Charlotte J Green

Katherine Macrae

Sarah Fogarty

D Grahame Hardie

Kei Sakamoto

Professor Harinder S Hundal
email: h.s.hundal@dundee.ac.uk

Counter modulation of fatty acid-induced proinflammatory NFκB signalling in rat skeletal muscle cells by AMPK

Keywords: PKB, Protein kinase B, AMPK, AMP-activated protein kinase, ACC, Acetyl Co-A carboxylase, NFκB, Nuclear factor kappa-B, IKK, Inhibitor of nuclear factor kappa-B kinase, IκB, Inhibitor of nuclear factor kappa-B, MAPK, Mitogen-activated protein kinase, MEK, Mitogen

Sustained over-supply of saturated non-esterified free fatty acids has been shown to promote skeletal muscle insulin resistance, which may be driven, in part, by an increase in inflammatory signalling within this tissue. Here we show that exposure of L6 myotubes to palmitate, a saturated fatty acid, induces activation of the NFκB pathway (based on increased IKK phosphorylation, IκBα loss and elevated IL-6 mRNA expression) and that this was associated with enhanced phosphorylation/activation of p38 MAPK, JNK and ERK as well as impaired insulin-dependent activation of PKB/Akt and glucose transport. NFκB activation by palmitate was unaffected by pharmacological inhibition of p38 MAPK or JNK, but was suppressed significantly by inhibition of MEK/ERK signalling. The importance of ERK with respect to downstream NFκB signalling was underscored by the finding that PMA, a potent ERK activator, enhanced IKK phosphorylation. Strikingly, both palmitate-and PMAinduced activation of IKK/NFκB were antagonised by AMPK activators because of reduced ERK signalling. Whilst palmitate-induced activation of NFκB was repressed by AMPK activation and by cellular over-expression of a mutated IκBα (S32A/S36A) super-repressor, this did not ameliorate the loss in insulin-stimulated PKB activation or glucose transport. Our data indicates that ERK plays a pivotal role in palmitate-induced activation of the IKK/NFκB signalling axis and that AMPK can restrain the activity of this proinflammatory pathway. The finding that insulin resistance persists in myotubes in which NFκB signalling has been repressed implies that palmitate and/or its lipid derivatives retain the capacity to impair insulin-regulated events independently of the increase in inflammatory signalling.

INTRODUCTION

There is mounting evidence that chronic activation of proinflammatory signalling in tissues such as skeletal muscle and adipose is a significant contributing factor in the development and progression of metabolic disorders such as insulin resistance, obesity and Type II diabetes [1,2]. This inflammatory response is triggered by circulating proinflammatory cytokines such as interleukin-6 (IL-6), tumour necrosis factor-α (TNFα) and by sustained increases in the concentration of free fatty acids (FFAs), such as palmitate. The actions of these stimuli serve not only to further induce tissue expression and secretion of IL-6 and TNFα (via the NFκB pathway [3,4]), but impair control of numerous signalling pathways regulating skeletal muscle insulin signalling, glucose uptake and cellular oxidative capacity thereby impacting on energy balance [2,[5][6][7]. The precise mechanism that governs activation of proinflammatory signalling by FFAs is, however, poorly understood. Whilst evidence exists linking Toll-like receptors (TLRs) of the innate immune system, in particular TLR2 and TLR4 (for which FFAs may be ligands), to activation of NFκB [8,9] the extent of their involvement remains unclear. This uncertainty stems from the observation that the effect of FFAs on inflammatory signalling via TLRs is not observed in a number of different cell types [10] or may be delayed for many hours following cell exposure to the fatty acid, which is considerably longer than would otherwise be expected from receptor-mediated signalling events that typically are initiated within minutes of the ligand-receptor binding event. Such observations imply that palmitate may induce NFκB activation via alternative mechanisms that rely upon uptake and metabolism of the fatty acid and the capacity of fatty acid-derived metabolites (e.g. DAG and ceramide) to regulate proteins promoting proinflammatory signalling [5].

