Kathryn Geraghty

Shuai Chen

Jean E Harthill

Adel F Ibrahim

Rachel Toth

Nick A Morrice

Franck Vandermoere

Greg B Moorhead

D Grahame Hardie

Carol Mackintosh
email: c.mackintosh@dundee.ac.uk

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to insulin-like growth factor 1, EGF, PMA and AICAR

AS160 mediates insulin-stimulated GLUT4 translocation, but is widely expressed in insulininsensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography we found that binding of AS160 to 14-3-3 isoforms in HEK293 cells was induced by insulin-like growth factor 1 (IGF1), epidermal growth factor (EGF), phorbol ester (PMA), and to a lesser extent by AICAR. AS160/14-3-3 interactions were stabilised by chemical cross-linking, and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666, and Ser751) were phosphorylated differentially in response to IGF1, EGF, PMA and AICAR. The binding of 14-3-3s to HA-AS160 was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC kinases RSK1, SGK1 and PKB displayed distinct signatures of AS160 phosphorylation in vitro: All three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK preferentially phosphorylated Ser588, with lesser phosphorylation of other sites. In cells, the IGF1stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI 3-kinase inhibitors, while the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and use of AICAR in HEK293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF1-induced phosphorylation of the novel phosphoSer666-Pro site was suppressed by AICAR, and by combined mutation of a TOS-like sequence (FEMDI) and rapamycin. Thus, while AS160 is a common target of insulin, IGF1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.

INTRODUCTION

14-3-3s are C-shaped proteins that dock onto specific phosphorylated sites, forcing changes in the conformations of their targets and/or their interactions with other molecules [START_REF] Mackintosh | Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes[END_REF]. Hundreds of proteins that bind to 14-3-3s directly, or as components of multi-subunit complexes, have been purified from mammalian cells using their affinity for 14-3-3s [START_REF] Benzinger | Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer[END_REF][START_REF] Jin | Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization[END_REF][START_REF] Meek | Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins[END_REF][START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF].

Thus, binding of 14-3-3s to phosphoproteins is a common mechanism for linking signalling pathways to the regulation of cellular processes.

A sense of the global regulation of 14-3-3/target interactions can be gleaned by using Far Western (14-3-3 overlay) assays to observe how the patterns of phosphorylated 14-3-3 binding proteins change in extracts of cells stimulated in different ways [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF][START_REF] Harthill | Regulation of the 14-3-3-binding protein p39 by growth factors and nutrients in rat PC12 pheochromocytoma cells[END_REF]. Such experiments show that the phosphorylation of a substantial subset of 14-3-3 targets is promoted by the phosphatidylinositide 3-kinase (PI 3-kinase) pathway, including cardiac PFK-2 [START_REF] Rubio | 14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKBphosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase[END_REF], TSC2 [START_REF] Cai | Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning[END_REF][START_REF] Li | Regulation of TSC2 by 14-3-3 binding[END_REF], p27(Kip1) [START_REF] Sekimoto | 14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1)[END_REF], FOXO3a [START_REF] Rena | Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14-3-3 binding, transactivation and nuclear targetting[END_REF], and AS160/TBC1D4 [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF][START_REF] Howlett | Resistance exercise and insulin regulate AS160 and interaction with 14-3-3 in human skeletal muscle[END_REF][START_REF] Ramm | A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160[END_REF] AS160/TBC1D4, protein kinase B (PKB/Akt) substrate of 160 kDa [START_REF] Kane | A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain[END_REF], has been implicated in mediating the translocation of the GLUT4 glucose transporter to the plasma membrane in response to insulin, a process whose deregulation occurs early in the pathophysiology of insulin resistance and Type II diabetes [START_REF] Perseghin | Cellular mechanism of insulin resistance: potential links with inflammation[END_REF]. The emerging role of AS160 in insulin-stimulated GLUT4 trafficking is:-

1)

In the basal state, most GLUT4 is in intracellular GLUT4 storage vesicles (GSVs).

AS160 is a Rab GTPase activating protein (RabGAP) that promotes hydrolysis of GTP to GDP by Rabs 8A, 10 and 14 on the GSV [16][17][START_REF] Miinea | AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain[END_REF][19]. It is proposed [19][20][START_REF] Watson | Bridging the GAP between insulin signaling and GLUT4 translocation[END_REF] that in their inactive GDP-bound state the GSV-bound Rabs inhibit GLUT4 exocytosis.

2)

Upon insulin stimulation, AS160 is phosphorylated [16,22], which leads to its binding to 14-3-3s [START_REF] Ramm | A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160[END_REF] and the inactivation of the RabGAP activity of AS160 and/or its dissociation from GSVs [17,[START_REF] Peck | Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase[END_REF]. Thus, GSV-associated Rabs are thought to become loaded with GTP and promote events that lead to expression of GLUT4 at the cell surface, thereby mediating the influx of glucose.

The anti-diabetic drug metformin activates the LKB1/AMPK signalling pathway [START_REF] Musi | Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes[END_REF][START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF][START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF], which has an insulin-sensitizing effect on the liver leading to decreased hepatic glucose production [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF], and also enhances glucose uptake into tissues, possibly by altering the kinetics of GLUT4 trafficking to and from the plasma membrane. AICAR (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside), which also activates AMPK, has been reported to promote phosphorylation of AS160 [START_REF] Kramer | Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle[END_REF][START_REF] Thong | The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic[END_REF], raising the question of whether metformin also regulates AS160. Other PI 3-kinase-independent pathways have also been reported to regulate AS160 [START_REF] Thong | The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic[END_REF].

Together, these findings suggest that AS160 may be a convergence point for signalling pathways that promote GLUT4 translocation. The tissue distribution of AS160 is wider than GLUT4 however, suggesting that AS160 may also regulate trafficking of vesicles carrying other cargoes. Moreover, while AS160 is subject to multisite phosphorylation [22],

most regulatory studies have focussed on Thr642, whose phosphorylation is linked to insulin-mediated 14-3-3 binding [START_REF] Ramm | A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160[END_REF]17] and GLUT4 trafficking [22], and/or monitored phosphorylation of AS160 using the PAS antibody, which recognises sequences phosphorylated on a generic Akt/PKB phosphomotif [START_REF] Kramer | Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle[END_REF][START_REF] Thong | The Rab GTPase-activating protein AS160 integrates Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4 traffic[END_REF][START_REF] Bruss | Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity[END_REF][30]. Here, our initial aim was to define further mechanistic details of 14-3-3 binding to AS160. Our findings led to a wider study in which we showed that IGF1, EGF, PMA and AICAR induce distinctive patterns of multisite phosphorylation and 14-3-3 binding of AS160, involving at least four protein kinases.

EXPERIMENTAL

Materials

Antibodies and protein kinases

Sheep anti-HA was raised against the peptide YPYDVPDYA, and sheep anti-AS160 against KAKIGNKP (near the C-terminus of human AS160; Supplementary Fig 1 GRKRTSpSTCSNES (745 to 757, pSer751). The pSer318 and pSer751 peptides were conjugated via their N-termini, whereas the others were coupled via the added cysteines.

Peptides were coupled separately to BSA and keyhole-limpet haemocyanin, mixed and injected into sheep at Diagnostics Scotland (Penicuik, UK). The antibodies were affinitypurified on phosphopeptide-Sepharose columns, and in some cases were also passed through columns coupled to the cognate unphosphorylated peptides and the flow-throughs collected.

In each case, the antibodies recognised the synthetic phosphopeptide immunogen, but not the corresponding unphosphorylated peptide in dot blot assays (not shown). Anti-phosphoErk1/2 (pThr202/pTyr204), anti-phosphoThr308-PKB and anti-PKB/Akt were from Cell Signalling Technology. Western blots used the indicated antibodies at 1 µg/ml. Fig 1, all K19 Western blots, and blots of cell lysates were visualised by ECL (enhanced chemiluminescence reagent), whereas other Western blots used secondary antibodies that were detected using the Odyssey Infrared Imaging System (LI-COR, Inc.). 14-3-3 overlays (using DIG-labelled 14-3-3 in place of primary antibody) used ECL detection [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF]. for 30 min) prior to stimulation with IGF1 and other stimuli. After stimulation, cells were lysed in 0.2 ml ice-cold lysis buffer [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF]. For experiments using chemical crosslinker, cells were rinsed with cold PBS, lysed in 0.3 ml lysis buffer containing dithiobis[succinimidyl propionate] (DSP, 2.5 mg/ml from 250 mg/ml in DMSO) for 30 min on ice, and unreacted cross-linker was quenched with 75 µl of 1 M Tris-HCl pH7.4 with a further 30 min incubation [START_REF] Kim | mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[END_REF].

For immunoprecipitations with anti-AS160 and anti-HA, 4 µg antibody/mg lysate was mixed at 4 o C for 1 h, then protein G-Sepharose (30 µl of a 50% suspension in lysis buffer) added and mixed for a further hour. The suspension was centrifuged at 12,000 g for 1 min between washes.

The human HeLa cell-lines stably transfected with wild-type LKB1 and kinase-dead LKB1 were those derived by Sapkota et al [32].

RESULTS

IGF1 promotes the phosphorylation and binding of AS160 to 14-3-3 proteins

Previously, AS160 was isolated from HeLa cell extracts by 14-3-3-affinity chromatography [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF]. Western blotting confirmed that AS160 bound to 14-3-3-Sepharose and was specifically eluted by a canonical 14-3-3 binding phosphopeptide, ARAApSAPA, but In contrast to the 14-3-3 overlays, only small sub-stoichiometric amounts of 14-3-3 proteins were detected in anti-AS160 immunoprecipitates from IGF1-stimulated cells (not shown). However, 14-3-3s were co-immunoprecipitated with AS160 from IGF1-or calyculin A-stimulated cells when protein-protein interactions were stabilised by DSP, which (Supplementary Table 2) [22]. In addition, phosphoSer318 and phosphoThr642 (also identified in [22]) were detected in endogenous AS160 using phosphospecific antibodies (later results), and by mass spectral analysis of recombinant AS160 isolated from human cells (not shown).

