Ludwig Institute for Cancer Research, Box 595, SE-751 24 Uppsala, Sweden
Corresponding author (e-mail: Aris.Moustakas{at}LICR.uu.se)
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SUMMARY |
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Key words: Phosphorylation, Signal transduction, Smad, Transforming growth factor-ß, Ubiquitination
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Introduction |
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Smads: a conserved family of signal transducers |
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Regulation of Smad function by phosphorylation |
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Although 2D phosphopeptide maps of ectopically overexpressed R-Smads are rather simple (Abdollah et al., 1997; de Caestecker et al., 1998; Macías-Silva et al., 1996), analysis of endogenous mammalian Smads reveals >10 different phosphopeptides (Souchelnytskyi et al., 1997; Yakymovych et al., 2001). Other kinases might therefore phosphorylate the Smads. Indeed, the latter contain phosphorylation sites for Erk-family MAP kinases (Kretzschmar et al., 1997), the Ca2+/calmodulin-dependent protein kinase II (CamKII) (Wicks et al., 2000) and protein kinase C (PKC) (Yakymovych et al., 2001) (Table 1).
Erk phosphorylates serine residues in the linker regions of Smad1 (Kretzschmar et al., 1997), Smad2 and Smad3 (Kretzschmar et al., 1999), and substitution of these serines by negatively charged residues inhibits nuclear translocation of Smads and thus signalling. Similarly, CamKII can phosphorylate Smad2 in vitro at linker-region residues Ser240 and Ser260 (as well as at Ser110 of the MH1 domain), which again inhibits nuclear translocation and signalling. Significantly, phosphorylation of Ser240 was observed in vivo upon treatment of cells with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). PKC phosphorylates Smad2 in vivo and in vitro at Ser47 and Ser110, and Smad3 at the analogous Ser37 and Ser70 (Yakymovych et al., 2001). Phosphorylation of Smad3 by PKC blocks DNA-binding and consequently transcriptional regulation. At the cellular level, this inhibits TGF-ß-induced apoptosis and increases susceptibility of cells to loss of contact inhibition (Yakymovych et al., 2001).
In several other cases, the underlying mechanism of Smad phosphorylation remains to be determined. de Caestecker et al., for example, demonstrated that Erk phosphorylates Smad2 in response to EGF or hepatocyte growth factor (HGF) at the C-terminal SSXS motif and thereby activates the Smad pathway (de Caestecker et al., 1998). The molecular mechanisms of synergistic activation of Smad2/3-mediated transcriptional responses by two other kinases, MEKK-1 and Jun N-terminal kinase (JNK), which phosphorylate unknown residues outside the SSXS motif, also need further investigation (Brown et al., 1999; Engel et al., 1999).
Phosphorylation of the Co-Smad, Smad4, has not been reported in mammals. However, in Xenopus, one of two Smad4 isoforms, Smad4ß, is phosphorylated, whereas the other, Smad4, is not (Howell et al., 1999; Masuyama et al., 1999). The Smad4ß phosphorylation sites and their importance for signalling remain unknown.
The I-Smads, Smad6 and Smad7, are phosphorylated by as-yet uncharacterised kinases (Imamura et al., 1997; Pulaski et al., 2001). Smad6 phosphorylation sites and their importance for signalling remain unexplored, although phosphorylation may not be mediated by the TGF-ß and BMP receptor kinases (Imamura et al., 1997). Smad7 is phosphorylated at Ser249, and this depends on the proliferation status of cells but not on TGF-ß receptor signalling (Pulaski et al., 2001). Although phosphorylation of Ser249 regulates the transcriptional activity of Smad7 (Pulaski et al., 2001), its role in regulation of transcription during TGF-ß superfamily or independent signalling remains to be uncovered.
Thus, phosphorylation not only activates Smad proteins but also modulates their activity. This provides a mechanism for integration of the Smad pathway with other signalling pathways that modulate TGF-ß superfamily signal transduction.
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Smad oligomerisation and activation |
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Early experiments indicated that oligomeric Smads are trimers (Kawabata et al., 1998; Shi, 2001). Equilibrium centrifugation and crystallographic studies have confirmed this in the case of Smad3 (Chacko et al., 2001; Correia et al., 2001). However, Wu et al. have recently proposed a dimeric configuration for the Smad2-Smad4 complex (Wu et al., 2001). Thus, different R-Smad-Co-Smad oligomers with distinct stoichiometries are possible (Fig. 3). This notion is supported by a recent analysis of native cellular Smads using gel chromatography (Jayaraman and Massagué, 2000). No information regarding the oligomeric status of BMP-specific R-Smads is currently available. Structural studies of different R-Smad-Co-Smad complexes are needed to resolve the important problem of their stoichiometry.
