Nucleocytoplasmic Shuttling of Smad Proteins

Nucleocytoplasmic shuttling of Smad proteins

Caroline S Hill1

1Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

Nuclear accumulation of active Smad complexes is crucial for transduction of transforming growth factor β (TGF-β)- superfamily signals from transmembrane receptors into the nucleus. It is now clear that the nucleocytoplasmic distributions of Smads, in both the absence and the presence of a TGF-β-superfamily signal, are not static, but instead the Smads are continuously shuttling between the nucleus and the cytoplasm in both conditions. This article presents the evidence for continuous nucleocytoplasmic shuttling of Smads. It then reviews different mechanisms that have been proposed to mediate Smad nuclear import and export, and discusses how the Smad steady-state distributions in the absence and the presence of a TGF-β-superfamily signal are established. Finally, the biological relevance of continuous nucleocytoplasmic shuttling for signaling by TGF-β superfamily members is discussed. Keywords: Smad, nuclear import and export, TGF-β-superfamily signaling, karyopherin, nucleocytoplasmic shuttling Cell Research (2009) 19:36-46. doi: 10.1038/cr.2008.325; published online 30 December 2008

Introduction into three functional classes. The first class are the receptor-regulated Smads or R-Smads (Smad1, 2, 3, 5 The transforming growth factor b (TGF-b) superfam- and 8), which are phosphorylated by the type I receptor ily of ligands, which comprises TGF-bs, Activins, bone kinases on an SXS motif at their extreme C-termini. In morphogenetic proteins (BMPs) and growth and differ- general Smad1, 5 and 8 are activated by BMP and GDF entiation factors (GDFs), regulates many different cel- signals, whereas Smad2 and 3 are activated by TGF-b lular processes, including proliferation, apoptosis, adhe- and Activin/Nodal signals. However, it has recently be- sion, differentiation and specification of cell fate. These come clear that TGF-b can also activate Smad 1, 5 and ligands play a crucial role in establishing and patterning 8 [4-6]. This is thought to be mediated by a receptor the basic body plan during embryogenesis, and are also complex comprising the TGF-b type II receptor (TbRII), essential for tissue homeostasis, wound repair and im- the TGF-b type I receptor (ALK5) and the tissue-specific mune regulation in adult organisms [1]. Both the ligands type I receptor (ALK1) in endothelial cells [6], and and components of the downstream signaling pathways through a receptor complex comprising TbRII, ALK5 are highly conserved from worm to man [2]. Receptors and either ALK2 or ALK3 in epithelial cells [5]. Upon for these ligands are serine threonine kinases, and signal- phosphorylation, the R-Smads form either homomeric or ing occurs through heteromerization of type II receptors heteromeric complexes with Smad4, the only member in with type I receptors, which are brought together by the the second functional class of Smads, the common medi- ligand. The constitutively active type II receptors activate ator Smads or co-Smads. Smad complexes then accumu- the type I receptors by transphosphorylation. Activated late in the nucleus and regulate the transcription of target type I receptors in turn phosphorylate their substrates, the genes in conjunction with other transcription factors [7]. best characterized of which are a subset of Smad proteins Smad4 only accumulates in the nucleus upon ligand acti- [3]. vation in the context of R-Smad–Smad4 complexes and There are eight different Smad proteins, which fall consequently Smad4 is dispensable for R-Smad nuclear accumulation in response to TGF-b, which occurs with the same efficiency in Smad4 null cells [8-10]. The third functional class of Smads are the inhibitory Smads or Correspondence: Caroline S Hill Tel: + 44-207-269-2941; Fax: + 44-207-269-3093 I-Smads (Smad6 and 7) that regulate the pathway nega- E-mail: [email protected] tively by mediating receptor inactivation through recruit- npg Smad nucleocytoplasmic shuttling 37 ing either the E3 ubiquitin ligases Smurf1/2 [11, 12] or reside exclusively in the cytoplasm in the absence of a the catalytic subunit of protein phosphatase 1 to the re- signal. Upon active signaling, they were thought to be receptors [13]. leased from the cytoplasm and translocate into the nucle- The R-Smads and Smad4 have a common structure [3] us, where they would regulate gene transcription and be (Figure 1). In their N-termini there is a well-conserved degraded subsequently [15]. More recent evidence, how- domain called Mad-homology 1 (MH1) domain, which ever, challenges this simple, linear view of the pathway. in Smad1, 3, 4, 5 and 8 binds to DNA directly. At the We now know that the Smads are continuously shuttling C-terminus there is another well-conserved domain, the between the cytoplasm and the nucleus even in unstimu- MH2 domain, which is responsible for receptor–Smad lated cells. Moreover, there is compelling evidence that and Smad–Smad interactions and also for many interac- the bulk of the Smads that have accumulated in the nu- tions with co-activators and co-repressors in the nucleus. cleus in response to a signal are not degraded there, but The SXS motif that is phosphorylated in the R-Smads rather engage in highly dynamic shuttling between the in response to ligand stimulation is at the extreme C- nucleus and the cytoplasm. The evidence suggests that terminus of the MH2 domain and these phospho-serines such shuttling provides an elegant sensing mechanism are critical for stabilizing Smad–Smad complexes [14]. to ensure that the concentration of active Smads in the The MH1 and MH2 domains are separated by a less nucleus reflects the actual signal strength at any given well-conserved proline-rich linker region. Smad6 and 7 time. contain a recognizable MH2 domain, but have only weak This article reviews the evidence for Smad nucleocy- homology with the other Smads at their N-termini. toplasmic shuttling and outlines the different molecular The initial view of the TGF-b-superfamily signaling transport mechanisms that have been proposed to account pathways suggested that R-Smads and Smad4 would for nuclear import and export of Smads. It then discusses