In addition to known sites, a new phosphopeptide from endogenous AS160 was identified by tandem mass spectrometry as AQGVRpSPLLR (Fig 2). The phosphorylated Ser666 within this phosphopeptide lies within a pSer-Pro motif that is not a consensus site for PKB. The MS data indicated a marked increase in phosphorylation at Ser666 in response to IGF1, and the increased phosphorylation was blocked by LY294002 (Supplementary Table 3).

Mutation analysis indicates that 14-3-3 binds to phosphorylated Thr642 and Ser341

HA-AS160 with each of these seven phosphorylation sites individually mutated from Ser/Thr to Ala was tested for 14-3-3 binding using overlay assays and by co-precipitation of 14-3-3s with HA-AS160 from cell extracts. Only the Thr642Ala mutation had a marked effect in preventing the IGF1-induced binding of 14-3-3s to HA-AS160 (Fig 3A).

The generation of phosphospecific-AS160 antibodies to monitor the phosphorylation of seven sites on AS160

To examine the cellular regulation of these phosphorylations further, we raised phospho-specific antibodies against each of the seven phosphorylated residues on AS160 (pSer318-AS160, pSer341-AS160, pSer570-AS160, pSer588-AS160, pThr642-AS160, pSer666-AS160, and pSer751-AS160) (Supplementary Fig 2A).

Phosphorylation of AS160 in response to IGF1 and AICAR, but not phenformin, in HEK293 cells

The phosphospecific antibody data for endogenous AS160, and HA-AS160 from transfected cells, showed that the phosphorylation of each of the seven sites was enhanced when cells were stimulated with serum and IGF1. These phosphorylations were prevented

In vitro phosphorylation of GST-AS160 by PKB, AMPK, SGK1 and RSK1; identification of an eighth phosphorylation site at Thr568

Consistent with its proposed role in phosphorylation of AS160 [START_REF] Kane | A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPase-activating protein (GAP) domain[END_REF], PKB preferentially phosphorylated Ser318, Ser588 and Thr642, and also catalysed trace

IGF1-induced phosphorylation of Ser666 is partially inhibited by combined mutation of a TOS-like motif in AS160 and rapamycin, and blocked by AICAR

The IGF1-induced phosphorylation of the novel phosphoSer666-Pro site was not affected by SB203580, a specific inhibitor of SAPK2a/p38, or U0126, inhibitor of Erk1/2 activation (data not shown).

mTOR mediates the insulin and IGF1-induced phosphorylation of pSer/Thr-Pro motifs on 4E-BP1/PHAS (pT 37 PGGTLFSTpT 46 P) [36] and p70S6K (PVDS 371 P) [START_REF] Saitoh | Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site[END_REF].

mTOR signalling (TOS) motifs on these proteins act as docking sites for the mTOR/raptor (TORC1) complex, and are required for the phosphorylation of the Ser/Thr-Pro sites [START_REF] Schalm | Identification of a conserved motif required for mTOR signaling[END_REF].

Intriguingly, the AS160 protein contains a sequence, FEMDI, identical to the TOS motif on 4E-BP1. Consistent with a functional role for this motif, when the FEMDI motif was

DISCUSSION

We identified a specific and reversible interaction between phosphorylated AS160 and 14-3-3 proteins in cells stimulated with IGF1. The affinity of the phosphoAS160/14-3-3 interaction is sufficient for the phosphorylated AS160 to bind rapidly to the 14-3-3s inside the cell. However, it is poised at a point where the phosphorylated AS160 and 14-3-3s dissociate during the washing procedures of immunoprecipitation, unless stabilised by crosslinking. Such reversibility makes physiological sense for a regulatory interaction that must respond dynamically to changes in the stimulation and metabolic status of the cell. Cellular AS160 binds to at least six of the seven human isoforms of 14-3-3s (Fig 1F and

Supplementary Table 1). 14-3-3sigma was absent from the AS160 immunoprecipitates, and was not detected in cell extracts using anti-14-3-3sigma antibodies (not shown). The 14-3-3s that interact with AS160 may simply reflect the complement of isoforms in this particular cell type, although isoform selectivity cannot be ruled out.

We identified eight phosphorylated residues in AS160, of which six have been reported previously [22] and the pSer666-Pro and pThr568 sites are novel. However, studies of the responsiveness of AS160 to extracellular stimuli generally use the anti-PAS antibody that primarily detects phosphoThr642 ( [START_REF] Kramer | Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle[END_REF]30]; Supplementary Fig 2B phosphorylation mediates only a low affinity basal interaction with 14-3-3s, which is markedly enhanced when Thr642 is phosphorylated in response to insulin or IGF1.

Consistent with an essential role for 14-3-3s in GLUT4 translocation [START_REF] Ramm | A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160[END_REF], Thr642 is critical for mediating the translocation of GLUT4 and glucose uptake in response to insulin [22].

We suggest that a single 14-3-3 dimer might bind phosphoSer341 and phosphoThr642.

Intriguingly, these sites flank the second phosphotyrosine binding domain (PTB) on AS160

(Supplementary Fig 1), which was reported to bind to insulin-responsive aminopeptidase (IRAP; [START_REF] Peck | Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase[END_REF]) thereby docking AS160 onto the GSVs [17,[START_REF] Peck | Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase[END_REF]. Thus, 14-3-3 binding might disrupt the interaction between IRAP and the second PTB domain of AS160, thereby releasing the AS160 from the GSVs, enabling the Rabs to be loaded with GTP to promote translocation of the GSVs towards the cell surface. However, we have thus far failed to observe the IRAP/AS160 interaction either in vitro or cell extracts (not shown).

The potential for the AMPK to mediate the stimulatory effects of the nucleoside AICAR, as well as exercise, on GLUT4 translocation and glucose uptake into skeletal muscle and adipocytes has been studied [START_REF] Kramer | Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle[END_REF][START_REF] Bruss | Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity[END_REF]30] . We considered the possibility that AMPK phosphorylates Ser588 of AS160, but that (being metabolic poisons) metformin and phenformin cause another effect that inhibits AS160 phosphorylation. However, neither metformin nor phenformin blocked the IGF1-or AICAR-induced phosphorylations of AS160. Another possibility is that phenformin activates a phosphatase that can obscure phenformin-induced phosphorylation, but is insufficient to impact on IGF1-and AICAR-induced phosphorylations. We emphasise that the paradoxical effects of AICAR and phenformin do not seem to be unique to HEK293 cells. These were also seen for the AS160 from rat L6 myotubes (not shown), which are commonly used for studying the regulation of glucose trafficking by insulin. However, the majority of extractable AS160 in L6 cells was a truncated form lacking the regulatory phosphorylation sites (not shown).

LKB1 is the upstream kinase that mediates the activation of AMPK by AICAR and metformin/phenformin [39]. LKB1 also activates a number of AMPK-related protein kinases [39]. We found that Ser588 of AS160 was phosphorylated in HeLa cells containing active LKB1, but not in cells carrying the kinase-dead LKB1 (Fig 4B). Overall, these findings are consistent AS160 being phosphorylated by AMPK or another LKB1-dependent enzyme. Our findings agree with the lack of effect of metformin on basal or insulin-induced AS160 phosphorylation measured with the PAS antibody [START_REF] Karlsson | Effects of metformin and rosiglitazone treatment on insulin signaling and glucose uptake in patients with newly diagnosed type 2 diabetes: a randomized controlled study[END_REF]. To our knowledge, no other researchers have reported effects of metformin/phenformin on AS160. Perhaps, others made the same findings with AICAR and metformin/phenformin as we report here, and have been similarly puzzled by these paradoxical data.

The identity of the kinase(s) that phosphorylates Ser666 is unknown. The IGF1induced phosphorylation of Ser666 on AS160 was not blocked by rapamycin, unlike the IGF1-dependent phosphorylation of pSer-Pro sites on elongation factor 2 kinase (eEF2K), p70S6K, and 4EBP1 ( [START_REF] Knebel | A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta[END_REF]; Fig 7A). Moreover, phosphorylation of Ser666 was not inhibited by mutation of a putative TOS motif (FEMDI), which mediates mTOR /TORC1-dependent phosphorylation of downsteam targets. We cannot rule out a role for mTOR at this stage though, because the combination of mutation of the TOS-like motif and rapamycin together suppressed the IGF1-induced phosphorylation of Ser666 of AS160. The FEMDI sequence is conserved in AS160 proteins from all other mammalian species currently cited in sequence databases. Moreover, the IGF1-induced Ser666 phosphorylation was inhibited by AICAR.

Activation of mTOR in the TORC1 complex by insulin and IGF1 initially involves PKB, which inhibits the Rheb-GAP activity of the Tuberous Sclerosis heterodimer (TSC1/TSC2), and thereby increases the RhebGTP/RhebGDP ratio [START_REF] Sarbassov Dos | Growing roles for the mTOR pathway[END_REF]. AMPK inhibits insulin-induced mTOR/TORC1 signalling by mechanisms that include phosphorylation and activation of TSC2 [START_REF] Sarbassov Dos | Growing roles for the mTOR pathway[END_REF][45][START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF][START_REF] Shaw | The LKB1 tumor suppressor negatively regulates mTOR signaling[END_REF]. Therefore, the finding that AICAR blocks IGF1-induced phosphorylation of Ser666 of AS160 is consistent with a role for the mTOR family.

Recently, a mutation in the TBC1D1 gene, encoding a protein related to AS160, was linked with severe obesity in females [48]. TBC1D1 was isolated as a 14-3-3-binding protein [START_REF] Jin | Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization[END_REF]. Thr596 in TBC1D1, analogous to Thr642 in AS160, is phosphorylated in response to insulin in adipocytes [49] and some other AS160 phosphorylation sites are conserved in TBC1D1 (Supplementary Fig 1), and we found that 14-3-3s bind to TBC1D1 in response to IGF1 and insulin (S. Chen and C. MacKintosh, unpublished). Thus, dissecting further details of the control of AS160 and TBC1D1 by insulin/IGF1, and the respective roles of these proteins in glucose homeostasis, should give new insights into links between insulin signalling, obesity and other metabolic disorders. AS160 and TBC1D1 are both widely expressed though, and our findings also highlight AS160 as a potential mediator of growth factor-regulated vesicle trafficking beyond insulin regulation of GLUT4.

