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STAT signalling in cell proliferation and in development
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STAT signalling in cell proliferation and in development Jeffrey G Williams Recent advances in STAT signalling research include a better understanding of the roles of mammalian STAT proteins in cell proliferation and apoptosis, and of non-mammalian STAT proteins in morphogenesis. Two different ways in which STAT signalling pathways can interface with Smad signalling pathways significantly increasing combinatorial signalling possibilities, have also been described. Addresses Department of Anatomy and Physiology, University of Dundee, Wellcome Trust Building Complex, Dow Street, Dundee DD1 5EH, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:503–507 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations BMP2 bone morphogenetic protein-2 EGF epidermal growth factor ES embryonic stem JAK Janus kinase LIF leukemia inhibitory factor NLS nuclear localisation signal PDGF platelet-derived growth factor STAT signal transducer and activator of transcription β transforming growth factor factor-β TGF-β TNF tumor necrosis factor
Introduction The STAT (signal transducer and activator of transcription) proteins were discovered as mediators of cytokine signalling in mammals. STATs 1, 2, 4 and 6 have relatively restricted functions, centred around disease resistance (reviewed in [1–3]). STAT3 and STAT5, however, have broader functions and STATs have also been discovered in non-mammalian organisms. The STATs interact with a number of other cellular proteins, some of which act as specific negative regulators of their function. This is a very active area of research with considerable recent progress [3] but in this review I focus on the mechanism of STAT activation and on new cellular and developmental roles for a number of different STAT proteins.
STAT structure and the JAK-STAT pathway Mammalian STAT proteins contain three conserved domains: a DNA binding domain, an SH2 domain and a site of tyrosine phosphorylation (Figure 1). These are situated in the approximate centre of the protein. In their carboxy-terminal regions, most mammalian STAT proteins contain a transcriptional activation domain. The amino-terminal proximal region interacts with other cellular proteins and very near to the amino terminus there is a weakly conserved region that directs the oligomerisation of STAT dimers [3]. When a cytokine binds to its receptor (Figure 2), it induces conformational changes — most likely including
Figure 1 DNA binding domain
SH2 domain
Transcriptional activation
Y Site of tyrosine phosphorylation Current Opinion in Genetics & Development
A diagrammatic representation of a typical STAT protein. The functions of the marked domains are described in the text. The crystal structures of the STAT1 homo-dimer [39] and STAT3 homo-dimer [31] are known and, despite the relatively weak primary sequence conservation in their amino-terminal halves, the two structures are highly similar. The STAT dimer forms a plier-like structure, with the handles of the pliers wrapped around the DNA. Each partner STAT contacts the DNA independently and the interaction interface of the two STATs is in the nose of the pliers, linking the SH2 domain on one STAT and the phosphorylated tyrosine on the partner STAT molecule. The consensus DNA binding site for STAT dimers, for instance the site used by STAT1 when it is activated by interferon-γ (the ‘GAS element’), is the dyad TTCnGAA, where n from is variable from STAT to STAT.
oligomerisation or multimerisation — and this activates JAKs (Janus kinases). JAKs are tyrosine kinases, one of which is constitutively associated with each receptor chain. The activated JAKs autophosphorylate, and/or transphosphorylate, and then phosphorylate the receptors. Classically the phosphorylated receptor tyrosine motifs act as docking sites for the SH2 domains of STATs, although recruitment of certain STATS at least can also occur independently of the SH2 domains and through the JAKs. In the case of the interferon-γ pathway, a STAT1 molecule is recruited to each of two IFNGR1 subunits in the minimally tetrameric (2 times IFNGR1/2) receptor. In other cases (e.g. interferon-α), two different STAT proteins, STAT1 and STAT2, are recruited. The JAKs then tyrosine phosphorylate the STAT molecules and this triggers a re-shuffle; the STAT monomers dissociate from the receptor and dimerise with one another via reciprocal SH2 domain:phosphotyrosine interactions. The STAT homo- or heterodimers migrate to the nucleus and, with or without interaction with additional specificity modulating factors (e.g. p48 in the case of interferon-α), interact with transcriptional co-activators and the target DNA to activate transcription.
The evolution of STAT signalling: some recent additions to the STAT family and alternative ways of activating STATs Although the STAT proteins were discovered in studies of cytokine signalling in mammalian cells, they have now been discovered in other eukaryotic lineages. These include organisms as widely diverged as Drosophila and
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Differentiation and gene regulation
Figure 2
(a) Ligand
Receptor Plasma membrane JAK
JAK
their appearance before the divergence of the animals, fungi and the Dictyostelids [5]. It is therefore very surprising that neither of the yeasts for which a total genome sequence is available, Schizosaccharomces pombe and Saccharomyces cerevisiae, has a recognisable homologue of the STAT proteins. Indeed, neither of these two ‘lower’ fungi possesses an SH2 domain. One possibility is that the common ancestor of the higher and lower fungi utilised SH2 domain signalling and that these pathways were lost during the evolution of modern lower fungi. Another possibility, revealed by the recently determined structure of the Cbl signalling protein, is that we are failing to recognise SH2 domains when they are highly diverged in primary sequence [6].
(b)
Ligand
Receptor
JAK
JAK
P
P
STAT monomer
Just as the range of organisms known to possess STATs has extended, so too has the range of upstream signalling pathways known to activate them. Cytokine receptors are single membrane-pass receptors but seven trans-membrane pass (serpentine) receptors are also now known to activate STATs (reviewed in [7]). These receptors normally function by activating heterotrimeric G proteins but, in this case, a G-protein-independent-pathway seems to be utilised. There is also no obligate requirement for a JAK, because other kinases, such as the EGF and PDGF receptors, can phosphorylate STATs in vitro (reviewed in [8]). A recent in vivo analysis, however, suggests that the c-Src tyrosine kinase is recruited to the ErbB1 receptor upon EGF stimulation and that it is c-Src that actually activates STAT1, STAT3 and STAT5 [9].
