Moreover, high cellular levels of CC3 and apoptosis needs to be tightly coordinated and regulated. result in slower rates of nuclear import and an increased sen- Hence, the tight coupling of these cellular events might keep sitivity to cell death (King and Shtivelman 2004). Distinct some more surprises in store in the future. karyopherins, in particular importin b, transportin, and exportin-4, could be identified as CC3-interacting proteins, Acknowledgements and this interaction is insensitive to RanGTP. Moreover, CC3 Ueli Aebi is gratefully acknowledged for critically read- co-immunoprecipitates and co-localizes with nucleoporins. ing the manuscript. This work is supported by a research Recombinant and endogenous CC3 causes a dramatic de- grant from the Swiss National Science Foundation, the M.E. crease in the nuclear import of transportin and importin a/b Mu¨ller Foundation, and the Kanton Basel Stadt. reporter cargoes, and it inhibits the translocation of transportin into the nucleus, thereby leading to an accumulation References of transportin at the NPCs and in the cytoplasm (King and Altieri, D.C. 2004. Molecular circuits of apoptosis regulation and Shtivelman 2004). Hence, docking of import receptors to cell division control: the Survivin paradigm. J. Cell. Biochem. the NPC by CC3, where they are trapped unproductively 92: 656–663. doi:10.1002/jcb.20140. 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