STAT signalling

European Journal of Cell Biology 91 (2012) 524–532

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Review

Dynamics and non-canonical aspects of JAK/STAT signalling Anne Mohr, Nicolas Chatain, Tamás Domoszlai, Natalie Rinis, Michael Sommerauer, Michael Vogt, Gerhard Müller-Newen ∗ Institut für Biochemie und Molekularbiologie, RWTH Aachen University, Aachen, Germany

a r t i c l e

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Article history: Received 27 May 2011 Received in revised form 5 September 2011 Accepted 12 September 2011 Keywords: Cytokines Receptors Signal transduction JAK STAT

a b s t r a c t The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway directly links ligand-binding to a membrane-bound receptor with the activation of a transcription factor. This signalling module enables the cell to rapidly initiate a transcriptional response to external stimulation. The main components of this evolutionary conserved module are cytokines that specifically bind to cytokine receptors leading to the activation of receptor-associated Janus tyrosine kinases (JAKs). The receptorbound JAKs activate STAT transcription factors through phosphorylation of a single tyrosine residue. Activated STAT dimers translocate into the nucleus to induce target gene expression. In this article we will review current opinions on the molecular mechanism and on intracellular dynamics of JAK/STAT signalling with a special focus on the cytokine receptor glycoprotein 130 (gp130) and STAT3. In particular we will concentrate on non-canonical aspects of Jak/STAT signalling including preassembled receptor complexes, preformed STAT dimers, STAT trafficking and non-canonical functions of STATs. © 2011 Elsevier GmbH. All rights reserved.

Introduction The human genome encodes four JAKs (JAK1, JAK2, JAK3 and TYK2) and seven STATs (STAT1, 2, 3, 4, 5A, 5B and 6). The number of ligands and receptors that can activate the JAK/STAT pathway is much higher. All interferons, most interleukins, many growth factors and some hormones activate STAT transcription factors. STATs are not only activated by cytokine receptors that associate with JAKs but also by receptor tyrosine kinases and G protein-coupled receptors. Depending on the receptor, STAT activation from noncytokine receptors might be JAK-dependent or JAK-independent (Levy and Darnell, 2002). STAT transcription factors are mostly dedicated to hematopoiesis and immunity with the exception of STAT3 which is also involved in reproduction, embryonic development, mammary involution and wound healing (Leonard and O’Shea, 1998; Levy and Lee, 2002; Stewart et al., 1992; Takeda et al., 1997). Besides its central role in hematopoiesis STAT5A controls mammary gland differentiation during pregnancy including the production of milk proteins. The closely related STAT5B protein in combination with the glucocorticoid receptor controls expression of insulin-like growth factor (IGF-1) in the liver in response to growth hormone (Hennighausen and Robinson, 2008). Dysregulated activation of

∗ Corresponding author at: Institut für Biochemie und Molekularbiologie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany. Tel.: +49 241 80 88860; fax: +49 241 80 82428. E-mail address: [email protected] (G. Müller-Newen). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.09.005

STATs plays an important role in chronic inflammation and cancer (O’Sullivan et al., 2007; Yu and Jove, 2004).

Canonical and non-canonical JAK/STAT signalling Canonical JAK/STAT signalling as frequently depicted in textbooks and review articles describes the basic features of this signalling module but exhibits some oversimplifications. JAK/STAT signalling emanates from cytokine receptors at the plasma membrane. According to the canonical model (Fig. 1A) cytokine receptors are enforced to dimerize upon ligand-binding. At the dimerized receptors the associated JAKs transphosphorylate each other resulting in their activation. The activated JAKs phosphorylate cytoplasmic tyrosine residues in the membrane-distal part of the receptor. These phosphotyrosine motifs serve as docking sites for various proteins with SH2 domains including STATs. STAT monomers specifically bind through their SH2 domain to compatible phosphotyrosine motifs. Receptor-bound STATs become tyrosine phosphorylated at a single tyrosine residue by the receptor-associated JAKs. Tyrosine phosphorylated STATs are released from the receptor and dimerize through reciprocal, intermolecular SH2 domain/phosphotyrosine interactions. The phospho-STAT dimers translocate into the nucleus, bind specifically to accessible response elements of DNA and induce the transcription of target genes. Further posttranslational modifications of STATs such as serine phosphorylation, lysine acetylation, ubiquitination and SUMOylation modulate the transcriptional activity and significantly contribute to STAT-induced