Figure legends

Figure 1 Phosphorylation-dependent binding of AS160 to 14-3-3 proteins in response to

IGF1

A. Isolation of AS160 by 14-3-3-affinity chromatography of HeLa cell extracts. Crude extract, flow through, first wash fractions (40 g) and 5 l of the concentrated last wash, control peptide and final wash fractions (<1 g protein) from the 14-3-3 column were loaded on a 4-12% Bis-Tris SDS-gel, alongside 2 g of the proteins that were eluted with 14-3-3-binding phosphopeptide, ARAApSAPA (phosphopeptide elution). The gel was transferred to nitrocellulose, and anti-AS160

Western blot performed. Protein stains and 14-3-3 overlay signals for this column were similar to those in [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF].

B. Untransfected HEK293 cells that had been serum-fed, unstimulated, IGF1stimulated, IGF1 stimulated in the presence of LY294002, and treated with calyculin A, were lysed in the presence of DSP cross-linker. Endogenous AS160 was immunoprecipitated from lysates (6 mg), denatured and run on a 4-12% Bis-Tris SDS-gel, transferred to nitrocellulose, and the 14-3-3-binding ability of AS160 was tested by 14-3-3 overlay (middle panel). Cellular 14-3-3s that co-precipitated with endogenous AS160 were identified by Western blots with pan-14-3-3 antibodies (K-19 from Santa Cruz; bottom panel).

C. 14-3-3-Sepharose (20 l, prepared as in [START_REF] Rubio | 14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to regulation of cellular metabolism, proliferation and trafficking[END_REF]) was incubated with HEK293 lysate (6 mg) from cells that were serum-fed, unstimulated (serum-starved), IGF1-stimulated, IGF1-stimulated in the presence of LY294002, and treated with calyculin A. The 14-3-3-Sepharose was washed three times in a high salt buffer followed by two washes in low salt. AS160 bound to the 14-3-3-Sepharose was identified by Western blotting.

D. Endogenous AS160 was immunoprecipitated from 4 mg HEK293 cell extract, and incubated for 30 min at 30 o C in the presence of 50 milliunits/ml protein phosphatase 2A (PP2A), with or without the inhibitor microcystin-LR (MC-LR; 5 mM), as indicated. Proteins were separated by SDS-PAGE, transferred to nitrocellulose and the AS160 was probed for binding to 14-3-3s in the overlay assay.

E. HA-tagged AS160A (see Methods) was immunoprecipitated from lysates (3 mg) of transfected HEK293 cells that had been serum-fed, unstimulated, IGF1-stimulated, IGF1-stimulated in the presence of LY294002 and calyculin A treated. The immunoprecipitated protein was analysed by the 14-3-3 overlay. In addition, HA-AS160 was immunoprecipitated from cell lysates (1 mg), denatured in SDS sample buffer and run on a 4-12% Bis-Tris gel, which was stained with Colloidal blue (bottom panel).

F. HA-AS160A was immunoprecipitated from cell lysates (1 mg) prepared with and without DSP crosslinker, as indicated. The immunoprecipitated protein was run on a 4-12% Bis-Tris SDS-gel, which was stained with Colloidal blue, and the proteins indicated were identified by MS/MS analysis of tryptic digests. (See text and Supplementary Table 1 for details).

Figure 2

Precursor ion scan for the tryptic digest of endogenous AS160 from calyculin A-treated cells By liquid chromatography mass spectrometry on an Applied Biosystems 4000 Q-TRAP, a precursor ion scan identifies those parent masses that yield daughter ions with a m/z of 79kDa (PO3-), that is only the phosphopeptides indicated. The phosphorylated peptides were analysed further to identify the phosphorylated residues (Supplementary Table 2).

Figure 3

Identification of 14-3-3 binding sites on AS160

A. HEK 293 cells growing in the presence of serum were transfected with vectors to express wildtype HA-AS160A and mutants with the single Ser/Thr to Ala mutations indicated. The cells were unstimulated (U) or stimulated with IGF1 (I), and HAtagged proteins were immunoprecipitated from 500 g extract, and subject to anti-HA, anti-phosphoThr642-AS160, and anti-14-3-3 (K19) Western blots, and 14-3-3 overlays.

B. HEK 293 cells transfected with wild-type HA-AS160A, Thr642Ala-AS160A, and double mutants were unstimulated (U) or treated with IGF1 (I). The HA-tagged proteins were immunoprecipitated from 500 µg cell extract, one fifth of each immunoprecipitate was loaded onto each lane for immunoblotting, and blots were subject to anti-AS160, anti-HA, anti-phosphoThr642-AS160, anti-phospho-Ser341, and anti-14-3-3 (K19) Western blots, and 14-3-3 overlays.

The specificity of the phospho-specific antibodies is demonstrated in Supplementary Fig 2A.

In vitro phosphorylation of AS160 by AMPK and the AGC kinases, PKB, SGK1 and

RSK1

In the right hand panels, GST-AS160A was expressed and purified from E. coli, and phosphorylated in vitro with PKB (1 Unit/ml) and AMPK purified from rat liver (10 Units/ml). The phosphorylated proteins (300 ng per lane) were analysed by Western blots using phospho-specific-AS160 antibodies (Supplementary Fig 3A ,C) and 14-3-3 overlay, as indicated. Similarly, in the left hand panels, the GST-AS160A was phosphorylated in vitro by PKB, SGK1 and RSK1 (1 Unit/ml).

  Synthetic peptides were from Graham Bloomberg (University of Bristol), oligonucleotides from MWG-Biotech, IGF1 (insulin-like growth factor 1) was from Biosource, microcystin-LR from Linda Lawton (Robert Gordon's University, Aberdeen), frozen human HeLa cell pellets from 4c Biotech (Ghent, Belgium), Vivaspin concentrators from Vivascience, tissue culture reagents from Life Technologies, protease-inhibitor cocktail tablets (no. 1697498) and sequencing grade trypsin from Roche Molecular Biochemicals, 5aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) from Toronto Research Chemicals (North York, ON, Canada), precast SDS-polyacrylamide gels from Invitrogen, and dithiobis[succinimidyl propionate] (DSP) from Perbio. Protein G-Sepharose and other chromatographic matrices were from GE-Healthcare. All other chemicals were from BDH Chemicals or Sigma-Aldrich.

  Purified recombinant protein kinases, generated in the Division of Signal Tranduction Therapy (DSTT), were His-PKBalpha-S473D (residues 118 to 480 of human protein) expressed in insect cells and activated by PDK1; His-RSK1 (1 to 735 of rat) expressed in insect cells and activated by p42MAPK and PDK1; SGK1-S422D (60 to 431 of human) expressed in insect cells and activated by PDK1; and bacterially-expressed GST-AMPK-T172D (3 to 308 of rat protein

  not a control peptide (Fig 1A). When endogenous AS160 was immunoprecipitated from extracts of HEK293 cells that had been stimulated with serum, IGF1 or the protein phosphatase inhibitor calyculin A, the isolated AS160 bound directly to 14-3-3 proteins in an overlay assay (Fig 1B, middle panel). There was only a trace 14-3-3-binding signal for AS160 from unstimulated cells, or cells stimulated with IGF1 in the presence of the PI 3-kinase inhibitor LY294002 (Fig 1B, middle panel). Similarly, AS160 bound to 14-3-3-Sepharose in extracts of cells that had been stimulated with serum, IGF1 and calyculin A (Fig 1C, lower panel). The IGF1-induced 14-3-3-binding signal was unaffected by the mTOR inhibitor rapamycin, or U0126, which inhibits the activation of the classical MAPK pathway (data not shown).

  reversibly links primary amines (Fig 1B, bottom panel). The co-precipitation of 14-3-3s with AS160 was specifically induced by IGF1 and calyculin A, and blocked by LY294002 (Fig 1B, bottom panel). The ability of AS160 from IGF1-induced cells to bind to 14-3-3s was abolished by dephosphorylation of the AS160 with protein phosphatase 2A (PP2A) in vitro, and this was prevented by the PP2A inhibitor microcystin-LR (Fig 1D).IGF1 also induced the binding of 14-3-3s to HA-AS160 in transfected cells, as determined by binding of the extracted HA-AS160 to 14-3-3s in the overlay assay (Fig 1E,middle), and co-precipitation of 14-3-3s (Fig 1Ebottom panel and 1F). MS analysis of tryptic digests showed that at least six of the seven human 14-3-3 isoforms were bound to the HA-AS160 extracted from IGF1-stimulated cells in the presence of DSP (Supplementary Table1): The upper Coomassie-stained band (Fig1F) contained peptides that are unique to the 14-3-3epsilon. The lower band (Fig1F) contained peptides specific to 14-3-3theta, gamma, alpha/beta, eta, and delta/zeta. No peptides specifying 14-3-3sigma were identified.Identification of phosphorylated sites on AS160, including known sites and the novel pSer666-Pro siteDigests of endogenous AS160 were analysed by liquid chromatography mass spectrometry[START_REF] Williamson | Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer[END_REF] to identify IGF1-induced phosphorylations. Initially, precursor ion scans were run to detect phosphopeptides (Fig2and not shown). A Multiple Reaction Monitoring (MRM) analysis was then performed to look for the known phosphorylated sites on AS160

The 14 - 3 - 3

 1433 binding of Thr642Ala-AS160 was not completely abolished however (Fig 3A), indicating the existence of a second lower affinity 14-3-3-binding site. Therefore, double mutants of Thr642Ala combined with Ser to Ala mutations of each of the other phosphorylated sites were generated. Of the double mutants, the combination of Thr642Ala and Ser341Ala completely prevented the IGF1-induced binding of 14-3-3s to AS160 (boxed lanes in Fig 3B). Consistent with phosphorylation of Ser341 being responsible for the basal binding of 14-3-3s to AS160, the Ser341Ala mutation abolished the binding of 14-3-3s to AS160 in unstimulated cells (Fig 3B and data not shown). In combination with Thr642Ala, the mutation of Ser318 to Ala also decreased the binding of 14-3-3s to AS160 (Fig 3B), consistent with the Ser318Ala mutation causing a decrease in the phosphorylation of Ser341 (Fig 3B and Supplementary Fig 2A).