The role of tyrosine phosphorylation (c)
P P
STAT dimer
Nucleus Current Opinion in Genetics & Development
The JAK-STAT signalling pathway. The process is simplified and, for clarity of presentation, divided into three steps (but see the main text for more detail). (a) Ligand binding to the receptor multimerises the receptor chains and this activates the JAKs. The JAKs then tyrosine phosphorylate the receptor chains. (b) STAT monomers are recruited by interaction of their SH2 domains with the tyrosine phosphorylated receptor chains. The JAKs then tyrosine phosphorylate the STATs. (c) The STATs dissociate from the receptor and re-associate with each other to form a dimer. The STAT dimer translocates to the nucleus and binds to regulatory DNA elements.
Dictyostelium. There is also evidence, based upon sequence homology, for proteins with a STAT-like structure in higher plants [4]. Ancestral plants are believed to have made
The generally accepted scheme for the series of events after STAT tyrosine phosphorylation is that the resultant STAT dimerisation causes translocation from the cytoplasm to the nucleus. In a mutant form of STAT1, where the site of tyrosine phosphorylation is altered to phenylanine and dimerisation is thereby prevented, nuclear accumulation does not occur [10]. The key role of dimerisation is also supported by a recent study using a fusion protein in which the ligand-binding/dimerisation domain of the oestrogen receptor was fused to the carboxyl terminus of the STAT1 protein [11]. This protein dimerises, translocates to the nucleus and binds DNA upon addition of estrogen. As the fusion protein does not become tyrosine phosphorylated after estrogen treatment, it would seem that dimerisation is sufficient to generate a nuclear import signal. Confusingly, however, other observations argue against a generalised, obligate role of dimerisation in STAT nuclear translocation [12–14]. Human fibroblasts cells undergo apoptosis when treated with tumour necrosis factor (TNF). However, because STAT1 is required for the constitutive expression of several caspases that are essential for apoptosis, mutant cells in which STAT1 is absent are resistant to TNF [12]. When null cells are transformed with a construct expressing STAT1, they are rendered sensitive to TNF-induced
STAT signalling in cell proliferation and in development Williams
apoptosis. Surprisingly, however, a form of STAT1 in which the site of tyrosine phosphorylation is mutated also restores apoptotic sensitivity. Thus a STAT1 function that presumably requires nuclear localisation seems to be occurring independently of the ability to dimerise. Furthermore, EGF treatment has been shown to induce nuclear translocation of STAT2 without an increase in STAT2 tyrosine phosphorylation [13]. Again, therefore, tyrosine phosphorylation seems not to be essential for nuclear translocation. Nor, apparently, is it sufficient, because src tyrosine kinase induces tyrosine phosphorylation of STAT5A without causing it to translocate to the nucleus [14].
Nuclear compartmentalisation of STAT proteins What the above observations imply is that there is no general pathway of STAT nuclear translocation. The STAT family members can behave quite differently from one another and an individual STAT can behave differently in different contexts. They also indicate the need for a better understanding of the precise mechanism of STAT nuclear localisation. The nuclear localisation signal (NLS) is a short lysine-rich sequence that is the binding site for an importin α, the class of adaptor proteins that mediates interaction of the NLS with the nuclear-import machinery (reviewed in [15]). This signal is present in proteins that are targeted to the nucleus but mutational analysis suggests that STAT1 does not contain a lysine-rich NLS [16]. Proving the absence of a short protein motif, of variable sequence composition, is inherently very difficult but Sekimoto et al. [16] show that different parts of importin α are necessary for STAT binding than are required for NLS binding. This strongly suggests that the importin α binding site in STAT1 is not a classic NLS. The importin α binding site has not been defined but in STAT5B, a conserved region (VVVI) within the DNA-binding domain is essential for nuclear import [17]. There is also a mechanism that recycles STAT molecules, back from the nucleus to the cytoplasm, and this has been shown to involve tyrosine dephosphorylation within the nucleus [18•]. Again, however, the mechanism of STAT nuclear export has not been elucidated in any detail.
STAT signalling and non-vertebrate development In Drosophila there is a genetically well characterised STAT signalling pathway comprising a JAK, encoded by the hopscotch gene, and a STAT encoded by the stat92E gene [19,20]. Female flies with mutations in either gene produce embryos with highly characteristic segmentation defects. Mutation of the unpaired gene yields embryos with similar segmentation defects and the properties of the Unpaired protein suggest very strongly that it is the ligand that activates the pathway; it is a glycoprotein of the extracellular matrix and, when ectopically expressed in cultured Drosophila cells, it directs activation of Hopscotch in co-cultured cells [21].
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The Drosophila JAK–STAT pathway is now also known to function in eye development, where a source of Unpaired activity at the midline of the developing eye causes a gradient of JAK–STAT activation [22•]. JAK–STAT signalling is also involved in Drosophila wing-vein formation [23] and in the determination of sexual identity [24]. A similar multiplicity of developmental functions for a single STAT signalling pathway has also recently been described for Dictyostelium. The Dd-STATa gene encodes a protein that is structurally homologous to animal STATs [25] and that rapidly translocates to the nucleus when extracellular cAMP binds to its serpentine receptor [26]. Our recent analysis of a null mutant has shown that the Dd-STATa protein is required for normal chemotaxis during early development, for the correct movement of prestalk cells during terminal differentiation and as a repressor regulating stalk-specific gene expression [27].