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Fig. 1. Canonical and non-canonical JAK/STAT signalling. (A) The main features of canonical JAK/STAT signalling are dimerization of receptors after ligand-binding, STAT monomers that dimerize upon phosphorylation by receptor-bound JAKs and nuclear entry of phosphorylated STATs only. (B) Non-canonical JAK/STAT signalling includes preformed dimers of receptors as well as STATs. Unphosphorylated STATs in the nucleus may contribute to the regulation of gene expression. The schemes do neither display further posttranslational modifications of STATs, nor other signalling pathways emerging from activated cytokine receptors such as MAPK and PI3K/Akt signalling nor mechanisms of signal attenuation such as phosphatases and SOCS proteins. For these topics see the article of Eulenfeld and Schaper in this issue.

gene responses (Lim and Cao, 2006). Among the numerous STAT target genes are those encoding suppressor of cytokine signalling (SOCS) proteins that act as feedback inhibitors at the receptor level through various mechanisms reviewed elsewhere (Alexander and Hilton, 2004). Non-canonical JAK/STAT signalling adds some refinements to the above outlined signalling mechanism (Fig. 1B). Cytokine receptors might be dimerized in the absence of the ligand. Instead of inducing dimerization the ligand stabilizes a preformed dimer and/or triggers a conformational change from an inactive to an active dimer. As proposed for the receptors, there is strong evidence for STAT dimers in the absence of the activating tyrosine phosphorylation. Again, activation would result from the change of the conformation of a preformed STAT dimer upon phosphorylation. In the canonical model STATs enter the nucleus in response to activation. In the non-canonical model a fraction of latent STATs is consistently located in the nucleus as a result of a steady state of constant nuclear import and export. These non-phosphorylated nuclear STAT molecules might also contribute to gene regulation (Li, 2008). Furthermore, STATs exert other non-canonical functions outside of the nucleus. Tyrosine phosphorylation shifts the nucleocytoplasmic distribution toward nuclear accumulation of STATs. In this article these non-canonical aspects of JAK/STAT signalling will be discussed in more detail.

Activation of cytokine receptors Cytokine receptors that are associated with JAKs are the most prominent activators of STATs. The ectodomain of these receptors contains at least one cytokine-binding module (CBM) consisting of two fibronectin type III-like domains (FNIII-like domain). According to characteristic sequence motifs in the CBM, class 1 and class 2 cytokine receptors can be discriminated (Bazan, 1990). Class 1 cytokine receptors are also known as hematopoietic receptors (Wells and de Vos, 1996), class 2 cytokine receptors bind interferons and cytokines of the interleukin (IL)-10 family (Pestka et al., 2004). While the extracellular domains of class 1 and class 2 cytokine receptors are mainly formed by ␤-sheets the structures of the ligands are dominated by ␣-helices. Therefore, these mediators are often designated as helical cytokines. All interferons and most interleukins are helical cytokines. Prominent exceptions are the IL-1 family (including IL-18, IL-33 and IL-37) and IL-17 family (including IL-25) of cytokines as well as IL-8, the latter being a chemokine known as CXCL-8. Furthermore, some colonystimulating factors and classical hormones such as granulocyte colony-stimulating factor (G-CSF), erythropoietin (Epo), growth hormone (GH), prolactin and leptin share the structure of helical cytokines and signal through the JAK/STAT pathway.