NLS DBM NES2 NES1 P P Smad1 MH1 Linker MH2 SSVS

CAN/Nup214 NLS Nup153 P P Smad2 Ex3 Linker MH2 SSMS

CAN/Nup214 Nup153 P P Smad3 MH1 Linker MH2 SSVS

NLS DBM Exportin 4

NLS NES

Smad4 MH1 Linker MH2

DBM

Figure 1 Smads have a conserved domain structure and harbor nuclear localization signals (NLS) and nuclear export signals (NES). R-Smads and Smad4 share two conserved domains, the N-terminal Mad-homology 1 (MH1) domain and the C-terminal MH2 domains. These domains are connected by a less well-conserved proline-rich linker region. In response to a TGF-β-superfamily ligand, the R-Smads, e.g. Smads1, 2 and 3, become phosphorylated at the C-terminal SXS motif, as indicated. The positions of NLSs and NESs as well as the DNA-binding motif (DBM) are indicated. An NLS is present in Smad2, but has not been shown to be functional. This is thought to be due to the presence of the additional exon 3 (Ex 3), which also interferes with DNA binding. A “hydrophobic corridor” in the MH2 domain of Smad2 and 3 is involved in binding to the nucleoporins CAN/Nup214 and Nup153, but full-length Smad4 seems to be required for interaction with CAN/Nup214 (not shown). The region of Smad3 that interacts with exportin 4 is also shown.