STAT signalling and the regulation of cell proliferation and apoptosis in mammalian cells Among the mammalian STAT proteins, STAT3 plays the most diverse roles [1,2] and two recent studies, both using ectopic activation strategies, have provided additional insights into its functioning. Embryonic stem (ES) cells can be maintained in an undifferentiated state by the addition of leukemia inhibitory factor (LIF) but expression of a dominant negative form of STAT3 leads to the differentiation of ES cells, even when LIF is present [28]. Conversely, expression of a fusion protein containing the ligand-binding domain of the estrogen receptor fused to STAT3 generates an ES cell derivative that is, in the presence of estrogen, independent of LIF for continued cell proliferation [29]. Thus, STAT3 signalling is both necessary and sufficient to maintain ES cells in an undifferentiated state. In order to generate a constitutively active form of the STAT3 protein, Bromberg et al. [30••] used a knowledge of its tertiary structure [31]. The authors noted the presence of a region within the SH2 domain that becomes closely juxtaposed to the dimeric partner in the STAT3:DNA complex. They reasoned that replacement of two residues within this region by cysteine might generate a protein that is constitutively dimeric and therefore constitutively active. This prediction was fulfilled and the cysteine-substituted STAT3 protein was found to act as an oncogene when expressed in immortalised fibroblasts. This adds to a growing body of evidence that links aberrant STAT signalling to oncogenesis (reviewed in [32]). Many cytokines induce expression of members of the antiapoptotic Bcl-2 family of proteins and STAT3 represses apoptosis in human myeloma cells by stimulating expression of Bcl-XL [33]. Although, the precise transcriptional regulatory mechanism for STAT3 is not yet known, two groups have now shown that the erythropoeitin receptor activates the binding of STAT5 to specific Bcl-XL promoter sequences, thus protecting erythroid precursor cells from apoptosis [34•,35].
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Cross-talk between the STAT and Smad signalling pathways Smad proteins become serine phosphorylated and activated when they associate with receptors of the TGF-β superfamily (reviewed in [36]). The STAT and the Smad proteins are sometimes referred to as ‘fast-track’ signalling molecules because members of both families of proteins can directly transduce a signal from the plasma membrane to the gene. An exciting link between the STATs and the Smads has now been discovered with evidence for co-operative signalling between LIF and BMP2 (bone morphogenetic protein-2, a TGF-β superfamily member). This is mediated by an indirect interaction between STAT3 and Smad1, with the transcriptional co-activator CBP/p300 acting as the bridging molecule [37••]. A different form of cross-talk between a STAT and a Smad pathway now explains the opposing effects that TGF-β and interferon-γ signalling exert on a number of cellular functions [38•]. These authors showed that interferon-γ, acting via JAK1 and STAT1, induces the expression of Smad7. Smad 7 is a repressor of TGF-β signalling that functions by preventing interaction of Smad3 with the TGF-β receptor.
Conclusions The simple paradigm for JAK–STAT signalling set by interferon-γ holds generally true but, as the STAT protein family itself has expanded and as many other signalling pathways are found to contain STATs, some of the ground rules seem to break down. This is particularly apparent when the role of tyrosine phosphorylation and dimerisation is considered and this highlights the need for more information on the fundamental mechanisms that determine the nuclear localisation of STATs. This will require delineation of the sequence elements within STAT molecules that direct their nuclear import and export. It will also require an understanding of their in vitro binding to Importin α and Exportin α. Because of the complications introduced by the dimerisation of STATs, the availability of constitutively and conditionally active STAT proteins will also be extremely valuable in studying their mechanism of nuclear compartmentalisation. Such constructs are also providing important insights into the biological functions of STATs. The link to the Smad-signalling pathway should be of great interest to the developmental biologist because TGF-β superfamily members are key components of many morphogenetic signalling pathways.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Heim MH: The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res 1999, 19:75-120.
2.
Akira S: Functional roles of STAT family proteins: lessons from knockout mice. Stem Cells 1999, 17:138-146.
3.
Chatterjee-Kishore M, van den Akker F, Stark GR: Association of STATs with relatives and friends. Trends Cell Biol 2000, 10:106-111.
4.
Peng J, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE, Beales J, Fish LJ, Worland AJ, Pelica F et al.: ‘Green revolution’ genes encode mutant gibberellin response modulators. Nature 1999, 400:256-261.
5.
Baldauf SL, Doolittle WF: Origin and evolution of the slime molds. Proc Natl Acad Sci USA 1997, 94:12007-12012.
6.
Kuriyan J, Darnell JE Jr: An SH2 domain in disguise. Nature 1999, 398:22-25.
7.
Williams JG: Serpentine receptors and STAT activation: more than one way to twin a STAT. Trends Biochem Sci 1999, 9:333-334.
8.
Leaman DW, Leung S, Li X, Stark GR: Regulation of STATdependent pathways by growth factors and cytokines. FASEB J 1996, 10:1578-1588.
9.
Olayioye MA, Beuvink I, Horsch K, Daly JM, Hynes NE: ErbB receptorinduced activation of STAT transcription factors is mediated by Src tyrosine kinases. J Biol Chem 1999, 274:17209-17218.