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Fig. 2. gp130 receptor complexes and the structure of STATs. (A) Left scheme: Domain structure of gp130. Conserved disulfide bonds in D2 and the WSXWS-motif in D3 are indicated. Right scheme: IL-6 and IL-11 signal through gp130 homodimers. For these cytokines to act, non-signalling accessory receptors (IL-6R␣ and IL-11R␣, respectively) are required. gp130 can also signal as a heterodimer with other tall cytokine receptors such as LIFR, OSMR or IL-27R. Leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1) and neuropoietin (NP) form heterodimers of gp130 and LIFR. Cardiotrophin-like cytokine (CLC) and ciliary neurotrophic factor (CNTF), in conjunction with another accessory receptor, CNTFR␣ (which is linked to the plasma membrane through a glycosylphosphatidylinositol (GPI)-anchor) also signal through gp130/LIFR heterodimers. Oncostatin M (OSM) and IL-27 signal through heterodimers of gp130 with OSMR and IL-27R (also known as WSX-1), respectively. (B) Conserved domains of STATs. STAT proteins share a common domain structure with an N-terminal domain followed by a coiled-coil domain, a DNA-binding domain, a linker domain, an SH2 domain and finally a C-terminal transactivation domain. The tyrosine residue that becomes phosphorylated upon activation is located between the SH2 and the transactivation domain. Activity of the transactivation domain is modulated by phosphorylation of a serine residue. C-terminally truncated ␤-isoforms of STATs that lack the transactivation domain are not shown. (C) The nonphosphorylated STAT3 “core fragment” lacking the N-terminal and the transactivation domain is a monomer (Ren et al., 2008) (left) supporting the importance of the N-terminal domain in the formation of unphosphorylated STAT3 dimers (Vogt et al., 2011). The phosphorylated core fragment is shown as a dimer bound to DNA (right) (Becker et al., 1998) plus the N-terminal domain of STAT4. The structure of the N-terminal domain of STAT4 (Vinkemeier et al., 1998) is shown as the structure of the N-terminal domain of STAT3 is not solved yet. No structural data are available on the transactivation domains of STATs. The colour code is equivalent to (B), Y705 important for activation of STAT3 is drawn in yellow (modified from Chatterjee-Kishore et al., 2000).

Cytokine receptors can act as homodimers, heterodimers or even higher order receptor complexes. The most simple receptor systems are formed by homodimers of class 1 cytokine receptors with ectodomains consisting of a single CBM represented by the receptors for growth hormone (GHR), erythropoietin (EpoR) and prolactin. Therefore, it is not surprising that the first crystal structure of the extracellular part of a class 1 cytokine receptor complex is derived from GH and GHR. The structure reveals that a single GH molecule binds with two separate sites (site 1 and site 2) to the two GHRs. This structure is an early example for redundancy and

plasticity in protein-protein interactions because largely identical surface areas of the two GHRs bind to the two structurally distinct sites of GH. This feature is of considerable importance for shared cytokine receptors discussed below. From this structure and supporting experimental data (Cunningham et al., 1991) a sequential mode of receptor activation was derived, meaning that site 1 of GH binds to the first GHR and this complex recruits the second GHR through site 2 (Fig. 1A). These interactions are stabilised by an additional contact of the membrane proximal parts of the two GHRs.

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This general view of receptor activation was challenged by the observation that the ectodomains of unliganded EpoR crystallise as a homodimer suggesting that the EpoR exists as a preformed dimer at the plasma membrane (Livnah et al., 1999). Moreover, the conformation of the unliganded EpoR dimer markedly differs from the EpoR dimer in the Epo/EpoR complex (Syed et al., 1998). From these and other observations it was concluded that activation of a cytokine receptor includes stabilisation of a (preformed) receptor dimer and adjustment of a conformation that allows initiation of signal transduction through activation of the associated JAKs (Ballinger and Wells, 1998) (Fig. 1B). Compared to sequential receptor activation (Fig. 1A) preformed receptor complexes should result in increased sensitivity because no intermediate complexes are formed which could dissociate prior to the encounter with a second receptor. Instead, the ligand is immediately captured in a high-affinity interaction with the preformed receptor dimer.