In unstimulated cells, cytoplasmic NFκB exists in an inactive state complexed with its accessory inhibitory protein, IκBα [11]. Activation of NFκB involves phosphorylation and degradation of IκBα, which permits nuclear translocation of NFκB and subsequent regulation of its target genes. Phosphorylation of IκBα is directed by a heteromeric kinase (IKK) complex consisting of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, NEMO/IKKγ [11]. Activation of IKK itself is dependent on serine phosphorylation by upstream kinases and is considered to be crucial in obesity-induced inflammatory signalling [12,13]. Although the upstream elements involved in lipid-induced IKK activation in skeletal muscle still remain poorly defined the NFkB pathway can serve as a target for mitogenactivated protein kinases (MAP kinases) [14]. Three subfamilies of MAP kinase whose members respectively include the extracellular signal-regulated kinases (ERKs), c-Jun NH 2terminal kinases (JNKs) and p38 MAPK have thus far been characterised. All three subfamilies have been implicated in the generation of proinflammatory mediators in numerous cell types [15][16][17][18][19] and there is also strong evidence linking their activation to the pathogenesis of insulin resistance [20]. It is therefore not surprising, given the increasing recognition of a link between lipid-induced inflammatory signalling and the development of insulin resistance, that there is considerable interest in defining potential anti-inflammatory strategies that counter the loss in insulin sensitivity induced by obesity and circulating lipid overload [12,13,21,22]. Recent work has implicated the AMP-activated protein kinase (AMPK) as a modulator of inflammatory responses based on observations that its activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses LPS-induced nuclear translocation and DNA binding of NFκB as well as expression of proinflammatory cytokines such as IFN-γ, TNFα, IL-6 and IL-8 in different cell types [23][24][25][26]. Precisely how AMPK promotes this anti-inflammatory response and whether it is as effective in antagonising the associated reduction in insulin sensitivity caused by fatty acid excess in skeletal muscle cells is currently unknown. In an attempt to address this issue we have investigated the role played by MAP kinases in promoting NFκB activation in response to palmitate in L6 skeletal muscle cells and explored what effect manipulating AMPK activity has upon palmitate's proinflammatory and insulin-desensitising potential. We show here that whilst palmitate induces activation of p38 MAPK, JNK and that of the ERKs, only inhibition of the ERK pathway results in suppression of fatty acid-induced NFκB signalling. We also demonstrate that activation of AMPK antagonises the fatty acid-induced activation of NFκB and critically show that this anti-inflammatory effect involves attenuating the increase in ERK-signalling. However, despite suppressing the palmitate-induced increase in inflammatory signalling, AMPK activation was unable to correct the reduction in insulin sensitivity caused by fatty acid overload.

EXPERIMENTAL PROCEDURES

Materials -α-MEM (α-Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), FBS (Foetal Bovine Serum), DCS (Donor Calf Serum) and antibiotic/antimycotic solution were from Life Technologies (Paisley, UK). All other reagent-grade chemicals, including insulin and Bovine Serum Albumin (BSA), were obtained from Sigma-Aldrich (Poole, UK) or BDH (Poole, UK) unless otherwise stated. Fraction V fatty acid free BSA was from Roche (Basel, Switzerland), phorbol 12-myristate 13-acetate was from Ascent Scientific (Bristol, UK), U0126, SB202190 and SP600125 were purchased from Calbiochem-Novabiochem (CA, U.S.A.), PD184352 and BIRB 796 were sourced in house from the Division of Signal Transduction and Therapy (DSTT), University of Dundee. A769662 was synthesised in house as described previously [27]. Complete protein phosphatase inhibitor tablets were purchased from Boehringer-Roche Diagnostics (Basel, Switzerland). 2-deoxy-D-[ 3 H]-glucose was from PerkinElmer Life Sciences (Boston, MA, U.S.A.). Phospho-PKB (Thr 308 ), phospho-PKB (Ser 473 ), phospho-ERK1/2 (Thr 202 /Tyr 204 ), phospho-IKKα/β (Ser 180/181 ), phospho-AMPK (Thr 172 ), phospho-ACC (Ser 79 ), phospho-MEK1/2 (Ser 217/221 ), phospho-SAPK/JNK (Thr 183 /Tyr 185 ), phospho-p38 MAPK (Thr 180 /Tyr 182 ), anti-β-actin, anti-PKB, and anti-HA antibodies were from Cell Signaling Technology (Beverley, MA). Anti-IκBα antibody was from Santa Cruz. Anti total pan-AMPKα1/α2 were generated as described previously [28].