Cell Research | Vol 19 No 1 | January 2009 Caroline S Hill npg 38 how the subcellular localization of Smads is regulated the receptors have been inactivated? The most likely ex- during signaling, and finally it evaluates the functional planation is that continuous nucleocytoplasmic shuttling role of nucleocytoplasmic shuttling of Smads in TGF-b- of Smads, even in actively signaling cells, which are in superfamily signaling. a state of maximal nuclear Smad accumulation, connects events at the plasma membrane with the nucleus. This Evidence for Smad nucleocytoplasmic shuttling notion has now been directly demonstrated by photo- bleaching and photoactivation experiments using GFP The first evidence for nucleocytoplasmic shuttling of fusions of Smad2 and 4 [21-23]. Smads came from studies on Smad4. In the absence of a The Smad2 that relocalizes to the cytoplasm upon in- TGF-b-superfamily ligand, Smad4 is distributed equally activation of the signal is unphosphorylated, suggesting between the nucleus and the cytoplasm. Treatment of that nuclear export is accompanied by dephosphorylation cells with leptomycin B (LMB), a specific inhibitor of [19, 24]. Furthermore, if cells are treated with TGF-b for the prototypic export receptor CRM1 or Exportin 1 [16], 1 h to allow maximal nuclear accumulation of R-Smad– led to rapid nuclear accumulation of Smad4 [17, 18]. Smad4 complexes, and then treated with LMB to block This simple experiment showed that CRM1 activity is CRM1 activity, as well as with the receptor inhibitor SB- required for nuclear export of Smad4, and demonstrated 431542 to block receptor activity, Smad4 as expected, that Smad4 must be continuously shuttling between the stays trapped in the nucleus but Smad2 still redistributes nucleus and the cytoplasm in the absence of a TGF-b to the cytoplasm [24]. As it is the C-terminal phospho- signal. The R-Smads in contrast were insensitive to treat- rylated serines in the R-Smads that are responsible for ment with LMB, indicating that CRM1 was not respon- stabilizing the R-Smad–Smad4 complexes [14], these sible for their nuclear export [18, 19]. This also meant results suggested that R-Smad–Smad4 complexes dis- that more sophisticated approaches were required to sociate in the nucleus as a result of R-Smad dephos- determine whether they also shuttle between the nucleus phorylation, and that monomeric R-Smads and Smad4 and the cytoplasm in unstimulated cells, and this was are then exported separately by distinct mechanisms. In subsequently shown for Smad2 [19] using a quantitative agreement with this idea, CRM1-dependent Smad4 ex- nuclear transport reporter system [20]. The same study port is thought to be inhibited by complex formation of used a heterokaryon assay to demonstrate the shuttling Smad4 with R-Smads, which physically prevents Smad4 ability of Smad2 and to map the export activity to a from interaction with CRM1 [27]. Moreover, R-Smad region comprising the linker and MH2 domain. Fluores- C-terminal phosphatases have recently been identified. cence perturbation and photoactivation experiments with Smad1 phosphatases include pyruvate dehydrogenase GFP-fusions of Smad2 have, more recently, provided phosphatase (PDP) and small C-terminal phosphatases direct visual proof of the continuous exchange between (SCP1, 2 and 3) [28, 29], while PPM1A can dephospho- the nuclear and the cytoplasmic pools of Smad2 both in rylate both subsets of R-Smads [30]. tissue culture cells, in tissue explants of Xenopus laevis Thus Smad shuttling during active signaling involves embryos, and in whole living zebrafish embryos [21-23]. continuous (but low level) R-Smad dephosphoryla- Thus there is compelling evidence that Smad2 and 4 con- tion, which leads to dissociation of Smad complexes tinuously shuttle between the cytoplasm and the nucleus in the nucleus and export of monomeric Smads to the in unstimulated cells. cytoplasm. If the receptors are still active the R-Smads TGF-b stimulation triggers a relatively slow nuclear are rephosphorylated, form complexes and return to the accumulation of Smad2, 3 and 4, which reaches a maxi- nucleus. If the receptors are no longer active, the Smads mum approximately 45 min after ligand stimulation [24]. will reaccumulate in the cytoplasm [24]. Once accumulated, the bulk of Smads remain maximally nuclear for at least 4 to 5 h, after which they start to General principles of nucleocytoplasmic transport slowly relocalize to the cytoplasm [18]. The kinetics of Smad relocalization to the cytoplasm mirrors receptor Nucleocytoplasmic transport occurs through the nu- inactivation and hence the slow fading of the signal. This clear pore complex (NPC), which forms a hydrophobic has been demonstrated directly because if signaling is channel through the nuclear envelope. In mammalian terminated prematurely by adding a specific type I recep- cells, NPCs are made up of multiple copies of approxi- tor inhibitor SB-431542 [25, 26], nuclear accumulation mately 30 different nucleoporins [31]. Only relatively of Smads begins to decrease immediately, causing the small molecules can pass through the NPC by diffusion bulk of Smads to relocalize to the cytoplasm within 2 h and thus proteins larger than ~20–30 kD need to be ac- [24]. How do the nuclear Smads “know”, however, that tively transported through the nuclear pore. Interaction www.cell-research.com | Cell Research npg Smad nucleocytoplasmic shuttling 39 of transport substrates with the NPC and their subse- molecules and can interact with repeats of FG-dipep- quent translocation are frequently mediated by transport tides, which are a common structural motif found in the receptors, karyopherins [32]. These proteins bind cargo majority of nucleoporins. Such interactions enable the