10. Sekimoto T, Nakajima K, Tachibana T, Hirano T, Yoneda Y: Interferongamma-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran/TC4. J Biol Chem 1996, 271:31017-31020. 11. Milocco LH, Haslam JA, Rosen J, Seidel HM: Design of conditionally active STATs: insights into STAT activation and gene regulatory function. Mol Cell Biol 1999, 4:2913-2920. 12. Kumar A, Commane M, Flickinger TW, Horvath CM, Stark GR: α-induced apoptosis in STAT1-null cells due to low Defective TNF-α constitutive levels of caspases. Science 1997, 278:1630-1632. 13. Johnson LR, McCormack SA, Yang CH, Pfeffer SR, Pfeffer LM: EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells. Am J Physiol 1999, 276:419-425. 14. Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA, Rosen JM: Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A. J Biol Chem 1999, 274:22484-22492. 15. Sekimoto T, Yoneda Y: Nuclear import and export of proteins: the molecular basis for intracellular signaling. Cytokine Growth Factor Rev 1998, 9:205-211. 16. Sekimoto T, Imamoto N, Nakajima K, Hirano T, Yoneda Y: Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear pore-targeting complex formation with NPI-1, but not Rch1. EMBO J 1997, 16:7067-7077. 17.
Herrington J, Rui L, Luo G, Yu-Lee LY, Carter-Su C: A functional DNA binding domain is required for growth hormone-induced nuclear accumulation of Stat5B. J Biol Chem 1999, 274:5138-5145.
18. Haspel RL, Darnell JE Jr: A nuclear protein tyrosine phosphatase is • required for the inactivation of Stat1. Proc Natl Acad Sci USA 1999, 96:10188-10193. In this paper, it is shown that there is a mechanism that dephosphorylates the STAT1 protein within the nucleus, rather than a cytoplasmic inactivation process that interrupts nucleo-cytoplasmic shuttling. 19. Yan R, Small S, Desplan C, Dearolf CR, Darnell JE Jr: Identification of a Stat gene that functions in Drosophila development. Cell 1996, 84:421-430. 20. Hou XS, Melnick MB, Perrimon N: Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 1996, 84:411-419. 21. Harrison DA, McCoon PE, Binari R, Gilman M, Perrimon N: Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev 1998, 12:3252-3263. 22. Zeidler MP, Perrimon N, Strutt DI: Polarity determination in the • Drosophila eye: a novel role for unpaired and JAK/STAT signaling. Genes Dev 1999, 13:1342-1353. This paper extends the range of function for the Drosophila JAK-STAT pathway to include eye development and shows there to be a gradient of the ligand, Unpaired. This is also very important for future research because very sophisticated genetic tools can be used to study Drosophila eye development which may yield new JAK-STAT-interacting proteins.
STAT signalling in cell proliferation and in development Williams
23. Yan R, Luo H, Darnell JE Jr, Dearolf CR: A JAK-STAT pathway regulates wing vein formation in Drosophila. Proc Natl Acad Sci USA 1996, 93:5842-5847. 24. Jinks TM, Polydorides AD, Calhoun G, Schedl P: The JAK-STAT signalling pathway is required for the initial choice of sexual identity in Drosophila melanogaster. Mol Cell 2000, 5:581-587. 25. Kawata T, Shevchenko A, Fukuzawa M, Jermyn A, Totty NF, Zhukovskaya NV, Sterling A, Mann M, Williams JG: SH2 signalling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in Dictyostelium. Cell 1997, 89:909-916. 26. Araki T, Gamper M, Early AE, Fukuzawa M, Abe T, Kawata T, Kim E, Firtel RA, Williams JG: Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor. EMBO J 1998, 17:4018-4028. 27.
Mohanty S, Jermyn KA, Early A, Kawata T, Aubry L, Ceccarelli A, Schaap P, Williams JG, Firtel RA: Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation. Development 1999, 126:3391-3405.
28. Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998, 12:2048-2060. 29. Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T: STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999, 18:4261-4269. 30. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, •• Albanese C, Darnell JE Jr: Stat3 as an oncogene. Cell 1999, 98:295-303. This is a conceptually and technically elegant paper that shows how precise protein engineering can be used to study a biological problem. In order to render the STAT3 protein dimeric and therefore constitutively active, the authors replaced two residues (A661 and N663) by cysteine. These two residues lie within a region of very close contact between the STAT3 monomers. Within the cell, the cysteine-substituted STAT proteins are constitutively active. The biological consequence is that the cells become oncogenically transformed and will induce tumours when injected into nude mice. There is a growing body of indirect evidence that links aberrant STAT signalling to oncogenesis (reviewed in [32]). By showing that a constitutively active form of STAT3 can directly induce tumour formation, this paper adds considerably to the evidence implicating STAT gene mis-expression in cancer. 31. Becker S, Groner B, Muller CW: Three-dimensional structure of the β homodimer bound to DNA. Nature 1998, 394:145-151. Stat3β 32. Catlett-Falcone R, Dalton WS, Jove R: STAT proteins as novel targets for cancer therapy. Curr Opin Oncol 1999, 11:490-496.