  The recognition of AS160 by anti-PAS was largely abolished by the Thr642Ala mutation indicating that phosphoThr642 is the prime anti-PAS recognition site on AS160 from cells stimulated with serum, IGF1 and insulin (Supplementary Fig 2B and data not shown).

  by LY294002 (Fig 4A and data not shown), except for phosphoSer588 whose phosphorylation was partly inhibited by LY294002 (Fig 4A and not shown). There was a high basal phosphorylation of Ser341 and Ser666 in unstimulated cells, which was also abolished by LY294002 (Fig 4A). The AMPK activator AICAR promoted phosphorylation of Ser588 of AS160 (Fig 4A), with much weaker phosphorylations of pSer341, Thr642 and Ser751 (Fig 4A and later results). Consistent with the much lesser phosphorylation of Ser341 and Thr642 compared to that seen with IGF1, AICAR promoted only weak binding of 14-3-3s to AS160 that was more easily detected in the overlay assay (Fig 4A) than by co-immunoprecipitation of AS160 with 14-3-3s from cell extracts (not shown). Phenformin promoted a much stronger phosphorylation of Thr172 in the activation loop of AMPK, and of the AMPK target acetyl CoA carboxylase (ACC), compared to AICAR and metformin (Fig 4A). Despite its strong activation of AMPK however, phenformin did not stimulate phosphorylation of AS160 (Fig 4A). Metformin promoted only weak phosphorylation of several sites on AS160 (Fig 4A), and, in particular, metformin did not stimulate phosphorylation of Ser588 to the same extent as AICAR (Fig 4A). We also noticed that phosphorylation of both AMPK and ACC was strongly promoted by LY294002 in HEK293 cells (Fig 4A), but not in other cells tested (not shown). Neither phenformin nor metformin inhibited any of the IGF1-and AICAR-stimulated phosphorylations of AS160 in HEK293 cells (not shown). In view of the paradoxical effects of AICAR and phenformin, we examined whether LKB1, an upstream activator of AMPK has any role in regulating phosphorylation of AS160. LKB1 is absent from HeLa cells [32] and AICAR did not promote phosphorylation of AS160 in these cells (data not shown). In HeLa cells that were stably transfected with wildtype LKB1, the presence of the active LKB1 was itself sufficient to induce phosphorylation of Ser588 of endogenous AS160, even in the absence of any AMPK activators (Fig 4B, left side). Kinase-dead LKB1 did not induce phosphorylation of AS160 at Ser588 (Fig 4B, right side).

  phosphorylation of Ser570 and Ser751 in vitro (Fig 5, right and left hand panels). In contrast, AMPK preferentially phosphorylated Ser588 of AS160, phosphorylated Ser570 to a similar degree as did PKB, and phosphorylated Thr642 to a lesser extent (Fig 5, right side). Compared with PKB, other AGC kinases displayed distinct signatures of AS160 phosphorylation in vitro (Fig 5, left side; and data not shown). In common with PKB, SGK1 and RSK1 phosphorylated Ser318, Ser588 and Thr642 (Fig 5, left side). In addition, RSK1 also phosphorylated Ser341 and Ser751; and SGK phosphorylated Ser751. MS analysis of tryptic digests of in vitro-phosphorylated AS160 also revealed a new phosphorylated site at Thr568, within the sequence KAKRSLpT 568 SSLENI, two residues N-terminal to the phosphoSer570 site (data not shown). A phospho-specific pThr568-AS160 antibody was raised and tested for phospho-and sequence specificity (Supplementary Fig 2C), and using this antibody it was found that SGK1 was the best Thr568 kinase of those tested, though this site was also phosphorylated by RSK1 (Fig 5, left side panel). These kinases displayed no or barely detectable phosphorylation of Ser666 (Fig 5). Phosphorylation and 14-3-3 binding of AS160 in response to EGF and PMA in HEK293 cells: a role for RSK in mediating the response to PMA We next assessed whether Thr568 of AS160 is phosphorylated in cells, and whether RSK is a physiological AS160 kinase. Alongside IGF1, AICAR and phenformin, cells were treated with EGF and the phorbol ester PMA, which stimulate RSK isoforms via the mitogen-activated protein kinase family members ERK1 and ERK2 ([34] and references within) (Fig 6 and Supplementary Fig 3).HA-AS160 and 14-3-3s were co-immunoprecipitated, and AS160 was phosphorylated on several sites and could bind to 14-3-3s in overlay assays, when extracted from cells stimulated with serum, IGF1, EGF and PMA (Fig 6 and Supplementary Fig 3). Of these stimuli, PMA gave the weakest 14-3-3-binding signals for AS160, consistent with its lesser stimulation of Thr642 phosphorylation (Fig 6 and Supplementary Fig 3). In contrast, PMA induced the strongest phosphorylation of Thr568, showing that this site is phosphorylated in cells (Fig 6) as well as in vitro (Fig 5). When PMA-induced and PKC-mediated phosphorylation of Erk1 and Erk2 was blocked by Go6983 (Fig 6, [35], this inhibitor largely abolished phosphorylation of AS160 in response to PMA (Fig 6). In addition, the RSK inhibitor BI-D1870 [34] markedly suppressed the responses to PMA, except the weak Thr642 phosphorylation (Fig 6). Go6983 and BI-D1870 also inhibited PMA-induced binding of AS160 to 14-3-3s (data not shown). Wortmannin had no effect on PMA-induced phosphorylations of AS160 (Fig 6). In contrast, wortmannin inhibited EGF-induced phosphorylation of Ser318 and Thr642, with little or no effect on the other EGF-regulated sites (Fig 6), though the RSK inhibitor BI-D1870 inhibited all the EGF-stimulated phosphorylations to different extents (Fig 6). In contrast to PMA, Go6983 inhibited phosphorylation of neither Erk1/2 nor AS160 in response to EGF. As before, the IGF1-stimulated phosphorylations were blocked by LY294002, and largely inhibited by wortmannin (Fig 6).

  mutated to AEMDI the IGF1-stimulated phosphorylation of Ser666 was more inhibited by rapamycin, compared with the wild-type HA-AS160 (Fig 7A, last lane). However, neither rapamycin alone, nor mutation of the FEMDI motif alone, had any obvious effect on Ser666 phosphorylation in IGF1-stimulated cells (Fig 7A). The IGF1-induced phosphorylation of a TOS-dependent site on p70S6K was prevented by rapamycin alone, as expected (Fig 7A, bottom panel). Amongst other effectors tested, the AMPK activator AICAR abolished both basal and IGF1-stimulated phosphorylation of Ser666 of AS160 (Fig 7B).

  using the anti-PAS antibody that primarily detects phosphoThr642 (Supplementary Fig 2B). However, we find that Ser588 is most strongly phosphorylated by AMPK, while Thr642 is a secondary site of phosphorylation by this kinase in vitro (Fig 5) and in cell responses to AICAR (Fig 4A). Intriguingly, Sano et al [22] pinpointed the double Ser588Ala/Thr642Ala-AS160 mutant as having a greater inhibitory effect on insulin-stimulated GLUT4 translocation than the single Thr642Ala-AS160 mutant, suggesting that Ser588 and Thr642 can both influence GLUT4 translocation. The high pSer588/pThr642 phosphorylation ratio induced by AICAR (Fig 4A), mirrors the specificity of AMPK towards AS160 in vitro (Fig 6), consistent with AMPK mediating the AICAR-induced phosphorylations. AICAR in cells is converted by adenosine kinase into the monophosphorylated nucleotide, ZMP, which mimics the effects of AMP on the AMPK system [39]. However, the AICAR-induced phosphorylation of AS160 occurred with barely detectable AMPK phosphorylation and activation (Fig 4A and not shown). This lack of effect of AICAR on total AMPK in HEK293 cells is consistent with previous findings [40]. AMPK is an alpha/beta/gamma heterotrimer and, theoretically, twelve forms of AMPK could exist comprising combinations of the two alpha, two beta and three gamma subunit isoforms. AICAR activates AMPK alpha2 complexes in mouse muscle [41], whereas AMPK alpha2 and gamma3 subunits are required for AICAR-induced AS160 phosphorylation detected by the PAS antibody [30]. The alpha2 subunit of the AMPK, which is a less major form than alpha1 in HEK293 cells (D.G. Hardie laboratory, personal observations), may therefore be the AS160 kinase. If so, one would have to explain how both metformin and phenformin promoted a marked phosphorylation of the AMPK and its downstream target ACC, and yet have little effect on phosphorylation of AS160, in contrast to the effects of AICAR. Metformin and phenformin inhibit Complex I of the mitochondrial respiratory chain and are thought to activate AMPK by increasing the intracellular AMP/ATP ratio [39]

Figure 4 Phosphorylation

 4 Figure 4Phosphorylation of seven sites on AS160 in response to different stimuli in HEK293cells, and effect of LKB1 on the phosphorylation of Ser588A. HEK293 cells transfected with HA-AS160A were stimulated as indicated. Crude lysates (60 µg per lane) were probed for binding to the antibodies as indicated along the left hand side of the upper panels. The cells transfected with empty vector were serum-starved before harvesting. In the lower four panels, extracts (40 g) of HEK293 cells that had been stimulated as indicated were analysed by Western blotting with antibodies to monitor the phosphorylation status of the activation loop on AMPK, the AMPK site on ACC (pSer79 on the rat protein, pSer80 in the human), and the activating (pThr308) phosphorylation site on PKB. B. Endogenous AS160 was immunoprecipitated from HeLa cells stably transfected with either LKB1 or kinase-dead LKB1 [32] and Western blotting and 14-3-3 overlays performed, as indicated.