Cytoplasm Nucleus

Ran EXP GTP Cargo

Ran EXP Ran GTP EXP Cargo GTP Cargo Export

RanGAP RanBP1

EXP EXP Cargo Cargo + Ran Ran Pi + GTP GDP

Ran Ran RCC1+GTP GDP GDP NTF2 NTF2

Ran Ran Pi + GDP GTP

IMP Cargo Cargo IMP

IMP Cargo Cargo Import Cargo Ran GTP IMP

RanGAP Ran RanBP1 GTP IMP

Cytoplasm Nucleus

Figure 2 Nuclear import and export cycles and the involvement of the RanGTPase system. For details see text. Adapted from [32].

Cell Research | Vol 19 No 1 | January 2009 Caroline S Hill npg 40 transporter-cargo complexes to pass through the nuclear the nucleus in a Ran- and energy-dependent manner [36]. pore. According to the transport direction, karyopherins Both groups found that nuclear import of the isolated are classified as importins or exportins [32]. MH1 domain was much stronger than import of the full- The directionality of nucleocytoplasmic transport is length protein in an in vitro import assay, and that im- established by the RanGTP gradient [32] (Figure 2). Ran portin b1 interacts much more strongly with the isolated is a small GTPase, which is mainly in its GTP-bound MH1 domain than with the full-length protein in GST- state in the nucleus, but in its GDP-bound state in the cy- pulldowns. In the same assays, TGF-b-dependent phos- toplasm. This is because RanGAP resides exclusively in phorylation of Smad3 seemed to enhance both its inter- the cytoplasm, which, together with RanBP1, catalyzes action with importin b1 and its nuclear import [36]. The cytoplasmic RanGTP hydrolysis. Conversely, the GDP- conserved NLS-like motif is non-functional in Smad2, exchange factor RCC1 is bound to chromatin and thus perhaps because of an insertion encoded by exon 3 of resides exclusively in the nucleus. Importins bind their Smad2, which is close to the NLS-like motif and might cargo in the cytoplasm without the need for RanGTP. In impair its accessibility for importin b1 binding [37] the classical pathway, the cargo is bound by one of the (Figure 1). The proposed mechanism deviates from the several variants of importin a through a basic nuclear lo- classical import mechanism by avoiding the need for one calization signal (NLS), which in turn recruits the actual of the several importin a proteins as a bridging factor transport receptor, importin b, forming a trimeric im- between importin b1 and the cargo. An NLS-like motif port complex. After transport into the nucleus, RanGTP in the MH1 domain of Smad1 has also been shown to be disrupts the import complex and the cargo protein is important for nuclear import of this BMP-responsive R- released into the nucleoplasm (Figure 2). Exportins in Smad [38]. contrast bind their cargo only in the presence of RanGTP In contrast to Smad3, Smad4 has been proposed to use (i.e. in the nucleus) by forming a heterotrimeric exportin- the classical pathway for nuclear import. An extended, cargo-RanGTP complex. After translocation of the export bipartite NLS has been identified in Smad4 (amino complex into the cytoplasm, RanGTP hydrolyzes its GTP acids 45-110), which overlaps with the corresponding with the help of RanGAP1 and RanBP1, causing dis- sequence motif responsible for Smad1 and 3 import, but sociation and release of the exported cargo into the cy- additionally extends into the DNA-binding region of the toplasm [32] (Figure 2). In addition to the karyopherin- Smad4 MH1 domain. The isolated Smad4 MH1 domain dependent transport, some proteins have been reported interacts with importin a1 through this motif [39]. to contact the NPC directly and enter or exit the nucleus In contrast to this karyopherin-dependent nuclear im- without the need for transport receptors [33]. port of Smad3 and 4, a very different mechanism for the import of Smad3, 4 and also 2 has also