Tall and shared cytokine receptors gp130, another class 1 cytokine receptor, differs from the above discussed receptors in several ways. First, the ectodomain consists of an N-terminal Ig-like domain and three additional membrane-proximal FNIII-like domains besides the CBM (Fig. 2A, left). Cytokine receptors with such an extended ectodomain architecture have been designated as ‘tall cytokine receptors’. This subfamily includes among others the receptors for G-CSF, leptin, and IL-12. Second, gp130 belongs to the group of shared cytokine receptors which are utilised by several ligands and can form heteromers with other receptors. Other important shared receptors are the common ␤-chain for the IL-3 family of cytokines (IL-3, IL5, granulocyte macrophage (GM)-CSF) and the common ␥-chain for the IL-2 family of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21). gp130 is the most promiscuous class 1 cytokine receptor serving nine cytokines with the help of six other receptors (Müller-Newen, 2003; Wang et al., 2009) (Fig. 2A, right). How can gp130 interact with so many different ligands and receptors? It has been shown that the ligand-binding site in the CBM of gp130 can adapt to the various receptor binding sites of the different cytokines through a mechanism termed ‘thermodynamic plasticity’ (Boulanger et al., 2003a). The interaction is dominated by an exposed hydrophobic residue in the CBM of gp130 (Phe191) located in a loop connecting two ␤-strands. This hydrophobic residue interacts with a hydrophobic patch on the cytokine surface (Kurth et al., 1999). The surrounding residues which are also located in conformable loops can be engaged in different ways (main chain atoms or side chain atoms, salt bridges or hydrogen bonds) depending on the bound cytokine. The promiscuity of gp130 challenges the view of preformed receptor complexes. Stable preformed gp130 homodimers would support IL-6 and IL-11 signalling but discriminate against the other cytokines that require heterodimers of gp130 with another tall cytokine receptor, such as the leukemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) (Fig. 2A). Therefore, if preformed gp130 homo- or heterodimers exist on the cell surface they must be of low affinity and transient to allow signalling of gp130 with other receptors. Indeed higher order complexes of gp130 are only detectable in lysates from stimulated cells (Fig. 3). Thus, if preformed receptor complexes exist the interactions might be too weak to survive solubilisation of membranes with detergents. To detect gp130 receptor complexes on the membranes of intact cells, fluorescence resonance energy transfer (FRET) studies with fluorescently labelled receptors have been performed (Giese et al., 2005; Tenhumberg et al., 2006). With this approach, preformed gp130 receptor complexes have been detected at the plasma membrane. However, using soluble receptors, no interactions of gp130 with

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Fig. 3. Analysis of cytokine receptor complexes by blue-native PAGE (bn-PAGE). HEK293T cells were transfected with fluorescent gp130-YFP and IL-6R␣-YFP (left panel) or fluorescent gp130-YFP and non-fluorescent LIFR (right panel). Forty-eight hours after transfection cells were stimulated for 30 min with IL-6 (25 ng/ml) or LIF (20 ng/ml) as indicated. Cellular lysates were prepared with 0.5% Triton X-100. Proteins were separated by blue-native polyacrylamide gel electrophoresis as described (Metz et al., 2007). Left to the gel, the polyacrylamide gradient or concentration is indicated. The wet gels were analysed using a Typhoon fluorescence scanner detecting only the YFP-labelled proteins. Higher molecular mass receptor complexes are only detectable upon stimulation of cells.