Figure 6 Phosphorylation

 6 Figure 6Phosphorylation of all eight sites on AS160 in response to IGF1, EGF and PMA in HEK293 cells, and effects of protein kinase inhibitors HEK293 cells transfected with HA-AS160A were stimulated after pre-exposure to inhibitors, as indicated, and HA-AS160A was analysed by Western blots. In addition, cell lysates (60 µg per lane) were analysed by Western blotting to detect phosphorylation of Erk1 and Erk2, pThr308 of PKB, and total PKB.

Figure 7 Effect

 7 Figure 7 Effect of FEMDI to AEMDI mutation, rapamycin and AICAR on phosphorylation of Ser666 A. HEK293 cells transfected with HA-AS160A and HA-AEMDI-AS160A were stimulated with IGF1, in the presence or absence of LY294002 and rapamycin, as indicated. Crude lysates (60 g) were probed with antibodies against phospho-Thr308-PKB and phosphoThr389-p70S6K. HA-tagged proteins were immunoprecipitated, run on SDS-PAGE, transferred to nitrocellulose and probed with the antibodies indicated. B. HEK293 cells transfected with HA-AS160A were stimulated with IGF1, in the presence and absence of AICAR, as indicated. HA-tagged proteins were immunoprecipitated, run on SDS-PAGE, transferred to nitrocellulose and probed with the antibodies indicated.

  Fig 4A

  Fig 6

  Fig 7

  ).

	Phosphospecific antibodies were raised against the following phosphopeptides:-
	FRSRCSpSVTGVQR (residues 311 to 324, where pS represents phosphorylated Ser318);
	CPRRRHApSAPSHVQ (Cys + 335 to 347, pSer341); CKAKRSLpTSSLENI (Cys + 562 to
	574, where pT is pThr568); CKRSLTSpSLENIFS (Cys + 564 to 576, pSer570);
	CMRGRLGpSVDSFER (Cys + 582 to 594, pSer588); CFRRRAHpTFSHPPS (Cys + 636 to
	648, pThr642); CAQGVRpSPLLRQS (Cys + 661 to 672, pSer666); and

Cell stimulations, cross-linking, lysis and immunoprecipitations

	Crosstide	(GRPRTSSFAEG)	for	PKB	and	SGK1,	Long-S6
	(KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) for RSK1, and AMARA peptide
	(AMARAASAAALARRR) for AMPK.
	Molecular biology: splice variants of AS160
	Two forms of AS160 appear to be products of one locus (NM_014832; gene id:
	ENSG00000136111). One form (termed AS160B) is identical to Z25171 from a skeletal
	muscle cDNA library. A shorter form (AS160A), more commonly represented in the
	sequence databases, is identical to AS160B except for deletion of residues 679 to 733
	encoded by exon 11. Constructs to generate HA-AS160A (pCMV5.HA AS160A) and HA-
	AS160B (pCMV5.HA AS160B) were generated by RT-PCR amplification from RNA
	extracted from HeLa cells and human skeletal muscle, respectively. Bacterial expression
	plasmids for GST-AS160 fusions (pGEX6P AS160A and pGEX6P AS160B) were generated
	by standard procedures. Site-directed mutagenesis used the Quickchange protocol
	(Stratagene) and KOD HotStart DNA polymerase (Novagene). Sequences of DNA
	constructs were checked (www.dnaseq.co.uk).
	Results are shown for the HA-AS160A and GST-AS160A form, though experiments
	in Fig 7 were repeated with AS160B and gave similar results (not shown). Features of
	AS160 and the related TBC1D1 are in Supplementary Fig 1. Unless stated, residue numbers
	given correspond to AS160B.
	Human HEK293 cells cultured on 10-cm diameter dishes in medium containing 10%
	(v/v) serum were used untransfected, or 16 to 30 h after transfection with the indicated
	plasmids. Cells were serum-starved for a further 4 to 12 h (unstimulated), then stimulated
	for 20 min with 50 ng/ml IGF1. Where indicated, calyculin A was used at 100 nM for	~3
	min, and AICAR at 2 mM for 60 min, phenformin at 2 mM for 60 min, EGF at 50 ng/ml for
	15 min, and PMA at 100 ng/ml for 30 min. Where indicated, cells were incubated with
	LY294002 (LY; 100 M for 1 h), wortmannin (100 nM for 1 h), U0126 (U; 10 M for 1 h),
	rapamycin (Ra; 50 nM for 30 min), Go6983 (1 M for 30 min) and BI-D1870 (BI; 10 M

). AMPK was purified from rat liver by Kevin Green in Grahame Hardie's laboratory. Unless indicated, the protein kinases were used at 1 Unit/ml, where 1 Unit is nmole phosphate per min at 30 o C incorporated into the substrate peptides

Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070649 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by the U.K. Medical Research Council, Diabetes UK and the companies who support the Division of Signal Transduction Therapy (DSTT) at the University of Dundee, namely AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck and Co, Merck KGaA, and Pfizer. DGH was supported by the EXGENESIS Integrated Project (LSHM-CT-2004-005272) of the European Commission. Thanks to Colin Bell and Claire Balfour for tissue culture support, and Greg Stewart and the DSTT antibody production team coordinated by Dr James Hastie for affinity purification of antibodies. We thank Rachel Naismith for secretarial assistance, and Kei Sakamoto for helpful discussions.


Takeshi Murakami

Robert Walczak

Sandrine Caron

Christian Duhem

Vincent Vidal

Raphaël Darteil

Bart Staels
email: bart.staels@pasteur-lille.fr.

The farnesoid X receptor induces fetuin-B gene expression in human hepatocytes

Keywords: farnesoid X receptor, fetuin-B, cystatin superfamily, alternative promoter, anti-cancer Biochemical FXR, farnesoid X receptor, RXR, retinoid X receptor, BAs, bile acids, CDCA, chenodeoxycholic acid, IR, inverted repeat, Cyp7A1, cholesterol-7α-hydroxylase, SHP, small heterodimer intestinal bile acid-binding protein, BSEP, bile salt export pump, NTCP, Na + -taurocholate cotransporting polypeptide, apo, apolipoprotein, PLTP, phospholipid

Farnesoid X receptor (FXR), a nuclear receptor activated by bile acids, is a key factor in the regulation of bile acid, lipid and carbohydrate metabolism. The recent development of synthetic FXR agonists and knock-out mouse models has accelerated the discovery of FXR target genes. In this study we identify human Fetuin-B as a novel FXR target gene. Treatment with FXR agonists increased Fetuin-B expression in human primary hepatocytes and in the human hepatoma HepG2 cell line. In contrary, Fetuin-B expression was not responsive to FXR agonist treatment in murine primary hepatocytes. Fetuin-B induction by FXR agonist was abolished upon FXR knockdown by siRNA. In addition to the previously described P1 promoter, we show that the human Fetuin-B gene is also transcribed from an alternative promoter, termed P2. Transcription via the P2 promoter was induced by FXR agonist treatment, whereas P1 promoter activity was not sensitive to FXR agonist treatment.

Two putative FXR response elements (IR-1) were identified in the region -1.6kb upstream of the predicted P2 transcriptional start site. Both motifs bound FXR/RXR complexes in vitro and were activated by FXR in transient transfection reporter assays. Mutations in the IR-1 sites abolished FXR/RXR binding and activation. Taken together, these results identify human Fetuin-B as a new FXR target gene in human hepatocytes.