Cell culture and fatty acid treatment -L6 muscle cells were cultured to myotubes as described previously [29] in α-MEM containing 2% (v/v) FBS and 1% (v/v) antibiotic/antimycotic solution (100 units/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin B) at 37 °C with 5% CO 2 . L6 muscle cells were exposed to fatty acids that had been conjugated to BSA (fraction V) for times and at concentrations indicated in the figure legends (controls were incubated with vehicle containing BSA but lacking the fatty acid) and incubated with insulin (100 nM) in the penultimate 10 min incubation period for immunoblotting analysis or 30 min for glucose uptake assays. L6 myotubes were routinely serum deprived for 2 h prior to any treatment with insulin.

Adenoviral constructs and cell infection -Recombinant replicative-deficient adenovirus harbouring dominant negative-AMPK D157A (DN-AMPK) were generated as described previously [30,31]. Adenovirus harbouring IκBαS32A/S36A was kindly provided by Dr Harry Heimberg (Vrije Universiteit Brussel, Belgium). Adenoviruses were propagated in HEK293 cells and stored at -80ºC. The viral titre was determined by standard plaque assay in A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

HEK293 cells. Confluent mononucleated L6 myoblasts were infected with adenovirus at 20 pfu/cell for DN-AMPK or 5.5 pfu/cell for IκBαS32A/S36A in serum free α-MEM for 2 h at 37 o C. Cells were subsequently maintained in fresh α-MEM containing 2% FBS at 37 o C and allowed to differentiate into myotubes prior to experimental analysis.

Cell Lysis -L6 myotubes were incubated for times and with the appropriate amount of effectors described in the figure legends. After treatment, cells were rinsed twice with icecold phosphate-buffered saline (PBS) and then lysed using lysis buffer (50 mM Tris/HCl, pH 7.4, 0.27 M sucrose, 1 mM sodium orthovanadate, 1 mM EDTA, 1 mM EGTA, 10mM sodium β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1% (w/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol and protease inhibitor (1 tablet/50 ml)). Whole-cell lysates were centrifuged (15,000 g at 4 °C for 10 min), and supernatants removed for storage at -80°C until required.

Immunoblotting -Cell lysates were subjected to SDS-PAGE and immunoblotted as previously reported [29]. Nitrocellulose membranes were probed with primary antibodies against proteins of interest as indicated in the figure legends. Detection of primary antibodies was performed using an appropriate peroxidase-conjugated IgG and protein signals visualized using enhanced chemiluminescence by exposure to Kodak autoradiographic film. Quantification of immunoblots was done using Image J software.

Immunoprecipitation -Protein G-Sepharose beads were washed 3 times in PBS and incubated with anti-AMPKα1/α2 for 1 h at 4 °C on an orbital platform shaker. Bead-antibody mix was then incubated with 500 μg of L6 cell lysate protein for 2 h at 4 °C before washing. The immunoprecipitates were washed twice with 1 ml of lysis buffer containing 0.5M NaCl and twice with Hepes assay buffer (50 mM Na Hepes, pH 7.0, 1 mM DTT, 0.02% Brij-35).

AMPK activity assay -AMPK were immunoprecipitated from 500 μg of muscle cell lysates using the pan-AMPK α1/α2 antibody and kinase activity towards SAMS peptide (HMRSAMSGLHVKRR) was measured as described previously [32].