LIFR or the IL-6 receptor ␣-subunit (IL-6R␣) have been observed in the absence of the ligands (Boulanger et al., 2003b; Skiniotis et al., 2008; Ward et al., 1994; Zhang et al., 1997). These observations point to a contribution of the transmembrane and cytoplasmic parts of the receptors, possibly including the associated JAKs, in the formation of receptor complexes on the plasma membrane in the absence of ligand. A recent study provides a structural snapshot of the entire gp130/IL-6R␣/IL-6/JAK1 complex reconstituted in lipid nanodiscs (Lupardus et al., 2011). This fascinating structure reveals intimate interactions between the transmembrane and juxtamembrane segments of the gp130 homodimer including the associated JAKs that could drive low-affinity interactions even in the absence of ligand. As gp130 does not signal in the absence of ligand even upon forced overexpression, the preformed, possibly transient receptor dimers must remain signalling incompetent. How then is signalling initiated upon ligand binding? A possible explanation comes from studies with monoclonal antibodies. Interestingly, unlike e.g. EpoR, gp130 cannot be efficiently activated with a single monoclonal antibody (mab) but requires the simultaneous action of two mabs (Autissier et al., 1998). One intact mab and the antigen-binding fragment (Fab-fragment) of a second mab turned out to be the minimal requirement for gp130 activation (Müller-Newen et al., 2000). These pairwise agonistic mabs bind to the same receptor epitopes as the natural ligand does. From these findings it was concluded that both, enforced stabilization of dimers and adjustment of a well-defined, signalling competent conformation are required for activation of gp130. Further studies suggested that the correct adjustment of the juxtamembrane domains and transmembrane helices are critical for the activation of cytokine receptors (Constantinescu et al., 2001; Greiser et al., 2002; Kurth et al., 2000; Seubert et al., 2003). Constitutively active gp130 mutants causing inflammatory hepatocellular carcinoma have been recently discovered (Rebouissou et al., 2009). In these mutants a few amino acids in the ligand-binding site of gp130 are deleted. These findings support the view that minor structural changes in the gp130 homodimer might be sufficient for activation. However, it should be noted that the exact mechanism of receptor activation might vary within the family of cytokine receptors as has been demonstrated for the functionally related family of receptor tyrosine kinases (Lemmon and Schlessinger, 2010).

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Fig. 4. Phosphorylation and nuclear translocation of STAT3 in response to IL-6 does not require an intact cytoskeleton. (A) HepG2 cells were transiently transfected with STAT3 fused to the cyan fluorescent protein (STAT3-CFP). Forty-eight hours after transfection cells were preincubated for 30 min with cytochalasin D (2 ␮M), nocodazole (5 ␮M) or a DMSO control as indicated. Cells were stimulated for 30 min with IL-6 (20 ng/ml). Cellular lysates were prepared and analysed by Western blot for STAT3 phosphorylation at Y705 (pSTAT3) and total STAT3 as indicated. (B) MEF cells lacking endogenous STAT3 were stably transfected with STAT3 fused to the yellow fluorescent protein (STAT3-YFP) (Herrmann et al., 2007). These cells were preincubated for 30 min with cytochalasin D (2 ␮M) or nocodazole (5 ␮M) as indicated. Cells were stimulated for 30 min with IL-6 (5 ng/ml) and sIL-6R (1 ␮g/ml). Cells were fixed and the actin or tubulin cytoskeleton was stained with phalloidin-TRITC or a tubulin antibody plus a rhodamine conjugated secondary antibody, respectively. The samples were analysed by confocal microscopy (Zeiss LSM 510). Activated STAT3 accumulates in dot-like structures within the nucleus as described earlier (Herrmann et al., 2004; Watanabe et al., 2004). According to a recent report these subnuclear structures might represent STAT paracrystals (Droescher et al., 2011). In this report it has been shown that paracrystal formation of STAT1 is prevented by SUMOylation at lysine 703. Scale bars represent 10 ␮m.