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33. Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R, Ciliberto G, Moscinski L, Fernandez-Luna JL, Nuñez G et al.: Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999, 10:105-115. 34. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF: Fetal • anemia and apoptosis of red cell progenitors in Stat5a–/–5b–/– mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 1999, 98:181-191. This paper uses doubly nulli-zygous transgenic mice to show that STAT5 is essential for normal fetal erythropoiesis but not for adult erythropoiesis. It is also important because it shows there to be interaction of STAT5 protein with a ‘GAS element’ (see legend to Figure 1) in the first intron of the Bcl-XL gene that directs erythropoietin-specific gene expression. 35. Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, Nuñez G, Fernandez-Luna JL: Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 1999, 274:22165-22169. β signaling. Genes Dev 36. Massagué J, Chen YG: Controlling TGF-β 2000, 14:627-644. 37. ••
Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T, Kawabata M, Miyazono K, Taga T: Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. Science 1999, 284:479-482. The authors show that LIF and BMP2 act co-operatively to induce primary foetal neural progenitor cells to differentiate as astrocytes. LIF activates STAT3 whereas BMP2 activates Smads 1, 5 and 8. Expression of a dominant-negative form of STAT3 or of Smad6, a negative regulator of Smad activity, respectively decreased either the LIF response or the BMP2 response. Co-immunopreciptation failed to show an interaction between STAT3 and Smad1 and the authors therefore sought an adaptor molecule that might act as a bridge. They found that the transcriptional co-activator p300 constitutes the adaptor and present evidence that STAT3 binds at the amino terminus of p300 in a cytokine-independent manner and that p300 binds Smad1 at its carboxyl terminus in a cytokine-dependent manner. These observations forge a novel link between these two very important ‘fast track’ signalling pathways. 38. Ulloa L, Doody J, Massagué J: Inhibition of transforming growth • factor-beta/SMAD signalling by the interferon-gamma/STAT pathway. Nature 1999, 397:710-713. The authors show how the STAT and Smad pathways interact at a transcriptional level, to downregulate TGF-β signalling in response to interferon-γ. 39. Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE Jr, Kuriyan J: Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell 1998, 93:827-839.
STAT signalling in cell proliferation and in development Jeffrey G Williams Recent advances in STAT signalling research include a better understanding of the roles of mammalian STAT proteins in cell proliferation and apoptosis, and of non-mammalian STAT proteins in morphogenesis. Two different ways in which STAT signalling pathways can interface with Smad signalling pathways significantly increasing combinatorial signalling possibilities, have also been described. Addresses Department of Anatomy and Physiology, University of Dundee, Wellcome Trust Building Complex, Dow Street, Dundee DD1 5EH, UK; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:503–507 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations BMP2 bone morphogenetic protein-2 EGF epidermal growth factor ES embryonic stem JAK Janus kinase LIF leukemia inhibitory factor NLS nuclear localisation signal PDGF platelet-derived growth factor STAT signal transducer and activator of transcription β transforming growth factor factor-β TGF-β TNF tumor necrosis factor
Introduction The STAT (signal transducer and activator of transcription) proteins were discovered as mediators of cytokine signalling in mammals. STATs 1, 2, 4 and 6 have relatively restricted functions, centred around disease resistance (reviewed in [1–3]). STAT3 and STAT5, however, have broader functions and STATs have also been discovered in non-mammalian organisms. The STATs interact with a number of other cellular proteins, some of which act as specific negative regulators of their function. This is a very active area of research with considerable recent progress [3] but in this review I focus on the mechanism of STAT activation and on new cellular and developmental roles for a number of different STAT proteins.
STAT structure and the JAK-STAT pathway Mammalian STAT proteins contain three conserved domains: a DNA binding domain, an SH2 domain and a site of tyrosine phosphorylation (Figure 1). These are situated in the approximate centre of the protein. In their carboxy-terminal regions, most mammalian STAT proteins contain a transcriptional activation domain. The amino-terminal proximal region interacts with other cellular proteins and very near to the amino terminus there is a weakly conserved region that directs the oligomerisation of STAT dimers [3]. When a cytokine binds to its receptor (Figure 2), it induces conformational changes — most likely including
Figure 1 DNA binding domain
SH2 domain
Transcriptional activation
Y Site of tyrosine phosphorylation Current Opinion in Genetics & Development
A diagrammatic representation of a typical STAT protein. The functions of the marked domains are described in the text. The crystal structures of the STAT1 homo-dimer [39] and STAT3 homo-dimer [31] are known and, despite the relatively weak primary sequence conservation in their amino-terminal halves, the two structures are highly similar. The STAT dimer forms a plier-like structure, with the handles of the pliers wrapped around the DNA. Each partner STAT contacts the DNA independently and the interaction interface of the two STATs is in the nose of the pliers, linking the SH2 domain on one STAT and the phosphorylated tyrosine on the partner STAT molecule. The consensus DNA binding site for STAT dimers, for instance the site used by STAT1 when it is activated by interferon-γ (the ‘GAS element’), is the dyad TTCnGAA, where n from is variable from STAT to STAT.
oligomerisation or multimerisation — and this activates JAKs (Janus kinases). JAKs are tyrosine kinases, one of which is constitutively associated with each receptor chain. The activated JAKs autophosphorylate, and/or transphosphorylate, and then phosphorylate the receptors. Classically the phosphorylated receptor tyrosine motifs act as docking sites for the SH2 domains of STATs, although recruitment of certain STATS at least can also occur independently of the SH2 domains and through the JAKs. In the case of the interferon-γ pathway, a STAT1 molecule is recruited to each of two IFNGR1 subunits in the minimally tetrameric (2 times IFNGR1/2) receptor. In other cases (e.g. interferon-α), two different STAT proteins, STAT1 and STAT2, are recruited. The JAKs then tyrosine phosphorylate the STAT molecules and this triggers a re-shuffle; the STAT monomers dissociate from the receptor and dimerise with one another via reciprocal SH2 domain:phosphotyrosine interactions. The STAT homo- or heterodimers migrate to the nucleus and, with or without interaction with additional specificity modulating factors (e.g. p48 in the case of interferon-α), interact with transcriptional co-activators and the target DNA to activate transcription.