RNA extraction and PCR -Total RNA was extracted from L6 myotubes using TRI reagent according to the manufacturer's instructions (Sigma-Aldrich, Dorset, U.K.). Quantitative real-time PCR was carried out using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA), SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich) and primers targeting IL-6, CINC-1 and GAPDH as a control. The sequences for these primers are as follows: IL-6 forward 5'-GACTGATGTTGTTGACAGCCA-3', IL-6 reverse 5'-ATGCTTAGGCATAACGCACTAGGTT -3', CINC-1 forward 5' -ACCCGCTCGCTTCTCTGTGC -3', CINC-1 reverse 5' -CAGCGCAGCTCATTGGCGAC -3', GAPDH forward 5′-TGGAAAGCTGTGGCGTGAT-3′ and GAPDH reverse 5′-GCTTCACCACCTTCTTGAT-3′. PCR amplification was performed with an initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 68°C for 1 min. The ratio of IL-6 and CINC-1 mRNA expression to GAPDH mRNA expression were calculated as described previously [33]. In some experiments semi-quantitative PCR was performed for analysis of IL-6 expression. For these, cDNA was amplified using rat specific primers for IL-6 using the 5'-primer AGCCACTGCCTTCCCTACTT and the 3'-primer GCCATTGCACAACTCTTTTCTC, designed and synthesised in house. PCR was performed using a Hybaid thermal cycler under the following conditions: initial denaturation was for 2 min at 94°C for one cycle, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 1 min A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

72°C followed by a final extension for 5 min at 72°C. The products were separated and visualized by ethidium bromide staining on a 1% (w/v) agarose gel.

Glucose Uptake -L6 myotubes were incubated with reagents for times and at concentrations indicated in the figure legends. Cells were washed 2 times with Hepes-buffered saline (140 mM NaCl, 20 mM Hepes, 5 mM KCl, 2.5 mM MgSO 4 , and 1 mM CaCl 2 , pH 7.4). Glucose uptake was assayed by incubation with 10 μM 2-deoxy-D-[ 3 H]glucose (1 μCi/ml) for 10 min as described previously [29]. Non-specific binding was determined by quantifying cellassociated radioactivity in the presence of 10 μM cytochalasin B. Medium was aspirated before washing adherent cells 2 times with 0.9% (w/v) ice-cold NaCl. Cells were subsequently lysed in 50 mM NaOH, and radioactivity was quantified using a Beckman LS 6000IC scintillation counter. Protein concentration in cell lysates was determined using the Bradford reagent [34].

Statistical Analyses -For multiple comparisons, statistical analysis was performed using oneway analysis of variance (ANOVA). For individual comparisons statistical analysis was performed using a t-test. Data analysis was performed using GraphPad Prism software and considered statistically significant at P values < 0.05.

RESULTS

Effects of palmitate upon NFκB and MAP kinase signalling in L6 skeletal muscle cells -

We initially monitored the effect of palmitate on the phosphorylation of IKKα/β and the associated loss in its downstream target, IκBα, serving as a readout for NFκB activation. Figure 1 shows that palmitate induces a dose-and time-dependent increase in IKKα/β phosphorylation which is associated with a concomitant loss in cellular IκBα protein levels. In an attempt to understand the contribution played by MAP kinases towards activation of the NFκB pathway we initially assessed the activation/phosphorylation status of ERK1/2, JNK and p38 MAPK. Figure 1 shows that all three MAP kinases were activated in response to palmitate at a dose and exposure period similar to that seen for activation of IKKα/β. Maximal activation of IKK, ERKs, p38 MAPK and JNK was observed at a dose of 750 μM palmitate and was detectable within 6-8 h of cell incubation with the fatty acid with phosphorylation of all four kinases being sustained in muscle cells incubated with palmitate for up to 16 h.

Effects of inhibiting p38 MAPK, JNK and ERK upon palmitate-induced activation of NFκB in L6 myotubes -

To establish what role, if any, activation of the MAP kinase subfamilies play with respect to regulation of the NFκB pathway we incubated muscle cells with palmitate for 16 h in the absence or presence of small molecule inhibitors that selectively target each kinase family. Since the three MAP kinase subfamilies were all activated following 6-8 h of palmitate treatment (Fig 1B), cells were exposed to kinase inhibitors for the last 8 h of the 16 h palmitate incubation period. BIRB 796 and SB202190 both target p38 MAPK and whilst neither prevents the stimulus-induced phosphorylation of p38 MAPK by its upstream kinase (Fig 2A), previous work from our group and that of others has shown that each compound potently suppresses the catalytic activity of p38 MAPK towards its downstream target, MAPKAPK2 [35,36]. The inhibition of MAPKAPK2 with BIRB 796 was verified as part of the present study (see supplementary Figure S1). SP600125 has been widely reported to inhibit JNK and we find that it significantly attenuates the palmitate-induced phosphorylation of JNK in our L6 muscle cells (Fig 2B). However, co-incubation of muscle cells with palmitate and BIRB 796, SB202190 or SP600125 did not suppress the fatty acid-induced activation of the IKKα/β complex or the associated loss in IκBα (Fig 2A and 2B). In contrast, PD184532 and U0126, both of which inhibit MEK [36] and thereby ERK, induced a marked reduction in the palmitate-driven activation of IKK and in the loss of IκBα (Fig 2C). This latter finding implies that in L6 myotubes ERK activation is likely to be an important upstream component of the NFκB proinflammatory response triggered by fatty acid excess.