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Organising Smad signalling centres |
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Several other proteins with possible roles in Smad anchoring have recently been described. Microtubules can anchor inactive Smads in the cytoplasm (Dong et al., 2000). Activation by a ligand results in dissociation of the Smads from the microtubule network. In fact, pharmacological disruption of microtubules leads to aberrant and constitutive activation of the Smad pathway. It is possible that microtubules serve as tracks for intracellular Smad movement. Filamin, an actin crosslinking factor and scaffolding protein, also associates with Smads and positively regulates transduction of Smad signals (Sasaki et al., 2001). Another example of a receptor- and Smad-associating scaffolding protein is caveolin 1, which interacts with the type I receptor and mediates localisation of the receptor complexes to caveolae, thus inhibiting Smad2-mediated signalling (Razani et al., 2001). Proteins of the sorting nexin (SNX) family of vesicle- and receptor-trafficking adaptors also interact with TGF-ß receptor complexes (Parks et al., 2001). Similarly, ARIPs (activin receptor interacting proteins) associate with Smad2 and enhance Smad2-mediated signalling in response to activin (Tsuchida et al., 2001). In addition, GIPC (GAIP-interacting protein, C-terminus) is a scaffolding protein for G subunits that associates with clathrin vesicles and interacts with the proteoglycan-like, type III TGF-ß receptor, enhancing Smad-mediated signalling (Blobe et al., 2001). Both ARIPs and GIPC are PDZ-domain-containing proteins that serve as multiprotein-complex organising centres (Harris and Lim, 2001). Finally, TRAP1 (TGF-ß receptor type I associated protein 1) associates with Smad4 and is proposed to serve as a Smad4 anchor that lies proximal to the receptor complex and might assist formation of R-Smad-Co-Smad oligomers (Wurthner et al., 2001).
The available data support the notion that interactions between TGF-ß superfamily receptors and Smads with adaptor/scaffolding proteins are an important regulatory mechanism. Proper receptor localisation in plasma membrane or endocytic vesicle microdomains, their proximity to cytoplasmic anchors that hold the Smads and the ability of such complexes to be mobilised between various cytoplasmic compartments are exciting new aspects of the regulation of Smad signalling (Fig. 4). Such mechanisms could provide cell-context specificity, allowing differential regulation of the basic Smad pathway.
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Nucleocytoplasmic shuttling |
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The nuclear import mechanisms of Smad1, Smad2 and Smad3 have been analysed in detail (Kurisaki et al., 2001; Xiao et al., 2000; Xiao et al., 2001; Xu et al., 2000). The MH1 domains of all eight Smads each contain a lysine-rich motif that in the case of Smad1 and Smad3 has been shown to act as a nuclear localisation signal (NLS) (Xiao et al., 2000; Xiao et al., 2001) (Fig. 2). In Smad3, C-terminal phosphorylation results in conformational changes that expose the NLS so that importin ß1 can bind and mediate Ran-dependent nuclear import (Kurisaki et al., 2001; Xiao et al., 2000) (Fig. 5). In contrast, Smad2, which has the same lysine-rich sequence in its MH1 domain, is released from the anchoring SARA after C-terminal phosphorylation and then translocates into the nucleus by a cytosolic-factor-independent import activity that requires a region of the MH2 domain (Xu et al., 2000) (Fig. 5). The difference between the two R-Smads of the TGF-ß and activin pathways is due to the presence of the unique exon 3 in the MH1 domain of Smad2 (Kurisaki et al., 2001) (Fig. 2). Thus, the lysine-rich sequence of the Smad MH1 domain may not be fully functional in all Smads, perhaps because of the unique structural determinants in each Smad. Whether the two different mechanisms of Smad2 and Smad3 nuclear import also reflect differences in their oligomerisation status remains unclear.