STAT dimers and STAT activation at receptors Despite the prevalence of the canonical model of JAK/STAT signalling it has been firmly established that STATs form dimers in the absence of the activating tyrosine phosphorylation (Braunstein et al., 2003; Novak et al., 1998; Stancato et al., 1996). For STAT3

it has been shown that the monomer/dimer ratio does not change upon phosphorylation (Haan et al., 2000; Kretzschmar et al., 2004; Schröder et al., 2004; Vogt et al., 2011). STAT proteins share a common modular structure (Fig. 2B and C) in which the N-terminal domain is of central importance for the formation of unphosphorylated STAT dimers (Vogt et al., 2011; Wenta et al., 2008). Thus,

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in analogy to what has been discussed above for cytokine receptors, STAT activation relies critically on the transition of an inactive to an active dimer. For STAT1 and STAT5A a gross rearrangement from an anti-parallel to a parallel homodimer upon activation has been proposed (Mertens et al., 2006; Neculai et al., 2005). The N-terminal domain is of central importance in this transition. Furthermore, the N-terminal domain of STAT1 is of importance for the dephosphorylation and inactivation in the nucleus (Mertens et al., 2006). However, deletion of the N-terminal domain in STAT3 only marginally affects activation and inactivation (Vogt et al., 2011; Zhang et al., 2006). Interestingly, only for STAT4 it has been unequivocally demonstrated that N-terminal domain-dependent formation of non-phosphorylated dimers is required for activation at the receptor (Ota et al., 2004). Zhang et al. reported that beside the SH2 domain the coiled-coil domain is of importance for STAT3 activation at the receptor (Zhang et al., 2002). Thus, as noted for cytokine receptors, mechanistic details of activation and inactivation vary between individual STATs. After ligand-binding most receptor complexes are internalised. In the endosomes which are formed after endocytosis the formerly extracellular part of the receptor together with the bound ligand extends into the lumen of the organelle whereas the cytoplasmic domain with the associated JAKs remains cytoplasmic. As a consequence, these vesicles should be capable of initiating signalling and have therefore been termed “signalling endosomes” (Howe and Mobley, 2004). Signalling endosomes have been firmly established in retrograde neuronal signalling mediated by receptor tyrosine kinases (Wu et al., 2009) as well as in transforming growth factor (TGF)-␤ (Hayes et al., 2002) and tumour necrosis factor (TNF)-␣ signal transduction (Schütze et al., 2008). The importance of signalling endosomes in JAK/STAT signalling and in particular for nuclear accumulation of STATs is still a matter of debate as outlined below.

Nucleocytoplasmic shuttling of STATs In the canonical model latent STATs are cytoplasmic and accumulate in the nucleus upon tyrosine phosphorylation and subsequent dimer formation. The non-canonical model accounts for the nuclear presence of STATs in the absence of the activating phosphorylation. In fact, in non-stimulated cells the nucleocytoplasmic distribution of latent STATs is perpetuated by a steady state of constant nuclear import and export. Disturbance of this equilibrium by, e.g. inhibitors of exportin-1 (also known as CRM1)-mediated nuclear export leads to nuclear accumulation of unphosphorylated STATs (Bhattacharya and Schindler, 2003; Frahm et al., 2006; Zeng et al., 2002). The steady state distribution as well as the shuttling dynamics differ between individual STATs. A real-time analysis with fluorescently labelled STAT3 (Pranada et al., 2004) revealed that nucleocytoplasmic shuttling is quite slow with a half-life of recovery after bleaching in the range of 20–25 min (Herrmann et al., 2007). Shuttling of STAT5A (Iyer and Reich, 2008), STAT5B (Zeng et al., 2002, and our unpublished observations) and STAT6 (Chen and Reich, 2010) shows a similar time-course. The involvement of importins in this slow shuttling of STATs is still a matter of debate (Liu et al., 2005; Ma and Cao, 2006; Vogt et al., 2011). Alternatively, nuclear import of latent STATs might be driven by direct contact with constituents of the nuclear pore complex (Marg et al., 2004). In contrast, latent STAT2 shuttles remarkably fast, driven by a strong export sequence leading to a very low nuclear presence in the steady state (Frahm et al., 2006). A recent study demonstrated that dimerisation of latent STAT3 is not required for nucleocytoplasmic shuttling (Vogt et al., 2011). Nuclear latent STATs might act as transcriptional cofactors in gene regulation (Li, 2008). Upon formation of phosphorylated STAT dimers importindriven nuclear import leads to nuclear accumulation. However, it