been described Mechanism of Smad transport between the cyto- [19]. Using mainly in vitro experiments, these authors plasm and the nucleus demonstrated that the main import activity for Smad2 and 3 resided in the linker-MH2 domain and import Smad import into the nucleus seemed entirely independent of karyopherins [19, 40]. A number of studies over the past years have ad- Rather, these Smads were thought to interact directly dressed the translocation mechanism of Smad nuclear with nuclear pore components, mainly with the nucleo- import and the ideas about how this process works have porin CAN/Nup214, which is located at the cytoplasmic changed over the past 8 years. In fact both karyopherin- side of the nuclear pore and Nup153, which is located at dependent import with the major import activity residing the nucleoplasmic side [19]. An identical mechanism was in the MH1 domain, and karyopherin-independent import proposed to be responsible for Smad4 import, except that mediated by direct contacts between nucleoporins and in this case the full-length protein was required for im- the MH2 domain, have been proposed. port [40]. A further follow-up study, aimed at assessing For Smad3, a karyopherin-dependent translocation the import mechanism by which TGF-b-induced homo- into the nucleus was described independently by two meric Smad3 and heteromeric Smad3–Smad4 complexes groups. One group identified a lysine-rich NLS-like are imported, demonstrated karyopherin-independent sequence in the MH1 domain of Smad3 (40KKLKK44), import for such complexes in an in vitro import assay [27]. which is conserved in all R-Smads [34]. In Smad3, this This view of Smad nuclear import has been revised motif was proposed to mediate karyopherin-dependent again more recently by some of the same authors as a nuclear import of Smad3 by binding to importin b1 di- result of a screen carried out in Drosophila cells look- rectly [35]. Similarly, another study demonstrated that ing for the importins required for nuclear accumulation Smad3 interacts with importin b1, and is imported into of Mothers against DPP (Mad), which is the Drosophila www.cell-research.com | Cell Research npg Smad nucleocytoplasmic shuttling 41 homolog of mammalian Smad1, in response to expres- A CRM1-dependent export mechanism identical to that sion of BMP receptors [41]. This screen identified the of Smad4 has been suggested for the BMP-responsive Drosophila protein mole-skin (Msk) as being important Smad1. First, a potential NES, termed NES1 was iden- for the nuclear import of phosphorylated Mad. These au- tified in the C-terminal region of the Smad1 MH2 do- thors also showed that knockdown of the mammalian or- main. Mutation of this motif caused Smad1 to localize thologs of Msk, importin 7 and 8 (Imp7 and Imp8) inhib- predominantly to the nucleus [38]. In addition, a second ited signal-induced nuclear accumulation of Smad1 and NES, termed NES2 and located in the linker adjacent to Smad2/3 in mammalian tissue culture cells [41]. Interest- the MH1 domain, was described [43]. NES2 partly over- ingly, knockdown of Imp7 and 8 either individually or in laps with the functional NES found in the corresponding combination had no effect on the subcellular localization region of Smad4 (Figure 1). Smad1 NES1 and NES2 are of Smad2/3 in unstimulated HeLa cells, leading the au- absolutely conserved among all the R-Smads, and al- thors to suggest that monomeric Smads are imported into though they seem to mediate CRM1-dependent export in the nucleus by a distinct mechanism from complexed the case of Smad1, they have no such activity in Smad2 Smads in ligand-induced cells, and that Smad monomers and 3, whose export is clearly CRM1-independent. There might be imported in a