The evolution of STAT signalling: some recent additions to the STAT family and alternative ways of activating STATs Although the STAT proteins were discovered in studies of cytokine signalling in mammalian cells, they have now been discovered in other eukaryotic lineages. These include organisms as widely diverged as Drosophila and
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Figure 2
(a) Ligand
Receptor Plasma membrane JAK
JAK
their appearance before the divergence of the animals, fungi and the Dictyostelids [5]. It is therefore very surprising that neither of the yeasts for which a total genome sequence is available, Schizosaccharomces pombe and Saccharomyces cerevisiae, has a recognisable homologue of the STAT proteins. Indeed, neither of these two ‘lower’ fungi possesses an SH2 domain. One possibility is that the common ancestor of the higher and lower fungi utilised SH2 domain signalling and that these pathways were lost during the evolution of modern lower fungi. Another possibility, revealed by the recently determined structure of the Cbl signalling protein, is that we are failing to recognise SH2 domains when they are highly diverged in primary sequence [6].
(b)
Ligand
Receptor
JAK
JAK
P
P
STAT monomer
Just as the range of organisms known to possess STATs has extended, so too has the range of upstream signalling pathways known to activate them. Cytokine receptors are single membrane-pass receptors but seven trans-membrane pass (serpentine) receptors are also now known to activate STATs (reviewed in [7]). These receptors normally function by activating heterotrimeric G proteins but, in this case, a G-protein-independent-pathway seems to be utilised. There is also no obligate requirement for a JAK, because other kinases, such as the EGF and PDGF receptors, can phosphorylate STATs in vitro (reviewed in [8]). A recent in vivo analysis, however, suggests that the c-Src tyrosine kinase is recruited to the ErbB1 receptor upon EGF stimulation and that it is c-Src that actually activates STAT1, STAT3 and STAT5 [9].
The role of tyrosine phosphorylation (c)
P P
STAT dimer
Nucleus Current Opinion in Genetics & Development
The JAK-STAT signalling pathway. The process is simplified and, for clarity of presentation, divided into three steps (but see the main text for more detail). (a) Ligand binding to the receptor multimerises the receptor chains and this activates the JAKs. The JAKs then tyrosine phosphorylate the receptor chains. (b) STAT monomers are recruited by interaction of their SH2 domains with the tyrosine phosphorylated receptor chains. The JAKs then tyrosine phosphorylate the STATs. (c) The STATs dissociate from the receptor and re-associate with each other to form a dimer. The STAT dimer translocates to the nucleus and binds to regulatory DNA elements.
Dictyostelium. There is also evidence, based upon sequence homology, for proteins with a STAT-like structure in higher plants [4]. Ancestral plants are believed to have made
The generally accepted scheme for the series of events after STAT tyrosine phosphorylation is that the resultant STAT dimerisation causes translocation from the cytoplasm to the nucleus. In a mutant form of STAT1, where the site of tyrosine phosphorylation is altered to phenylanine and dimerisation is thereby prevented, nuclear accumulation does not occur [10]. The key role of dimerisation is also supported by a recent study using a fusion protein in which the ligand-binding/dimerisation domain of the oestrogen receptor was fused to the carboxyl terminus of the STAT1 protein [11]. This protein dimerises, translocates to the nucleus and binds DNA upon addition of estrogen. As the fusion protein does not become tyrosine phosphorylated after estrogen treatment, it would seem that dimerisation is sufficient to generate a nuclear import signal. Confusingly, however, other observations argue against a generalised, obligate role of dimerisation in STAT nuclear translocation [12–14]. Human fibroblasts cells undergo apoptosis when treated with tumour necrosis factor (TNF). However, because STAT1 is required for the constitutive expression of several caspases that are essential for apoptosis, mutant cells in which STAT1 is absent are resistant to TNF [12]. When null cells are transformed with a construct expressing STAT1, they are rendered sensitive to TNF-induced
STAT signalling in cell proliferation and in development Williams
apoptosis. Surprisingly, however, a form of STAT1 in which the site of tyrosine phosphorylation is mutated also restores apoptotic sensitivity. Thus a STAT1 function that presumably requires nuclear localisation seems to be occurring independently of the ability to dimerise. Furthermore, EGF treatment has been shown to induce nuclear translocation of STAT2 without an increase in STAT2 tyrosine phosphorylation [13]. Again, therefore, tyrosine phosphorylation seems not to be essential for nuclear translocation. Nor, apparently, is it sufficient, because src tyrosine kinase induces tyrosine phosphorylation of STAT5A without causing it to translocate to the nucleus [14].
Nuclear compartmentalisation of STAT proteins What the above observations imply is that there is no general pathway of STAT nuclear translocation. The STAT family members can behave quite differently from one another and an individual STAT can behave differently in different contexts. They also indicate the need for a better understanding of the precise mechanism of STAT nuclear localisation. The nuclear localisation signal (NLS) is a short lysine-rich sequence that is the binding site for an importin α, the class of adaptor proteins that mediates interaction of the NLS with the nuclear-import machinery (reviewed in [15]). This signal is present in proteins that are targeted to the nucleus but mutational analysis suggests that STAT1 does not contain a lysine-rich NLS [16]. Proving the absence of a short protein motif, of variable sequence composition, is inherently very difficult but Sekimoto et al. [16] show that different parts of importin α are necessary for STAT binding than are required for NLS binding. This strongly suggests that the importin α binding site in STAT1 is not a classic NLS. The importin α binding site has not been defined but in STAT5B, a conserved region (VVVI) within the DNA-binding domain is essential for nuclear import [17]. There is also a mechanism that recycles STAT molecules, back from the nucleus to the cytoplasm, and this has been shown to involve tyrosine dephosphorylation within the nucleus [18•]. Again, however, the mechanism of STAT nuclear export has not been elucidated in any detail.