INTRODUCTION

The farnesoid X receptor (FXR, NH1H4) is a member of the nuclear receptor family that is expressed at highest levels in the liver, kidney, intestine and the adrenal gland [START_REF] Forman | Identification of a nuclear receptor that is activated by farnesol metabolites[END_REF][START_REF] Seol | Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors[END_REF]. FXR is activated by bile acids (BAs), such as chenodeoxycholic acid (CDCA) [START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF], or by synthetic FXR agonists, such as GW4064 [START_REF] Maloney | Identification of a chemical tool for the orphan nuclear receptor FXR[END_REF]. In most cases, FXR binds to inverted repeat elements on DNA (IR-1) as a heterodimer with the retinoid X receptor (RXR). FXR regulates the expression of a variety of genes related to bile acid, cholesterol, triglyceride and carbohydrate metabolism. For example, FXR negatively regulates bile acid synthesis by decreasing the expression of cholesterol-7α-hydroxylase (Cyp7A1), the rate limiting enzyme in the pathway of BA synthesis [START_REF] Lu | Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors[END_REF][START_REF] Goodwin | A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis[END_REF] through the induction of another nuclear receptor, SHP. Enterohepatic circulation of bile acids is also controlled by FXR, which modulates the expression of genes such as the intestinal bile acid-binding protein (I-BABP), bile salt export pump (BSEP) and Na + -taurocholate cotransporting polypeptide (NTCP) [START_REF] Grober | Identification of a bile acid-responsive element in the human ileal bile acid-binding protein gene. Involvement of the farnesoid X receptor/9-cis-retinoic acid receptor heterodimer[END_REF][START_REF] Ananthanarayanan | Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor[END_REF][START_REF] Denson | The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp[END_REF]. FXR is also an important factor in the control of cholesterol metabolism through the inhibition of apolipoprotein (apo) -AI expression and through the induction of phospholipid transfer protein expression (PLTP) [START_REF] Claudel | Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element[END_REF][START_REF] Urizar | The farnesoid X-activated receptor mediates bile acid activation of phospholipid transfer protein gene expression[END_REF]. Similarly, the expression of factors involved in the control of plasma triglyceride levels, such as apo-CII, apo-CIII and peroxisome proliferator-activated receptor α (PPARα), is under the control of FXR [START_REF] Kast | Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids[END_REF][START_REF] Claudel | Farnesoid X receptor agonists suppress hepatic apolipoprotein CIII expression[END_REF][START_REF] Pineda | Bile acids induce the expression of the human peroxisome proliferator-activated receptor alpha gene via activation of the farnesoid X receptor[END_REF]. As a consequence, FXR-deficient mice show increased liver and serum triglyceride levels [START_REF] Sinal | Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis[END_REF]. It has also been postulated that the lipogenic response is negatively regulated by FXR [START_REF] Watanabe | Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c[END_REF][START_REF] Duran-Sandoval | The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition[END_REF]. Several recent reports have demonstrated that FXR activity is essential to maintain normal glucose homeostasis [START_REF] Ma | Farnesoid X receptor is essential for normal glucose homeostasis[END_REF][START_REF] Zhang | Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice[END_REF][START_REF] Cariou | The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice[END_REF]. The role of FXR in the negative regulation of both hepatic glucose production and lipogenesis has been postulated [START_REF] Yamagata | Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1[END_REF][START_REF] Duran-Sandoval | The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition[END_REF]. Finally, fxr gene ablation in mice results in an impaired insulin sensitivity [START_REF] Ma | Farnesoid X receptor is essential for normal glucose homeostasis[END_REF][START_REF] Zhang | Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice[END_REF][START_REF] Cariou | The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice[END_REF]. These reports suggest that FXR is a critical factor involved in lipid and carbohydrate homeostasis and a potential clinical target to treat the metabolic syndrome Fetuin-A and fetuin-B are members of the fetuin family. Human fetuin-B (382 aa) shares 22% sequence similarity with fetuin-A. Also known as α2-Heremans and Schmid glycoprotein (Ahsg), fetuin-A is a major globular protein in foetal serum and is a member of the cystatin superfamily of cysteine protease inhibitors. Fetuin-A is the only known protein inhibitor of ectopic calcification that is systemic [START_REF] Schafer | The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification[END_REF]. Due to a high affinity for hydroxyapatite, fetuin-A accumulates in the skeleton, where it inhibits the formation and precipitation of the apatite precursor mineral, basic calcium phosphate (BCP) [START_REF] Schinke | The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis[END_REF]. In addition, Fetuin-A has been implicated in several processes related to osteogenesis, bone resorption and inhibition of vascular calcification [START_REF] Schafer | The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification[END_REF][START_REF] Szweras | alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling[END_REF]. In addition, it has been reported that fetuin-A acts as a negative regulator of insulin receptor tyrosine kinase activity [START_REF] Mathews | Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene[END_REF]. Fetuin-B is a paralogue of fetuin-A and functional analysis revealed that fetuin-B is able to act as calcium phosphate precipitation inhibitor albeit with lower affinity [START_REF] Olivier | Fetuin-B, a second member of the fetuin family in mammals[END_REF][START_REF] Denecke | Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A[END_REF]. Since fetuin-B concentration in human serum tends to be low, a major quantitatively systemic contribution to calcium phosphate precipitate removal is rather unlikely. Other authors have reported that fetuin-B expression is upregulated in certain primary skin tumors and in a subset of human cancer cell lines [START_REF] Hsu | Identification of Fetuin-B as a member of a cystatinlike gene family on mouse chromosome 16 with tumor suppressor activity[END_REF]. Indeed, overexpression of fetuin-B in skin squamous carcinoma cells led to suppression of tumor growth in nude mice [START_REF] Hsu | Identification of Fetuin-B as a member of a cystatinlike gene family on mouse chromosome 16 with tumor suppressor activity[END_REF]. Based on a number of recent reports suggesting a role for FXR in certain cancers [START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF][START_REF] Silva | Lipids isolated from bone induce the migration of human breast cancer cells[END_REF][START_REF] Kim | Spontaneous hepatocarcinogenesis in farnesoid X receptor-null mice[END_REF], we decided to search for novel FXR target genes in hepatocytes using a pharmacological approach and microarray technology. Our results identified the human fetuin-B gene as a FXR agonist regulated gene. Subsequently, we investigated the molecular mechanisms of fetuin-B regulation by FXR.

Biochemical

Biochemical

MATERIALS AND METHODS

Culture and treatment of HepG2 cells and human primary hepatocytes.

Human hepatoblastoma HepG2 cells (ATCC) were seeded in 6-well plates (5 x 10 5 cells/well) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, streptomycin/penicillin, sodium pyruvate, glutamine and non-essential amino acids (Invitrogen, Cergy-Pontoise, France) at 37°C in a humidified 5% CO 2 atmosphere. Human primary hepatocytes were purchased from Biopredic International (Rennes, France) and grown in the culture medium that was furnished by the supplier at 37°C in a humidified 5% CO 2 atmosphere.

To induce FXR target gene expression, HepG2 cells and human primary hepatocytes were treated for 24 hours with chenodeoxycholic acid (CDCA; 75µM) (Sigma, Saint-Quentin, France) or with the synthetic FXR agonist GW4064 (5µM) in DMEM supplemented with 1% fetal calf serum. Small interfering RNA (siRNA) mediated FXR knockdown was performed as previously described by Sirvent et al. [START_REF] Sirvent | The farnesoid X receptor induces very low density lipoprotein receptor gene expression[END_REF][START_REF] Sirvent | Farnesoid X receptor represses hepatic lipase gene expression[END_REF]. GW4064 was synthesized at GENFIT SA (Loos, France) according to Maloney et al. [START_REF] Maloney | Identification of a chemical tool for the orphan nuclear receptor FXR[END_REF] Culture and treatment of mouse primary hepatocytes.

Mouse primary hepatocytes were isolated as previously described [START_REF] Duran-Sandoval | The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition[END_REF]. Before the addition of collagenase (0.0025%) (Sigma), livers were perfused with Hank's balanced salt solution (Sigma) at a rate of 5ml/min via the portal vein. Cell viability was assessed by the trypan blue exclusion test and it was always higher than 60%. Hepatocytes were cultured as monolayers in the serum free DMEM (Invitrogen) supplemented with 0.1% BSA, 50nM dexamethasone, 1% Glutamine, 1% Gentamycine, 25mM Glucose and 2% ULTROSER (Biosepra) at 37°C in a humidified 5% CO 2 atmosphere. FXR target gene expression was studied in mouse hepatocytes after 24 hours of treatment with either CDCA (50µM) or GW4064 (5µM).

RNA extraction, PCR analysis and DNA microarray analysis.

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized by the reverse transcription reaction from 1µg of total RNA, 200 ng of random hexamer primers and 200 units of M-MLV reverse transcriptase (Invitrogen). PCR analysis was performed using the following reaction buffer : 0.125 mM dNTP, 1.25 mM MgCl 2 , 1 unit of GoldStar taq polymerase (Eurogentec, Seraing, Belgium) and 100 nM specific primers. Standard PCR conditions were denaturation at 95°C for 5 minutes, followed by 40 cycles of 1 minute at 95 °C, 1 minute at 55° C and 1 minute at 72° C. DNA microarry analysis was performed as described previously [START_REF] Sirvent | The farnesoid X receptor induces very low density lipoprotein receptor gene expression[END_REF][START_REF] Sirvent | Farnesoid X receptor represses hepatic lipase gene expression[END_REF].

Quantitative PCR analysis.

Real-time quantitative PCR analysis was performed using Brilliant SYBR Green QPCR master mix (Stratagene) and specific primers according to the manufacturer's instructions. QPCR Reporter plasmids, which contain three copies of each FXR response element were produced by the ligation of a synthetic dsDNA cassette into the TK-pGL3 vector cut with SmaI.

The following oligonucleotides were used to assemble 3xFXRRE cassettes. Reporter assays were performed in HepG2 cells. . The standard transfection procedure with the cationic lipid RPR 120535B was described in detail elsewhere [START_REF] Raspé | Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation[END_REF]. Shortly, reporter plasmids (100 ng) were cotransfected with both pcDNA3 human FXR (30 ng) and pSG5 human RXRα (30 ng). pSG5 (Stratagene, La Jolla, California, USA) and pcDNA3 (Invitrogen, Leek, The Netherlands) empty vectors were used as negative controls. All samples were complemented with pBSK + plasmid (Stratagene) to bring the total amount of DNA to 500 ng. Following three hours of incubation, transfection medium was replaced with a medium containing 1% FCS with or without CDCA.

Luciferase activity was measured 36 hours later using a TR717 luminometer (Applied Biosystems).

Transfection efficiency was monitored by a cotransfection with 10 ng of a CMV-driven βgalactosidase expression plasmid. Transfection experiments were done at least three times in triplicate.

Electrophoretic mobility shift assays (EMSA).

Human FXR and human RXR were produced in vitro using the TNT Quick Coupled Transcription/Translation System (Promega, Madison, Wisconsin, USA). Double stranded probes were produces by annealing sense and anti-sense oligonucleotides (5 µg each) in the following buffer (50mM Tris-HCl(pH8.0), 50mM KCl, 5mM MgCl 2 , 1mM dithiothreitol). Oligonucleotides were denatured at 95°C for 5 min, then the temperature was reduced to 65°C for 10 min and finally the mix was incubated at room temperature for 16hr. The probe was subsequently labeled with 32 P γ-ATP using T4-polynucleotide kinase. FXR and/or RXR proteins preps (2 µl) were incubated for 15 min at room temperature in a total volume of 20 µl with 1 µg poly(dI-dC) and 1 µg herring sperm DNA in binding buffer (10 mM HEPES, pH 7.6, 2.5 mM MgCl 2 , 10% glycerol, 2.5 mg/ml BSA, 50 mM NaCl, and 0.5mM DTT) before the radiolabeled probe (1 ng) was added. Binding reactions were further incubated for 15 min and resolved by 6% nondenaturing polyacrylamide gel electrophoresis in 0.25x Tris-Borate-EDTA (TBE) buffer at room temperature. Excess of the unlabeled probe was included in the binding reaction, just before adding the labeled probe, to perform competition assays.

Oligonucleotides used in the study :

RESULTS

FXR agonists induce Fetuin-B expression in human but not in murine primary hepatocytes.