A role for AMPK in modulating the proinflammatory potential of palmitate -Several reports have suggested that AMPK is able to antagonise the effect of numerous proinflammatory cytokines in various cell types [23][24][25][26]. We were therefore keen to assess whether AMPK could also oppose the proinflammatory potential of palmitate. Whilst there is evidence in the literature showing that acute palmitate treatment increases AMPK activity [37], sustained overloading of muscle cells with palmitate led to a significant (3-fold) reduction in basal AMPK phosphorylation and activity, which, in line with the data already presented, also induced an associated loss in the abundance of IκBα (Fig 3A). As expected, treatment of muscle cells with the specific AMPK activator, A769662 [27], significantly increased phosphorylation and activity of AMPK and, intriguingly, also promoted a modest increase in IκBα abundance. On the basis of these observations we hypothesised that activation of AMPK may protect the muscle cells against the proinflammatory effects of palmitate.

In order to test this hypothesis L6 muscle cells were treated with palmitate for 16 h in the absence or presence of AMPK activating drugs, phenformin or A769662. Fig 3B shows that when presented alone to muscle cells both drugs induced activation of AMPK based on the increased phosphorylation of ACC (Ser79), an AMPK target and thus a downstream readout for the kinase. Both phenformin and A769662 antagonised the palmitate-induced activation of IKK (Fig 3B), and consistent with this effect, both compounds ameliorated the fatty acidinduced loss in IκBα (Fig. 3B and3C). Treatment with palmitate or AMPK activators did not cause any detectable change in the cellular abundance of ACC, ERK1/2 or that of the α1 and α2 subunits of AMPK (Fig 3B). Our observations thus imply that AMPK activation exerts a suppressive effect upon palmitate-induced NFκB signalling.

To further substantiate this possibility, the effect of AMPK activation on the expression of IL-6 and CINC-1 (an IL-8 like cytokine), two NFκB-regulated muscle genes [38,39], was assessed using real-time quantitative PCR following cell incubation with palmitate. Fig 4 shows that palmitate induced a significant increase in both IL-6 and CINC-1 mRNA, although the magnitude with which IL-6 expression was enhanced was substantially greater than that of CINC-1. Cell treatment with A769662 for the penultimate 8-h incubation with the fatty acid significantly antagonised the increase in both IL-6 and CINC-1 expression. Given that AMPK activation opposes the palmitate-induced phosphorylation of ERK1/2 in a time dependent manner that correlates tightly with reduced phosphorylation of IKK (Fig 3B )  we postulated that AMPK may suppress the proinflammatory potential of palmitate via inhibition of ERK1/2 signalling.

Since AMPK antagonises the palmitate-induced phosphorylation of ERK1/2 (Fig 3B ) we assessed whether AMPK activation in combination with MEK inhibition exerted an additive effect with respect to suppression of IKK phosphorylation/activation and IκBα protein loss.

Effects of expressing a dominant negative AMPK on palmitate-induced IκBα degradation -

To further validate that AMPK acts a negative modulator of NFκB signalling, a myc-tagged dominant negative AMPK adenoviral construct (AdAMPK-DN) was expressed in L6 muscle cells prior to incubation with palmitate and A769662. AMPK was rendered dominant negative by mutating an aspartate residue at position 157 to an alanine in α1 AMPK as previously described [30]. Expression of this viral construct was validated by blotting cell lysates with an anti-myc antibody to detect the tagged protein and also by analysis of total AMPK levels, which were considerably elevated in the viral-infected cells compared with control cells that were mock-infected with virus lacking the AMPK construct (Fig 6A). Confirmation that the construct behaved in a dominant negative manner was established by demonstrating that whilst A769662 induced ACC Ser79 phosphorylation in wild type muscle cells, those expressing the AdAMPK-DN construct lacked this stimulus-induced phosphorylation of ACC (Fig 6A). Consistent with other data presented in this paper, AMPK activation antagonised the fatty-acid induced loss in IκBα in wild-type L6 muscle cells. However, this antagonism was not observed in cells expressing the AMPK-DN construct (Fig 6A and6B). Furthermore, the ability of A769662 to counter the palmitate-induced activation of ERK was lost in cells expressing the AMPK-DN construct (Fig 6A ).