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Along with other Smads, Smad1 has recently been shown to have both ligand-dependent import and constitutive export activities (Xiao et al., 2001). The latter depends on an NES in the MH2 domain, which is N-terminal to the L3 loop (Fig. 2). This is conserved among all Smads but proposed to be active only in certain Smads. Smad2 and Smad3 also exit the nucleus, but this occurs after prolonged treatment with TGF-ß (Pierreux et al., 2000). The putative NESs in Smad2 and Smad3 have not been identified. It is also unclear whether their export mechanisms depend on specific exportins. Finally, I-Smads are constitutively imported to the nucleus and are exported to the cytoplasm in response to TGF-ß or BMP signalling (Itoh et al., 1998; Itoh et al., 2001). The functional NLSs and NESs and the mechanisms of regulation of I-Smad nucleocytoplasmic shuttling have not been characterised yet.
The physiological significance of selective regulation of the subcellular distributions of different Smads is hard to understand at this point, when only limited comparative analyses of these mechanisms are available.
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Nuclear signalling |
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Smad3 and Smad4 also associate with GC-rich motifs in promoters of certain genes, which demonstrates a relaxed DNA-binding specificity of the Smad MH1 domain (Labbé et al., 1998). The BMP-responsive Smads also have highly conserved ß-hairpin loops and thus are predicted to bind to SBEs, as has been recently demonstrated in the case of Smad5 (Li et al., 2001). Alternatively, BMP-dependent R-Smads can directly, but very weakly, bind to GC-rich motifs in several Drosophila promoters and one mammalian (MADH6) promoter (Ishida et al., 2000; Kim et al., 1997).
All the above examples involve Smad-mediated activation of gene expression. Recently, however, the first examples of Smad-dependent gene repression were uncovered. The DNA elements involved do not resemble SBEs or GC-rich motifs (Alliston et al., 2001; Chen et al., 2001). Whether Smads associate directly with such elements remains to be examined.
The fact that GAL4-Smad chimeras exhibit transcriptional activity in mammalian and yeast cells indicated that Smads might associate with the basal transcriptional machinery (Liu et al., 1996). The transactivation function of Smads maps to the MH2 domain and is mediated by direct association of the MH2 domain with co-activators of the p300 and P/CAF (p300- and CBP-associating factor) families (Itoh et al., 2000a; Itoh et al., 2000b) (Fig. 6). Smad4 appears to play a crucial role in regulating the efficiency of transactivation of the Smad complexes in the nucleus. This is thought to involve the unique Smad-activation domain (SAD) of Smad4, which allows stronger association with the p300/CBP co-activators and confers a unique conformation on the Smad4 MH2 domain (Chacko et al., 2001; de Caestecker et al., 2000b) (Fig. 2).
As mentioned above, Smad signalling can also lead to repression of gene expression. Smad3 has been reported to associate with histone deacetylase (HDAC) activities through its MH1 domain, but whether Smads interact directly with HDACs remains unclear (Liberati et al., 2001). Alternatively, Smads can interact with co-repressors that recruit HDACs (Fig. 6). These co-repressors include the homeodomain DNA-binding protein TGIF (Wotton et al., 1999) and the proto-oncogene products Ski and SnoN (Liu et al., 2001). Such co-repressors appear to modulate the nuclear activity of Smads, and their levels of expression define the level of Smad transcriptional activity.
SnoN illustrates an interesting example of a nuclear feedback loop (Liu et al., 2001; Stroschein et al., 2001). SnoN basal levels have been proposed to maintain TGF-ß-responsive genes in a repressed state. When a cell is stimulated by TGF-ß, the incoming nuclear Smads target SnoN for ubiquitination and degradation (see below), thus relieving repression and possibly allowing other Smad complexes to activate transcription of target genes. One such gene is SnoN itself, which presumably re-represses target genes as soon as the nuclear Smad signal declines. Whether such a model applies generally to many TGF-ß superfamily gene targets or to selected groups of genes remains to be elucidated.
Therefore, the required transcriptional specificity of the Smad pathway is achieved through multiple SBE motifs in promoters of Smad-target genes, which confer higher Smad-binding affinity, and additional transcription factors that cooperate with the Smads (Fig. 6). The in vivo characteristics of such transcriptional complexes and their dynamic interaction with chromatin remain largely unexplored. However, the list of Smad-interacting transcription factors is large (Table 1), providing a mechanistic basis for cell-type- and context-specific gene regulation. Because Smad-interacting transcription factors have been recently reviewed exhaustively (Itoh et al., 2000b; Massagué and Wotton, 2000), we do not discuss all cases of such transcription factors here. The plethora of interacting proteins provides a mechanistic explanation for the documented crosstalk between the Smad pathway and many other signalling networks, which range from the Ras/MAPK pathway to the Wnt/ß-catenin and nuclear hormone signalling cascades (Itoh et al., 2000b; Massagué, 2000).