STAT signalling and non-vertebrate development In Drosophila there is a genetically well characterised STAT signalling pathway comprising a JAK, encoded by the hopscotch gene, and a STAT encoded by the stat92E gene [19,20]. Female flies with mutations in either gene produce embryos with highly characteristic segmentation defects. Mutation of the unpaired gene yields embryos with similar segmentation defects and the properties of the Unpaired protein suggest very strongly that it is the ligand that activates the pathway; it is a glycoprotein of the extracellular matrix and, when ectopically expressed in cultured Drosophila cells, it directs activation of Hopscotch in co-cultured cells [21].
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The Drosophila JAK–STAT pathway is now also known to function in eye development, where a source of Unpaired activity at the midline of the developing eye causes a gradient of JAK–STAT activation [22•]. JAK–STAT signalling is also involved in Drosophila wing-vein formation [23] and in the determination of sexual identity [24]. A similar multiplicity of developmental functions for a single STAT signalling pathway has also recently been described for Dictyostelium. The Dd-STATa gene encodes a protein that is structurally homologous to animal STATs [25] and that rapidly translocates to the nucleus when extracellular cAMP binds to its serpentine receptor [26]. Our recent analysis of a null mutant has shown that the Dd-STATa protein is required for normal chemotaxis during early development, for the correct movement of prestalk cells during terminal differentiation and as a repressor regulating stalk-specific gene expression [27].
STAT signalling and the regulation of cell proliferation and apoptosis in mammalian cells Among the mammalian STAT proteins, STAT3 plays the most diverse roles [1,2] and two recent studies, both using ectopic activation strategies, have provided additional insights into its functioning. Embryonic stem (ES) cells can be maintained in an undifferentiated state by the addition of leukemia inhibitory factor (LIF) but expression of a dominant negative form of STAT3 leads to the differentiation of ES cells, even when LIF is present [28]. Conversely, expression of a fusion protein containing the ligand-binding domain of the estrogen receptor fused to STAT3 generates an ES cell derivative that is, in the presence of estrogen, independent of LIF for continued cell proliferation [29]. Thus, STAT3 signalling is both necessary and sufficient to maintain ES cells in an undifferentiated state. In order to generate a constitutively active form of the STAT3 protein, Bromberg et al. [30••] used a knowledge of its tertiary structure [31]. The authors noted the presence of a region within the SH2 domain that becomes closely juxtaposed to the dimeric partner in the STAT3:DNA complex. They reasoned that replacement of two residues within this region by cysteine might generate a protein that is constitutively dimeric and therefore constitutively active. This prediction was fulfilled and the cysteine-substituted STAT3 protein was found to act as an oncogene when expressed in immortalised fibroblasts. This adds to a growing body of evidence that links aberrant STAT signalling to oncogenesis (reviewed in [32]). Many cytokines induce expression of members of the antiapoptotic Bcl-2 family of proteins and STAT3 represses apoptosis in human myeloma cells by stimulating expression of Bcl-XL [33]. Although, the precise transcriptional regulatory mechanism for STAT3 is not yet known, two groups have now shown that the erythropoeitin receptor activates the binding of STAT5 to specific Bcl-XL promoter sequences, thus protecting erythroid precursor cells from apoptosis [34•,35].
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Differentiation and gene regulation
Cross-talk between the STAT and Smad signalling pathways Smad proteins become serine phosphorylated and activated when they associate with receptors of the TGF-β superfamily (reviewed in [36]). The STAT and the Smad proteins are sometimes referred to as ‘fast-track’ signalling molecules because members of both families of proteins can directly transduce a signal from the plasma membrane to the gene. An exciting link between the STATs and the Smads has now been discovered with evidence for co-operative signalling between LIF and BMP2 (bone morphogenetic protein-2, a TGF-β superfamily member). This is mediated by an indirect interaction between STAT3 and Smad1, with the transcriptional co-activator CBP/p300 acting as the bridging molecule [37••]. A different form of cross-talk between a STAT and a Smad pathway now explains the opposing effects that TGF-β and interferon-γ signalling exert on a number of cellular functions [38•]. These authors showed that interferon-γ, acting via JAK1 and STAT1, induces the expression of Smad7. Smad 7 is a repressor of TGF-β signalling that functions by preventing interaction of Smad3 with the TGF-β receptor.
Conclusions The simple paradigm for JAK–STAT signalling set by interferon-γ holds generally true but, as the STAT protein family itself has expanded and as many other signalling pathways are found to contain STATs, some of the ground rules seem to break down. This is particularly apparent when the role of tyrosine phosphorylation and dimerisation is considered and this highlights the need for more information on the fundamental mechanisms that determine the nuclear localisation of STATs. This will require delineation of the sequence elements within STAT molecules that direct their nuclear import and export. It will also require an understanding of their in vitro binding to Importin α and Exportin α. Because of the complications introduced by the dimerisation of STATs, the availability of constitutively and conditionally active STAT proteins will also be extremely valuable in studying their mechanism of nuclear compartmentalisation. Such constructs are also providing important insights into the biological functions of STATs. The link to the Smad-signalling pathway should be of great interest to the developmental biologist because TGF-β superfamily members are key components of many morphogenetic signalling pathways.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
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Herrington J, Rui L, Luo G, Yu-Lee LY, Carter-Su C: A functional DNA binding domain is required for growth hormone-induced nuclear accumulation of Stat5B. J Biol Chem 1999, 274:5138-5145.