Effects of ceramide on PKB and NFκB signalling -We have shown previously that ceramide generated from palmitate or added exogenously to cells in a cell-permeable form promotes a targeted loss in the insulin-dependent activation of PKB by a mechanism involving atypical PKCs (λ/ζ) [6,40,41]. However, there is also evidence in the literature suggesting that in some cell types ceramide promotes activation of NFκB signalling, which may contribute to the reduction in insulin signalling [42]. To assess whether this might be the case in our muscle cells we investigated the effects of ceramide on both PKB and NFκB activation. Consistent with our previous work [6], Fig 7 shows that sustained incubation of muscle cells with palmitate inhibited PKB activation in response to an acute insulin challenge. This effect was mimicked by subjecting L6 cells to either a short (2h, 100 μM) or more chronic low grade (16h, 10 μM) incubation with C2-ceramide. Under both circumstances C2-ceramide reduced the insulin-dependent activation of PKB. In contrast, unlike palmitate treatment, C2ceramide did not induce phosphorylation/activation of IKK or promote loss of IκBα. Consistent with this latter finding, AMPK activation with A769662 also failed to antagonise the palmitate-induced loss in insulin-stimulated glucose uptake, which is widely accepted as being a key PKB-regulated process (Fig 8B). To further substantiate these findings L6 cells were infected with an adenoviral construct harbouring a mutant form of IκBα (S32A/S36A) that acts as a potent repressor of NFκB activity. Mutation of Ser32 and Ser36 to alanine renders IκBα resistant to IKK phosphorylation, thereby protecting it from proteosomal degradation and retaining its capacity to hold NFκB in an inhibited state. Fig. 8C shows that palmitate induces IKK activation, IκBα loss and expression of IL-6 (assessed using a semiquantitative PCR approach) in wild type (mock transfected) L6 cells and those infected with the empty viral vector. However, cells expressing the stable IκBαS32A/S36A protein do not exhibit a stimulus-induced increase in IL-6 expression despite the attendant increase in IKK activation (Fig 8C). Moreover, despite suppressing palmitate-induced NFκB signalling using the super repressor, this experimental approach did not antagonise the loss in insulinstimulated PKB activation caused by palmitate (Fig 8C ).

Effects of suppressing NFkB activation on insulin sensitivity of L6 muscle cells -Whilst the data presented in

DISCUSSION

There is growing recognition that an increase in tissue inflammation is a key feature associated with obesity and that this may, in part, be initiated by the sustained elevation and tissue delivery of circulating free fatty acids that prevail during this condition [43]. In skeletal muscle, fatty acid overload has been shown to enhance proinflammatory NFκB signalling and this has been linked to the development and progression of insulin resistance in this tissue [22,44]. Precisely how fatty acids, such as palmitate, promote an increase in inflammatory tone is not well understood and, although there is mounting interest of the role played by TLRs in this response, there is a large body of evidence that suggests that increased accumulation of fatty-acid derived metabolites (e.g. ceramide, DAG etc) play a crucial role in modulating the activity of numerous molecules, including those involved in insulin and inflammatory signalling [41,[45][46][47]. The major focus of the work described here was to (i) gain an insight into how palmitate induces proinflammatory NFκB signalling in cultured myotubes, with particular focus on the role played by members of the MAP kinase family and ceramide as potential protagonists of the inflammatory response, (ii) establish whether AMPK activation opposes the proinflammatory drive initiated by palmitate and (iii) assess whether activation of NFκB signalling underpins the loss in insulin sensitivity invoked by palmitate in L6 myotubes.