To identify new FXR target genes, we have performed DNA microarray analysis using human primary hepatocytes treated with 5µM GW4064 for 24hrs. The results revealed fetuin A (1.6 fold) and fetuin B (4.5 fold) were induced by FXR agonist (data not shown). To confirm these results, Fetuin-A and fetuin-B regulation by FXR was studied in a human hepatoma cell line HepG2 (Fig. 1A) and in primary human hepatocytes (Fig. 1B). Gene expression analysis by real-time quantitative PCR (Q-PCR) revealed that Fetuin-B but not Fetuin-A mRNA level was significantly increased in cells treated with either the natural (CDCA) or synthetic (GW4064) FXR agonist. Surprisingly, fetuin-B expression in mouse primary hepatocytes was completely insensitive to FXR agonist treatment (Fig. 1C). These results suggest that the effect of FXR agonists on fetuin-B expression is species-specific.

Induction of Fetuin-B expression by CDCA and GW4064 requires FXR expression.

Gene silencing by siRNA was used to determine whether the regulation of Fetuin-B expression by FXR agonists observed in human liver cells was dependent on FXR expression. HepG2 cells were therefore transfected with siRNA complexes (anti-FXR and/or negative control) followed by FXR ligand treatment. Basal Fetuin-B expression level was reduced by approximately 49% in FXR siRNA treated cells as compared to the negative control (Fig. 2, white columns). Moreover, CDCA and GW4064-dependent increase in Fetuin-B expression was strongly attenuated in FXR siRNA treated cells (Fig. 2, mesh and black columns). As control, FXR agonist induction of SHP expression was abolished by FXR siRNA and GAPDH expression was not affected by either FXR siRNA or the negative control siRNA in the same experiment [START_REF] Sirvent | The farnesoid X receptor induces very low density lipoprotein receptor gene expression[END_REF][START_REF] Sirvent | Farnesoid X receptor represses hepatic lipase gene expression[END_REF].

Induction of Fetuin-B through FXR occurs via the P2 but not the P1 promoter.

A previous report [START_REF] Hsu | Identification of Fetuin-B as a member of a cystatinlike gene family on mouse chromosome 16 with tumor suppressor activity[END_REF] has identified a fetuin-B promoter (Fig. 3A,P1) and three transcripts, which arise by differential splicing of 5'-terminal exons. We were unable to identify any putative FXR binding sites in the vicinity of this fetuin-B promoter. In addition, a 1.1kb reporter construct (sequence -1000 through +102 bp) derived from the published promoter sequence was not regulated by FXR over-expression and agonist treatment (not shown). We therefore suspected that the fetuin-B gene has other, alternative promoters. A data base search identified one expressed sequence tag (DA449793), which mapped both to the 5'-terminal fetuin-B exons and to a genomic sequence that is located 6 kb upstream of the published exon 1. Subsequent RT-PCR analysis confirmed the existence of transcripts which originate at an alternative promoter (Fig. 3 AB). Detailed analysis with both P1 or P2 specific primers revealed that each promoter produces three different transcripts that differ in size (Fig. 3B).

Sequencing of P1 promoter transcripts identified the Fetuin-B P1α, P1β and P1γ isoforms, which have been previously described by Hsu et al [START_REF] Hsu | Identification of Fetuin-B as a member of a cystatinlike gene family on mouse chromosome 16 with tumor suppressor activity[END_REF]. Three novel Fetuin-B forms, called P2α, β  and γ were identified by sequencing the PCR fragments obtained with the P2 specific primer (Fig. 3A). The fetuin-B P2α transcript contains the untranslated exon that follows the P2 promoter, the coding part of the Real-time quantitative PCR assays, specific for either P1-derived or P2-derived transcripts were developed to determine which of the two Fetuin-B promoters is responsive to FXR. As shown in Fig. 3C, the level of P2 specific transcripts was increased over three times by both CDCA and GW4064 in HepG2 cells, whereas P1 specific transcripts did not respond. Similarly, FXR agonist treatment increased the abundance of P2 specific transcripts 20-fold in human primary hepatocytes (Fig. 3D). The level of P1 specific transcripts was not affected by FXR agonist treatment in human primary hepatocytes.

Functional FXR response elements are present in the human Fetuin-B P2 promoter.

The P2 promoter flanking sequence, obtained from NCBI human genome resources (build 36.2 at http://www.ncbi.nlm.nih.gov/genome/guide/human/), was analyzed by VISTA tools (http://genome.lbl.gov/vista/index.shtml) for putative FXR binding sites. Two IR-1 motifs GGGTCATTGTCCT (-1592bp to -1577bp) and AGGTCAGGGACCT (-1559bp to -1546bp) were identified as putative FXR binding sites. Genomic sequence alignment with fetuin-B orthologs from different species (Fig. 4A) showed that only one half site was conserved in each FXRE motif in mice.

G to A conversion at the third position of an IR-1 half-site is usually detrimental for receptor binding.

The human Fetuin-B P2 promoter (positions : -1978bp / +263bp) was tested in transient transfection reporter assay in HepG2 cells. As shown in Fig. 4B, CDCA treatment increased the transcriptional activity of the reporter plasmid (8-fold over control) in the presence of cotransfected FXR and RXR expression plasmids. The control plasmid (pGL3-basic) was not affected. Mutations introduced in each IR-1 motif compromised this induction. These results demonstrate that both FXRE1 and FXRE2 motifs are important for the activation of the Fetuin-B P2 promoter by FXR.

Next, both IR-1 motifs were tested in the context of a heterogenous promoter to check if those motifs are sufficient to direct FXR-mediated regulation. HepG2 cells were transfected with reporter constructs, which carried three copies of either FXRE1 or FXRE2 motif in the TKpGL3 vector. As shown in Fig. 4C, CDCA-activated FXR/RXR strongly induced the FXRE1 motif (12-fold increase over the control), whereas the FXRE2 motif was only activated 2.5-fold in the same conditions. This difference in the response strength to FXR might probably be attributed to the presence of a guanine at the last position in the second half site in FXRE2. This position is usually occupied by an adenine in nuclear receptor binding motifs. Mutations introduced in either FXRE1 and FXRE2 motifs completely abolished FXR/RXR mediated regulation (Fig. 4C), which confirm that these motifs are functional FXREs mediating FXR specific regulation of the human fetuin-B promoter. Electrophoretic Mobility Shift Assays (EMSA) were performed to test direct binding of the FXR/RXR complexes to the FXRE1 and FXRE2 motifs. A complex formed by the in vitro translated hFXR/hRXR but not by FXR alone, was able to bind both FXRE 1 (Fig. 5A, lanes 8 through 17) and FXRE 2 (Fig. 5B, lanes 8 through 17) motifs. FXR/RXR binding was easily displaced with a wt (Fig. 5AB, lanes 9 through 12) but not with a mutant (Fig. 5AB, lanes 13 through 16) unlabeled probe.

FXRE1 and FXRE2 motifs identified in the human

The identity of the FXR/RXR complex was confirmed by supershift analysis (Fig. 5AB, lane 17) using a specific anti-FXR antibody. These results demonstrate that the FXR/RXR heterodimer is able to bind specifically to both FXRE motifs identified in the human fetuin-B promoter P2.

Biochemical

DISCUSSION

Fetuins are abundant foetal serum α-globulins, which belong to the cystatin family of cysteine protease inhibitors. The vast majority of physiological data available to date comes from studies on fetuin-A (mouse) or on its human orthologue α2-Heremans and Schmid glycoprotein (AHSG), which acts as a systemic inhibitor of ectopic calcification [START_REF] Schafer | The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification[END_REF][START_REF] Schinke | The serum protein alpha2-HS glycoprotein/fetuin inhibits apatite formation in vitro and in mineralizing calvaria cells. A possible role in mineralization and calcium homeostasis[END_REF]. In clinic, AHSG concentrations in serum were significantly lower in patients on haemodialysis than in healthy controls [START_REF] Ketteler | Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study[END_REF]. Low concentrations of the glycoprotein were associated with raised amounts of CRP and with enhanced cardiovascular and all-cause mortality [START_REF] Ketteler | Association of low fetuin-A (AHSG) concentrations in serum with cardiovascular mortality in patients on dialysis: a cross-sectional study[END_REF]. In addition, sera from patients on long-term dialysis with low AHSG concentrations showed impaired ex-vivo capacity to inhibit calcium phosphate precipitation. Reconstitution of sera with purified AHSG returned this impairment to normal. Similarly, low serum concentrations of AHSG in peritoneal dialysis patients are associated with valvular calcification, higher cardiovascular mortality and development of malnutrition, inflammation, atherosclerosis/calcification (MIAC) syndrome [START_REF] Wang | Associations of serum fetuin-A with malnutrition, inflammation, atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients[END_REF].

Fetuin-A is also implicated in a variety of other metabolic regulations, through the inhibition of the TGF-β pathway in relation to bone growth and remodelling [START_REF] Szweras | alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling[END_REF] and acting as a negative regulator of insulin receptor tyrosine kinase activity [START_REF] Szweras | alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling[END_REF][START_REF] Mathews | Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene[END_REF]. High levels of fetuin-A in subjects with coronary artery disease were associated with the metabolic syndrome and with a pro-atherogenic lipid profile in the Heart and Soul Study [START_REF] Ix | Association between human fetuin-A and the metabolic syndrome: data from the Heart and Soul Study[END_REF].

Fetuin-A and fetuin-B share overlapping but not identical tissue distribution patterns and functions.

At the RNA level, the highest expression of both fetuin-A and fetuin-B was reported in the liver, followed by kidney and tongue [START_REF] Denecke | Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A[END_REF]. Fetuin-B but not fetuin-A mRNA has been detected in mouse ovaries. Interestingly, unlike fetuin-A, fetuin-B concentration in serum is higher in females [START_REF] Denecke | Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A[END_REF]. On a structural basis, fetuin-B is clearly homologous to fetuin-A, as illustrated by the shared arrangement in three domains, including two cystatin-like domains D1 and D2 [START_REF] Olivier | Fetuin-B, a second member of the fetuin family in mammals[END_REF]. Mutational analysis showed that in the case of fetuin-A, the basic calcium precipitation (BCP) activity resides in the NH 2 -terminal cystatin-like domain D1 [38]. Functional experiments have shown that fetuin-B is indeed capable to inhibit BCP, although with lower efficiency [START_REF] Denecke | Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A[END_REF]. In addition, both fetuin-B expression in human hepatocytes and its protein level in the serum are low as compared to fetuin-A. Therefore, fetuin-B can potentially function as a BCP inhibitor in vivo, but it is unlikely to make a major systemic contribution.