karyopherin-independent man- is currently no explanation as to why the motifs are func- ner as had been earlier suggested [19, 40]. Very recently, tional in Smad1 but not in Smad2 and 3. further work has investigated the possible mechanism Nuclear export of Smad2 and 3 is clearly independent of Imp7/8-mediated Smad import. Nuclear import of of CRM1, in both the absence and the presence of a Smad4, both in the presence and absence of signal has TGF-b signal [18, 19, 24, 27]. For Smad2, a karyopher- been shown to require Imp7 and 8; and a KKLK motif in in-independent export mechanism mediated by direct the MH1 domain, which is part of the originally identi- binding to nucleoporins has been suggested [19], similar fied NLS in Smad4 [39], was shown to be required for to what was originally proposed for Smad2/3 import. Smad4 import, although not directly for binding to Imp8. However, Smad3 has recently been shown to require a Taken together it seems clear that in the context of karyopherin for nuclear export, in this case exportin 4 Smad complexes after ligand stimulation, all the Smads [44], suggesting that Smad2 export is also likely to be require karyopherins to import them into the nucleus. karyopherin-dependent. For Smad1, 3 and 4, the basic NLS-like motifs in the N-terminal MH1 domains are required, although the situ- Mechanisms underlying TGF-β-induced nuclear ation is less clear for Smad2. Knockdown experiments accumulation of Smads strongly implicate Imp7 and 8, but it will also be important now to reassess the role of importin a and b. More Irrespective of the exact mechanism by which Smads work is needed to resolve the issue of whether R-Smad translocate through the NPC, it has been of great interest import in the basal state is dependent on karyopherins, or to determine how the steady-state distribution of Smads independent. in both the resting state and the presence of a TGF-b superfamily ligand is established. Smad export from the nucleus It is generally accepted that, in the absence of a TGF-b The mechanism of Smad4 export from the nucleus signal, Smad4 is distributed equally between the nucleus was identified in 2000 and was shown to be mediated by and the cytoplasm [18]. Such distribution is thought to be CRM1 [17, 18]. As mentioned above these experiments established by the constitutive nuclear import and export made use of the specific CRM1 inhibitor, LMB [16]. of Smad4, which are thought to be of comparable rates. Treatment of unstimulated cells with LMB triggered nu- In contrast, the R-Smads reside predominantly in the cy- clear accumulation of Smad4, demonstrating that Smad4 toplasm in the absence of a signal, although they are by export is CRM1-dependent. A canonical nuclear export no means excluded from the nucleus. This has been most signal (NES) was identified in the N-terminal part of the thoroughly studied for Smad2. Given the continuous linker region of Smad4 (142DLSGLTLQ149), and deletion exchange of R-Smads between the two compartments, or mutation of critical residues in this NES enhanced their nucleocytoplasmic distribution directly reflects their nuclear localization of Smad4 [17, 18]. Interestingly, a mean residence times in both compartments. The mean variant of Smad4, XSmad4b that exists only in X. laevis residence times are determined by the rates of nuclear and in no other vertebrates, lacks this NES and is consti- import and nuclear export, and by possible interactions tutively nuclear [42]. Hence, this NES seems to be nec- with retention factors that could cause temporary se- essary to mediate nuclear export of Smad4. questration of Smads from the transport machinery. The The situation is more complicated for the R-Smads. Smad-interacting FYVE-domain protein SARA [45] has