18. Haspel RL, Darnell JE Jr: A nuclear protein tyrosine phosphatase is • required for the inactivation of Stat1. Proc Natl Acad Sci USA 1999, 96:10188-10193. In this paper, it is shown that there is a mechanism that dephosphorylates the STAT1 protein within the nucleus, rather than a cytoplasmic inactivation process that interrupts nucleo-cytoplasmic shuttling. 19. Yan R, Small S, Desplan C, Dearolf CR, Darnell JE Jr: Identification of a Stat gene that functions in Drosophila development. Cell 1996, 84:421-430. 20. Hou XS, Melnick MB, Perrimon N: Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 1996, 84:411-419. 21. Harrison DA, McCoon PE, Binari R, Gilman M, Perrimon N: Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev 1998, 12:3252-3263. 22. Zeidler MP, Perrimon N, Strutt DI: Polarity determination in the • Drosophila eye: a novel role for unpaired and JAK/STAT signaling. Genes Dev 1999, 13:1342-1353. This paper extends the range of function for the Drosophila JAK-STAT pathway to include eye development and shows there to be a gradient of the ligand, Unpaired. This is also very important for future research because very sophisticated genetic tools can be used to study Drosophila eye development which may yield new JAK-STAT-interacting proteins.
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23. Yan R, Luo H, Darnell JE Jr, Dearolf CR: A JAK-STAT pathway regulates wing vein formation in Drosophila. Proc Natl Acad Sci USA 1996, 93:5842-5847. 24. Jinks TM, Polydorides AD, Calhoun G, Schedl P: The JAK-STAT signalling pathway is required for the initial choice of sexual identity in Drosophila melanogaster. Mol Cell 2000, 5:581-587. 25. Kawata T, Shevchenko A, Fukuzawa M, Jermyn A, Totty NF, Zhukovskaya NV, Sterling A, Mann M, Williams JG: SH2 signalling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in Dictyostelium. Cell 1997, 89:909-916. 26. Araki T, Gamper M, Early AE, Fukuzawa M, Abe T, Kawata T, Kim E, Firtel RA, Williams JG: Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor. EMBO J 1998, 17:4018-4028. 27.
Mohanty S, Jermyn KA, Early A, Kawata T, Aubry L, Ceccarelli A, Schaap P, Williams JG, Firtel RA: Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation. Development 1999, 126:3391-3405.
28. Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998, 12:2048-2060. 29. Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T: STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999, 18:4261-4269. 30. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, •• Albanese C, Darnell JE Jr: Stat3 as an oncogene. Cell 1999, 98:295-303. This is a conceptually and technically elegant paper that shows how precise protein engineering can be used to study a biological problem. In order to render the STAT3 protein dimeric and therefore constitutively active, the authors replaced two residues (A661 and N663) by cysteine. These two residues lie within a region of very close contact between the STAT3 monomers. Within the cell, the cysteine-substituted STAT proteins are constitutively active. The biological consequence is that the cells become oncogenically transformed and will induce tumours when injected into nude mice. There is a growing body of indirect evidence that links aberrant STAT signalling to oncogenesis (reviewed in [32]). By showing that a constitutively active form of STAT3 can directly induce tumour formation, this paper adds considerably to the evidence implicating STAT gene mis-expression in cancer. 31. Becker S, Groner B, Muller CW: Three-dimensional structure of the β homodimer bound to DNA. Nature 1998, 394:145-151. Stat3β 32. Catlett-Falcone R, Dalton WS, Jove R: STAT proteins as novel targets for cancer therapy. Curr Opin Oncol 1999, 11:490-496.
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33. Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R, Ciliberto G, Moscinski L, Fernandez-Luna JL, Nuñez G et al.: Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999, 10:105-115. 34. Socolovsky M, Fallon AE, Wang S, Brugnara C, Lodish HF: Fetal • anemia and apoptosis of red cell progenitors in Stat5a–/–5b–/– mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 1999, 98:181-191. This paper uses doubly nulli-zygous transgenic mice to show that STAT5 is essential for normal fetal erythropoiesis but not for adult erythropoiesis. It is also important because it shows there to be interaction of STAT5 protein with a ‘GAS element’ (see legend to Figure 1) in the first intron of the Bcl-XL gene that directs erythropoietin-specific gene expression. 35. Silva M, Benito A, Sanz C, Prosper F, Ekhterae D, Nuñez G, Fernandez-Luna JL: Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 1999, 274:22165-22169. β signaling. Genes Dev 36. Massagué J, Chen YG: Controlling TGF-β 2000, 14:627-644. 37. ••
Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T, Kawabata M, Miyazono K, Taga T: Synergistic signaling in fetal brain by STAT3-Smad1 complex bridged by p300. Science 1999, 284:479-482. The authors show that LIF and BMP2 act co-operatively to induce primary foetal neural progenitor cells to differentiate as astrocytes. LIF activates STAT3 whereas BMP2 activates Smads 1, 5 and 8. Expression of a dominant-negative form of STAT3 or of Smad6, a negative regulator of Smad activity, respectively decreased either the LIF response or the BMP2 response. Co-immunopreciptation failed to show an interaction between STAT3 and Smad1 and the authors therefore sought an adaptor molecule that might act as a bridge. They found that the transcriptional co-activator p300 constitutes the adaptor and present evidence that STAT3 binds at the amino terminus of p300 in a cytokine-independent manner and that p300 binds Smad1 at its carboxyl terminus in a cytokine-dependent manner. These observations forge a novel link between these two very important ‘fast track’ signalling pathways. 38. Ulloa L, Doody J, Massagué J: Inhibition of transforming growth • factor-beta/SMAD signalling by the interferon-gamma/STAT pathway. Nature 1999, 397:710-713. The authors show how the STAT and Smad pathways interact at a transcriptional level, to downregulate TGF-β signalling in response to interferon-γ. 39. Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE Jr, Kuriyan J: Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell 1998, 93:827-839.