Here, we show that in human primary hepatocytes the expression of the gene coding for fetuin-B is directly up-regulated by agonist activated FXR through binding sites, which are located in the alternative P2 promoter. Three novel fetuin-B transcripts, which originate at the P2 promoter and arise by differential splicing of their 5'-terminal exons were identified in this study. Interestingly, the untranslated exon P2 is directly connected to the exon 4 in the fetuin-B P2γ transcript. This situation implies that the translation of the P2γ transcript is initiated at a different site. Indeed, a putative alternative start codon exists in the exon 4 at the position 21bp downstream from splicing site in exon Interestingly, fetuin-B expression in primary mouse hepatocytes was not affected by FXR agonist treatment. Genomic sequence alignment of fetuin-B promoters from different species revealed that neither the first, nor the second FXRE is conserved between mouse and human. In addition, those putative FXRE elements in the mouse promoter are positioned relatively far (3.9kb) from the nearest putative transcriptional start site. Finally, basal fetuin-B concentration in serum is 31 times higher in mouse than in humans [START_REF] Denecke | Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A[END_REF]. Therefore, we postulate that the mouse fetuin-B gene is regulated by different mechanisms than its human orthologue.

Recently reports show FXR regulates apoptosis and metastasis in breast cancer cells [START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF][START_REF] Silva | Lipids isolated from bone induce the migration of human breast cancer cells[END_REF]. FXR agonists induce apoptosis in human breast carcinoma cells and this effect is diminished by knockdown of FXR [START_REF] Swales | The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression[END_REF]. On the other hand, the transfection of fetuin-B expression plasmids suppresses the growth of carcinoma cell line, B9 cells [START_REF] Hsu | Identification of Fetuin-B as a member of a cystatinlike gene family on mouse chromosome 16 with tumor suppressor activity[END_REF]. These results suggest that the FXR-fetuin B pathway might regulate proliferation and apoptosis in cancer cells.

In summary, we have demonstrated that FXR agonist treatment results in the activation of fetuin-B gene expression in human hepatocytes. This effect is selectively mediated through the P2 promoter and is species-specific. Since the functional importance of fetuin-B protein has not been firmly (C) HepG2 cells were transfected with reporter constructs containing three copies of either FXRE1 or FXRE2 element. TK-pGL3 plasmid was used as a control. Mutations were introduced in the FXRE 1 or FXRE 2 elements (E1mt, E2mt) as described in the text. Electrophoretic mobility shift assays were performed using end-labeled fetuin-B FXREs (wild type probe, lanes 5-17; mutant probe, lanes [START_REF] Duran-Sandoval | The farnesoid X receptor modulates hepatic carbohydrate metabolism during the fasting-refeeding transition[END_REF][START_REF] Ma | Farnesoid X receptor is essential for normal glucose homeostasis[END_REF]). An IR-1 element, previously identified in the IBABP promoter (lanes 1-4), was used as a positive control. Unprogrammed reticulocyte lysate (lanes 1,5 and 18), hRXR (lanes 2 and 6), hFXR (lanes 3 and 7), hRXR and hFXR (lanes 4, 8-17 and 19).

Competition was performed by adding 10 (lanes 9 and 13), 50 (lanes 10 and 14), 100 (lanes 11 and 15),

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  A Fwd: 5'-ACAACGGCTCCAATTTTCAG-3' human Fetuin-A Rev: 5'-TACACTTGGCTGCCTCTGTG-3' human Fetuin-B P1 Fwd: 5'-AGTTGTAACAAAACCGCTCAA-3' human Fetuin-B P1 Rev: 5'-TCTGTGGAGAACAAGGCCCAG-3' human Fetuin-B P2 Fwd: 5'-AACCCTGAAGGTGTGACTGG-3' human Fetuin-B P2 Rev: 5'-GCACAGGACTAGGATGCAGA-3' human Fetuin-B ex7 Fwd: 5'-TGTGCATCTGGACCTAACCA-3' human Fetuin-B ex7 Rev: 5'-GCTTTTTCTTTGGGGAAAGG-3' human Fetuin-B ex5 Rev: 5'-GTCGGAGGACTGAAGTGAACA-3' mouse Fetuin-B Fwd: 5'-CCCCATTGACTTGTCAAACC-3' mouse Fetuin-B Rev: 5'-ACACCCACTGGTTCATAGCC-3' 28S Fwd: 5'-AAACTCTGGTGGAGGTCCGT-3' 28S Rev: 5'-CTTACCAAAAGTGGCCCACTA-3' cyclophilin Fwd: 5'-GCATACGGGTCCTGGCATCTTGTCC-3' cyclophilin Rev: 5'-ATGGTGATCTTCTTGCTGGTCTTGC-3' Plasmid cloning and site-directed mutagenesis. A human Fetuin-B promoter sequence (fragment between -1978 and +263 with respect to the transcriptional start site) was amplified from human genomic DNA by PCR with the following primers: (5'-TGTGGTACCGTTTAAATAACTTGTATTGAG-3') and (5'-TGTACGCGTCACAGTTGTGCTGGTTTATGA-3'). The resulting PCR product was cloned into the pGL3 basic reporter vector (Promega Corp., Madison, WI) using KpnI and MluI to yield the reporter plasmid phFetuin-B-pGL3. The sequence of the FXR response elements was mutated with the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The following oligonucleotides (5'-TAAATCAGTTCCCAGAATCATTGTTTTCTGATGTAAGCCCACCAG-3') and (5'-GTAAGCCCACCAGAAATCAGGGATTTTTGCGTTAGCAGAAGCTGG-3') were used to introduce mutations into the FXRE1(-1592) and FXRE2(-1559)sites, respectively.

FXRE1( 1592 ©

 1592 )wt oligo sense (5'-TTCCCAGGGTCATTGTCCTCTGATGTTCCC Biochemical Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 1559)wt oligo sense (5'-CACCAGAGGTCAGGGACCTTTGCGTCACCAGA GGTCAGGGACCTTTGCGTCACCAGAGGTCAGGGACCTTTGCGT-3') and oligo antisense (5'-ACGCAAAGGTCCCTGACCTCTGGTGACGCAAAGGTCCCTGA CCTCTGGTGACGCAAAGGTCCCTGACCTCTGGTG-3') FXRE2(-1559)mt oligo sense (5'-CACCAGAAATCAGGGATTTTTGCGTCAC CAGAAATCAGGGATTTTTGCGTCACCAGAAATCAGGGATTTTTGCGT-3') and oligo antisense (5'-ACGCAAAAATCCCTGATTTCTGGTGACGCAAAAATCCCTG ATTTCTGGTGACGCAAAAATCCCTGATTTCTGGTG-3') All constructs were verified by restriction enzyme digestion and sequence analysis Cell culture and transfection assays.

Biochemical

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  FXRE1wt sense : (5'-GTTCCCAGGGTCATTGTCCTCTGAT-3', FXRE1wt antisense : (5'-ATCAGAGGACAATGACCCTGGGAAC-3'), FXRE1mt sense : 5'-GTTCCCAGAATCATTGTTTTCTGAT-3', FXRE1mt antisense : (5'-ATCAGAAAACAATGATTCTGGGAAC-3'); FXRE2wt sense : (5'-ACCAGAGGTCAGGGACCTTTGCGTT-3'), FXRE2wt antisense : (5'-AACGCAAAGGTCCCTGACCTCTGGT-3'), FXRE2mt sense : (5'-ACCAGAAATCAGGGATTTTTGCGTT-3'), FXRE2mt antisense: (5'-AACGCAAAAATCCCTGATTTCTGGT). IBABP IR-1 sense : (5'-GATCTCAAGAGGTCATTGACCTT TTTG-3'), IBABP IR-1 antisense: (5'-CAAAAAGGTCAATGACCTCTTGAGATC -3'). A FXR-specific antibody SC-1204 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reaction (2 µl/tube) before addition of the probe, to perform supershift assays. Biochemical Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society exon 1 and exons 2 through 7. Fetuin-B P2β is missing exon 3. Finally, exons 4 through 7 are directly spliced to the P2 specific 5'-untranslated exon in the fetuin-B P2γ transcript.

  Fetuin-B P2 promoter bind FXR/RXR complexes in an EMSA assay. Biochemical Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society 4. This putative, P2γ derived protein should only contain the cystatin-like domain D2. The abundance and the functional importance of this new isoform remains to be explored.

Figure 1

 1 Figure legends

Figure 2 Figure 3 FXRFigure 4

 234 Figure 2 The induction of fetuin-B expression by CDCA and/or GW4064 is FXR dependent HepG2 cells were transfected with either small interfering RNA (siRNA) targeting FXR or with negative control siRNA. Twelve hours post siRNA transfection cells were treated with either 75µM CDCA or with 1µM GW4064 for the next 24 hours. Human fetuin-B mRNA levels were then measured by the real-time quantitative PCR and normalized to cyclophilin mRNA levels. Data are expressed as a relative expression versus DMSO in the control group; mean +/-S.E.(n=3)

Figure 5

 5 Figure 5 The FXR/RXR complex binds in vitro to the human fetuin-B FXRE 1 and FXRE 2 motifs.

reactions were Biochemical Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society

	run on MX4000 or MX3000 apparatus (Stratagene). Reaction conditions were denaturation at 95°C for
	10 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 55°C and 30 seconds at 72°C.
	Fetuin-B mRNA levels were normalized to 28S mRNA. Statistical significance was assessed using
	student's t-test.

ACKNOWLEDGEMENTS

This work was supported by grants from the EU Grant Hepadip (n° 018734) and the Agence Nationale de la Recherche (n° A05056GS).

Biochemical

Journal Immediate Publication. Published on 27 Jul 2007 as manuscript BJ20070658 © 2007 The Authors Journal compilation © 2007 Biochemical Society