Cell Research | Vol 19 No 1 | January 2009 Caroline S Hill npg 42 been suggested to act as a retention factor for Smad2 in toplasm did not indicate mobilization of GFP-Smad2 in the absence of a signal [46]. This was on the basis of the response to TGF-b, as would be expected for an anchor- observation that addition of the recombinant Smad-bind- release mechanism. Nuclear mobilities of GFP-Smad2 ing domain of SARA in a nuclear import assay inhibited and GFP-Smad4, however, are slightly diminished in nuclear import of Smad2 in vitro, and overexpression of TGF-b-induced cells, pointing to possible nuclear tether- SARA targeted Smad2 to early endosomes in vivo [46]. ing of active Smad complexes [23]. Compartment-spe- However, in cell lines expressing low levels of GFP- cific photoactivation revealed that export rates of GFP- Smad2 no localization to early endosomes is observed Smad2 are strongly decreased in TGF-b-induced cells and GFP-Smad2 shows a rather diffuse cytoplasmic when compared with uninduced cells, whereas import distribution [22], as does endogenous Smad2 [18]. Both rates are apparently unchanged (but see below). These findings together indicate that the amount of SARA is data suggest that neither increased import nor release limiting and is unlikely to bring about quantitative cyto- from cytoplasmic retention plays a predominant role in plasmic sequestration of Smad2. Alternatively, the pre- TGF-b-induced R-Smad nuclear accumulation in vivo. dominantly cytoplasmic localization of R-Smads could Rather, selective trapping of active Smad complexes in result from R-Smad import rates simply being weaker the nucleus from a cycling pool of monomeric Smads than the export rates. Quantitative kinetic studies on the is likely to be the most important mechanism [23]. The import and export of GFP-Smad2 have proven that this is nuclear trapping may be due to binding of Smad com- indeed the case [23]. plexes to nuclear structures, which would explain the How does ligand stimulation then lead to nuclear ac- observed decrease in mobility. For example, this could cumulation of active Smad complexes? Four distinct reflect weak binding to DNA, where Smad complexes mechanisms could theoretically account for the nuclear scan for their optimal binding sites in promoters of tar- accumulation of a continuously shuttling, predominantly get genes. Alternatively, Smad complexes may not be cytoplasmic protein in response to ligand stimulation. Li- exported simply because they are not recognized by the gand induction could lead to a release from cytoplasmic export machinery as has already been shown for Smad4 anchoring or to the establishment of nuclear anchoring. [27]. Interestingly, the transcriptional regulator TAZ has Alternatively, the presence of a signal could increase the very recently been shown to be involved in retaining rate of nuclear import or decrease the rate of nuclear ex- Smad complexes in the nucleus. When TAZ is knocked port, or both. All four mechanisms could lead to a change down Smad2/3–Smad4 complexes form, but fail to accu- in mean residence times in either compartment or in both mulate in the nucleus [47], and TAZ appears to be able to compartments, and thus cause a predominantly nuclear bind with active Smad complexes to promoter elements. steady-state distribution. Whether binding to TAZ in the nucleus is sufficient to As Smad4 is dispensable for nuclear accumulation of account for the measured decrease in nuclear export rate R-Smads and its own nuclear accumulation is strictly de- of Smad complexes relative to monomeric Smads re- pendent on R-Smad accumulation [8, 27, 37], the mecha- mains to be investigated. nism of R-Smad nuclear accumulation is of most interest. Fluorescence perturbation experiments in conjunc- An anchor-release model was originally suggested for tion with siRNA knockdown of different components, Smad2 based on the finding that overexpression of SARA inhibition of signaling by small molecules and protein sequesters Smad2 into clusters in the cytoplasm [45, 46], interaction studies in vitro can lead to a hypothesis of and that overexpression of the Smad2-interacting nuclear how ligand stimulation leads to nuclear accumulation protein FAST-1/FoxH1 traps Smad2 in the nucleus even of active Smad complexes. However, experimentally it in uninduced cells [19]. In this model, phosphorylation- is impossible to determine whether such a hypothesis is dependent disruption of the Smad2–SARA interaction consistent with the observed kinetics of nuclear accumu- [45] enables phosphorylated Smad2 to move into the lation of activated Smad complexes and their behavior nucleus, where it is retained by means of phosphoryla- after receptor inactivation, as many critical reactions tion-dependent interactions with the transcription factor within this complex signaling network are not amenable FoxH1/FAST-1 or related proteins [19]. However, no to experimental investigation. To overcome these prob- loss-of-function experiments have been performed to lems a mathematical model was developed [48] based on show that SARA and FoxH1/FAST-1 actually perform the kinetic measurements that had been made in epitheli- these functions when present at their endogenous levels. al cells, stably expressing GFP-tagged Smad2 [23], using More recently, a detailed kinetic analysis of Smad the network topology outlined in Figure 3. The model nucleocytoplasmic shuttling has led to a different view simultaneously fitted four independent datasets with ex- [23] (Figure 3). FRAP mobility measurements in the cy- cellent accuracy and moreover fitted a dataset not used in www.cell-research.com | Cell Research npg Smad nucleocytoplasmic shuttling 43