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is not clear whether STATs interact directly with importins (Liu et al., 2005; Ma and Cao, 2006; Nardozzi et al., 2010) or through an adapter such as MgcRacGAP (Kawashima et al., 2009). For persistently activated STAT3 a steady state of constant import and export that involves an activation/inactivation cycle with phosphorylation at the receptor and dephosphorylation in the nucleus was postulated (Darnell, 2005). Indeed a rapid nucleocytoplasmic shuttling of persistently activated STAT3 has been demonstrated with fluorescently labelled STAT3 in the presence of v-Src as the activating kinase (Herrmann et al., 2007). Besides the reciprocal intermolecular SH2 domain/phosphotyrosine interaction, dimerisation of STAT3 in response to IL-6 or OSM has been reported to be stabilised by acetylation at lysine 685. Acetylation is catalysed by p300/CREB-binding protein (CBP) histone acetyltransferase leading to enhanced DNA-binding and gene induction (O’Shea et al., 2005; Wang et al., 2005; Yuan et al., 2005). A postulated phosphorylation/acetylation switch that regulates the activity of STAT1 (Krämer et al., 2009) has recently been critically re-evaluated (Antunes et al., 2011). The role of the cytoskeleton in activation and nuclear accumulation of STATs is also controversially discussed. Bild et al. reported that cytoplasmic transport of STAT3 in response to epidermal growth factor (EGF) stimulation is an active process that requires receptor-mediated endocytosis and cytoplasmic transport (Bild et al., 2002). Another report describes the sequestration of STAT3 to endosomes in response to IL-6 stimulation (Xu et al., 2007). Kermorgant et al. discriminated between weak STAT3 activators such as the receptor tyrosine kinase c-met (the hepatocyte growth factor (HGF) receptor) that require receptor trafficking for nuclear accumulation of STAT3 and strong STAT3 activators such as the OSMR that trigger nuclear accumulation independently of receptor trafficking (Kermorgant and Parker, 2008). Endosomal trafficking depends on an intact cytoskeleton. Lillemeier et al. reported that activation and nuclear translocation of STAT1 in response to interferons does neither require an intact actin cytoskeleton nor microtubules (Lillemeier et al., 2001). We made similar observations for nuclear translocation of STAT3 in response to IL-6 and related cytokines. Treatment of cells with cytochalasin D or nocodazole to destroy the actin filaments or microtubules, respectively, did neither affect STAT3 phosphorylation (Fig. 4A) nor nuclear translocation (Fig. 4B). In a recent study we covalently immobilised fluorescently labelled IL-6 or OSM on beads. These beads had a diameter of 3 ␮m and therefore were too big to be internalised. Stimulation of cells with these cytokine-loaded beads resulted in nuclear accumulation of STAT3 (Recker et al., 2011). Thus, there is cumulative evidence that in response to strong activators such as IL6 or OSM receptor trafficking or signalling endosome formation is not needed for nuclear accumulation of STAT3. This view is also supported by the observation that microinjection of phosphorylated STAT1 leads to nuclear accumulation without the involvement of any receptor (Meyer et al., 2003). However, these findings do not exclude that signalling endosomes might contribute to JAK/STAT signalling as has been shown in particular in the context of STAT1/2 signalling in response to type I interferons (Marchetti et al., 2006). Endosomal signalling might become even more important for cells with long protrusions such as neurons or dendritic cells. Activation of cytokine receptors at distant sites of axons or dendrites might require retrograde transport and endosomal trafficking of STATs for nuclear accumulation of the transcription factor.