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Smad degradation and roles in protein ubiquitination pathways |
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Finally, proteasomal degradation of Smad4 occurs in tumour cells, which either harbour deleterious mutations in MADH4 or express activated oncoproteins such as Ras; the specific ubiquitination mechanism is as yet elusive (Maurice et al., 2001; Morén et al., 2000; Saha et al., 2001; Xu and Attisano, 2000). Whether mediated by Smurfs or other E3 ligases, the ultimate degradation of nuclear Smads after prolonged ligand stimulation has been firmly established as a mechanism that shuts off the signalling pathway.
Recent findings underscore additional roles of proteasomal degradation in the control of Smad signalling. The Smurfs can also regulate ubiquitination and degradation of other target proteins, including the TGF-ß receptor complex and the transcriptional co-repressor SnoN (Bonni et al., 2001; Ebisawa et al., 2001; Kavsak et al., 2000; Stroschein et al., 2001) (Fig. 7). Smurf1 and Smurf2 associate with the nuclear I-Smad Smad7 after stimulation by TGF-ß (Ebisawa et al., 2001; Kavsak et al., 2000). The I-SmadSmurf complex is exported to the cytoplasm and ubiquitinates the receptors on the cell surface or endosomal membranes; these are then targeted for degradation in proteasomes and lysosomes. Whether Smad6 plays a similar adaptor role in ubiquitination remains to be examined. An additional inhibitor that recruits Smad7 to the receptor is STRAP, a WD domain protein that binds to both the MH2 domain of Smad7 and the type I receptor (Datta and Moses, 2000). It would be interesting to test whether STRAP participates in Smurf-mediated ubiquitination of the type I receptor. A search for Smad-interacting proteins uncovered the human enhancer of filamentation 1 (HEF1), whose levels are regulated by proteasomal degradation induced by the Smad pathway (Liu et al., 2000). Thus, the paradigm of Smads acting as mediators of ubiquitination of cellular proteins may be extensive and involve various different mechanisms and molecular partners (Fig. 7).
Alternatively, when entering the nucleus, activated Smad2 and Smad3 can interact with Smurf2 and with the anaphase-promoting complex (APC) and thus stimulate ubiquitination of SnoN, for example, with which the Smads and E3 ligases interact. This leads to the efficient and rapid elimination of the SnoN co-repressor (Bonni et al., 2001; Stroschein et al., 2001) (Fig. 7). The mechanisms by which TGF-ß signals might discriminate among Smurfs that target the Smads themselves, as opposed to other protein targets, such as SnoN or the receptors, when uncovered, may point to novel means of regulation of Smad signalling.
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Perspectives |
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The recent advent of functional genomics and the ability to globally monitor gene expression at the RNA and protein levels provides an important approach for the future. A major quest is to identify co-regulated groups of genes that respond to TGF-ß superfamily signals and classify them on the basis of their mode and kinetics of regulation, the functions of the encoded proteins and the cell type and developmental context. Indeed an effort exploiting oligonucleotide and cDNA microarray analysis has already revealed a large number of genes that are regulated by TGF-ß (Akiyoshi et al., 2001; Chen et al., 2001; Verrecchia et al., 2001; Zavadil et al., 2001). For this task, the use of cells derived from mice in which specific genes are inactivated will be valuable, as exemplified by recent reports that have assigned differential outputs to the Smad2, Smad3 and Smad4 signals (Piek et al., 2001; Sirard et al., 2000). In addition, since the Smad pathway provides a plethora of interacting signalling factors, proteomic screens will provide the complete repertoire of Smad-interacting proteins, and again a major goal should be to associate these factors and signalling networks with the physiology or pathology of specific cell types. To this end, systematic analysis of model organisms, especially invertebrates, will consolidate the biological relevance of complex signalling networks (Padgett and Patterson, 2001). The new technologies also hold promise for a better understanding of the contribution of Smads to various disease conditions and thus may provide novel drug targets.
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ACKNOWLEDGMENTS |
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