TGF-β

Plasma P membrane P TβRII TβRI/ALK5

Smad2 Smad2 Smad4 Smad4 P P

Nuclear envelope CRM1

P P Smad4 Smad2 Smad2 Smad4 Gene TF TAZ regulation

PPM1A

TAZ Nuclear retention

TAZ

Figure 3 A model for nucleocytoplasmic shuttling of Smads in uninduced and in TGF-β-stimulated cells. The shaded portion rep- resents Smad shuttling in unstimulated cells. Receptor activation by TGF-β ligand binding leads to R-Smad phosphorylation and complex formation with Smad4. In the absence of signaling, Smad2 appears cytoplasmic because its export rate is faster than its import rate. In these conditions Smad4 is distributed approximately equally between the cytoplasm and nucleus. Upon ligand induction Smad complexes accumulate in the nucleus, as a result of their import being faster than that of monomeric Smads and their export being inhibited, possibly as a result of nuclear retention. The transcriptional regulator TAZ has been shown to be required for ligand-induced Smad nuclear accumulation. Smad complexes regulate transcription in conjunction with other transcription factors (TF). The phosphatase, PPM1A has been proposed to be the Smad2 C-terminal phosphatase. Adapted from [23]. For further details, see text.

its construction [48]. In doing so, it verifies the plausibil- lated Smad3 interacts more efficiently with importin b1 ity and mechanistic relevance of the underlying network than unphosphorylated Smad3, and is imported into the topology. Most interestingly, the mathematical model has nucleus more efficiently [36]. The mathematical model- led to additional mechanistic insights. First, it was evi- ing also indicated that the C-terminal Smad phosphatase dent that although inhibition of Smad export on TGF-b must reside in the nucleus. stimulation was essential, it was not sufficient to cause the observed kinetics of Smad nuclear accumulation on The biological function of Smad nucleocytoplasmic TGF-b signaling. The mathematical model requires that shuttling the import of Smad complexes into the nucleus should be approximately 5 times faster than the import of mo- Appropriate reaction of cells to external stimuli re- nomeric Smads [48]. Although this increase in Smad quires exact timing of both the initiation and the dura- import rate was not measureable in vivo [23], it was tion of the specific response. It is also important that earlier shown using in vitro experiments that phosphory- the strength of the response in terms of levels of active

Cell Research | Vol 19 No 1 | January 2009 Caroline S Hill npg 44 nuclear Smads matches the level of receptor activation significance of Smad nucleocytoplasmic shuttling in the at all times. Activation of Smads by TGF-b in epithelial presence of signal is that it provides a mechanism where- cells proceeds relatively slowly and is maintained over by the intracellular transducers of the signal can continu- extended time periods. Maximal activation is reached ously monitor receptor activity, an essential requirement after about 45 min of active signaling in cell culture [18, of signaling in multicellular organisms. It is now appar- 24] and is characterized by plateauing levels of phospho- ent that this is a common mechanism employed also by rylated R-Smads and maximal nuclear accumulation of other signaling pathways. Since the original Smad nucle- Smads. Both features are maintained for several hours. ocytoplasmic shuttling hypothesis was proposed [24], a It is not until the signal ceases, i.e. the actively signaling very similar model has been suggested for the JAK/STAT receptors have been inactivated by either dephosphoryla- pathway and for serum-induced signaling via the serum tion or degradation after 4-5 h, that the bulk of the Smads response factor and its co-activator Mal [50-52]. redistribute into the cytoplasm. In some pancreatic cancer cells the duration of signaling was found to be at- Acknowledgments tenuated, and this correlated with lower receptor levels in these cells [10]. These observations, together with the I would like to thank Becky Randall and Mike Howell finding that premature termination of active signaling by (CRUK London Research Institute) for useful comments receptor inhibition causes the Smads to redistribute into on the paper and all members of the Hill lab past and the cytoplasm, led to the idea that nucleocytoplasmic present, in particular Bernhard Schmierer (Oxford centre Smad shuttling provides a sensing mechanism whereby for integrative systems biology), for stimulating discus- the Smads themselves directly monitor receptor activity sions about Smad nucleocytoplasmic shuttling. The Hill [24]. Thus, as the receptors are inactivated, the signal lab is funded by Cancer Research UK and the European fades and the pool of active Smads in the nucleus dimin- Commission. ishes. In contrast, if the receptors remain highly active for a long period of time, a pool of active nuclear Smads References will be maintained. 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Cell Research | Vol 19 No 1 | January 2009 Caroline S Hill npg 46 2008; 105:6608-6613. 51 Vartiainen MK, Guettler S, Larijani B, Treisman R. Nuclear 49 Schmierer B, Hill CS. TGFb-SMAD signal transduction: mo- actin regulates dynamic subcellular localization and activity of lecular specificity and functional flexibility. Nat Rev Mol Cell the SRF cofactor MAL. Science 2007; 316:1749-1752. Biol 2007; 8:970-982. 52 Vinkemeier U. Getting the message across, STAT! Design 50 Meyer T, Vinkemeier U. Nucleocytoplasmic shuttling of STAT principles of a molecular signaling circuit. J Cell Biol 2004; transcription factors. Eur J Biochem 2004; 271:4606-4612. 167:197-201.