Non-canonical functions of STATs The best documented function of STATs is the regulation of transcription as DNA-bound dimers (Fig. 2C). Such STAT/DNA complexes are only formed upon activation of STATs through

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Fig. 5. Non-canonical functions of STATs and their regulation through phosphorylation. Serine (blue) and tyrosine (red) phosphorylation regulate the availability of STAT3 for its canonical function as a transcription factor and the diverse non-canonical functions such as stabilisation of microtubules through capturing of stathmin, support of the respiratory chain and promotion of heterochromatin formation. See text for more details.

phosphorylation of the critical tyrosine residue. Beside this canonical function some non-canonical functions are emerging for STAT3 which are independent of tyrosine phosphorylation. It has been shown that STAT3 binds to the COOH-terminal tubulin-interacting domain of the microtubule-destabilising protein stathmin leading to stabilisation of the microtubule network (Ng et al., 2006) which directly affects cell migration (Gao and Bromberg, 2006; Verma et al., 2009). The function of STAT3 as a regulator of oxidative phosphorylation within mitochondria is another challenging observation. The absence of STAT3 in mitochondria compromises the function of the respiratory chain (Wegrzyn et al., 2009). Instead of tyrosine phosphorylation and DNA-binding the serine phosphorylation site at position 727 is required for the mitochondrial activity of STAT3 and STAT3-dependent cell transformation by oncogenic ras (Gough et al., 2009). In mitochondria STAT3 interacts with complexes I and II of the respiratory chain. It has recently been argued that this interaction cannot fully account for the mitochondrial functions of STAT3 because of the low amount of mitochondrial STAT3 compared to the proteins of the respiratory chain (Phillips et al., 2010). Nevertheless, the mitochondrial activity of STAT3 in combination with its canonical function as a transcription factor might contribute to the metabolic switch to anaerobic glycolysis (Warburg effect) which is observed in cancer cells. Hypoxia-inducible factor-1␣ (HIF-1␣) is a central transcription factor for the induction of glycolytic enzymes. STAT3 increases the levels of HIF-1␣ through canonical transcriptional and noncanonical posttranslational activities (Darnell, 2010; Demaria et al., 2010). Non-canonical functions of STAT5A have been reported in the context of chronic myeloid leukemia (CML). In this disease

activated STAT5A is located in the cytoplasm of leukemic cells. There, STAT5A interacts with the regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI3K) and the adapter protein Gab2 resulting in the activation of the serine threonine kinase Akt which is essential for cell growth (Harir et al., 2007). Moreover, a localisation of activated STAT5A in podosomes which is dependent on the Src family kinase Hck has been described (Poincloux et al., 2007). The function of STAT5A in these structures is not known. Finally the nuclear presence of non-phosphorylated STATs implies a possible activity as a transcriptional coactivator which is independent from canonical DNA-binding (Li, 2008). A function as a regulator of heterochromatin stability has been reported for the STAT ortholog STAT92E in Drosophila (Shi et al., 2008). Loss of STAT92E affects heterochromatin formation. The same effect is observed upon expression of a constitutively active mutant of the JAK ortholog Hopscotch. Mutant Hopscotch leads to STAT92E phosphorylation indicating that non-phosphorylated STAT92E contributes to heterochromatin formation (Shi et al., 2006). In this context non-phosphorylated STAT92E acts through interaction with the heterochromatin protein 1 (HP1) (Shi et al., 2008). Whether STATs have a similar function in the regulation of heterochromatin in mammalian cells is not known yet. However, tyrosine phosphorylation of histone H3 by nuclear JAK2 in hematopoietic cells has been reported (Dawson et al., 2009). As a consequence HP1␣ is excluded from heterochromatin leading to its destabilisation. Some of the non-canonical activities of STATs and their regulation through phosphorylation are summarised in Fig. 5. These non-canonical functions open a wide field for future research on the multifaceted JAK/STAT signalling pathway.

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Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 542, projects B12 and Z1).

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