Our findings indicate that whilst sustained incubation of muscle cells with palmitate induces activation of all three MAP kinase subfamilies, only the classical ERK pathway appears to be important with respect to supporting activation of NFκB signalling in L6 myotubes (Fig 9A). We suggest that activation of ERK not only precedes that of IKK, but also lies upstream of the IKK/NFκB signalling pathway. This proposition is based on the following evidence. First, activation of ERK correlates temporally with that of IKK and the associated reduction that this causes in IκBα abundance. Secondly, PMA, which potently activates ERK [48], also induces activation of IKK in L6 myotubes and, thirdly, pharmacological inhibitors that selectively target the MEK/ERK signalling axis (i.e. PD184532 and U0126) significantly reduce palmitate's capacity to stimulate NFκB signalling (Fig 9B). The concept that ERK activation may contribute to an increase in inflammatory tone is not unprecedented. Previous studies in macrophages [18], epithelial cells [49,[START_REF] Zhou | Ras and mitogen-activated protein kinase kinase kinase-1 coregulate activator protein-1-and nuclear factor-kappaB-mediated gene expression in airway epithelial cells[END_REF] and vascular smooth muscle cells [19] have shown that a variety of proinflammatory stimuli (e.g. lipopolysaccharide (LPS), TNFα, IL-8) promote ERK-dependent NFκB activation, presumably via signalling initiated from binding to their respective cell surface receptors. Precisely how palmitate initiates proinflammatory signalling in L6 myotubes is not entirely clear, but is unlikely, in our view, to be initiated via TLRs based on the rather slow response time (6-8 h) with which the fatty acid induces IKK activation. Moreover, it is noteworthy that in separate studies we have found that bacterial LPS, a well established TLR2/4 agonist, fails to promote any detectable activation of IKK/NFκB signalling when incubated with L6 myotubes for periods of up 12 h yet, under identical incubation conditions, induces robust IKK activation and IκBα loss in mouse dendritic cells within 30 min (Macrae, Lipina and Hundal, unpublished data). The absence of an acute LPS-or palmitate-induced inflammatory signalling response in L6 myotubes implies that although TLR2 and TLR4 are expressed in skeletal muscle tissue [START_REF] Frost | Multiple Toll-like receptor ligands induce an IL-6 transcriptional response in skeletal myocytes[END_REF] and cells [8,[START_REF] Reyna | Elevated toll-like receptor 4 expression and signaling in muscle from insulin-resistant subjects[END_REF] they may be refractory to stimulation by LPS and palmitate, at least over the incubation periods deployed with the muscle cells used in the current study. It is currently unclear whether this observation represents a cell line specific effect, but it would be of interest to assess the effects of both LPS and palmitate upon IKK/NFκB signalling under circumstances when TLR2/4 and their adaptor proteins are either stably and moderately overexpressed or silenced in L6 muscle cells. Such studies may provide a more definitive assessment of whether TLRs are involved or not in the proinflammatory response triggered by palmitate in our experimental model.

If not TLRs, what might initiate the fatty acid-induced proinflammatory response? We have shown previously that accumulation of both DAG and ceramide is enhanced significantly in muscle cells following incubation with palmitate [6] and although our data indicate that ceramide is unlikely to contribute to activation of the ERK/NFκB signalling axis we believe that DAG may do so. An important consequence of accumulating this fatty acid derived molecule is the activation of DAG-sensitive PKC isoforms. It is noteworthy that one member of this family, PKCθ, has been implicated in palmitate-induced NFκB signalling in murine C2C12 myotubes [47]. Whether PKCθ acts directly to activate the IKK/NFκB pathway or mediates its effect via another route is currently unclear, but given that PMA, a DAG mimetic, is capable of inducing IKK activation in an ERK-dependent manner it seems plausible that DAG-sensitive PKCs may act via the MEK/ERK signalling pathway to promote NFκB signalling in L6 myotubes as has been suggested in macrophages [18].

Activated AMPK can target a panoply of signalling proteins that regulate metabolic pathways affecting cellular energy balance [START_REF] Hardie | AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy[END_REF]. In skeletal muscle, AMPK activation enhances glucose uptake and fatty acid oxidation [START_REF] Hardie | AMPK: a key sensor of fuel and energy status in skeletal muscle[END_REF] and it is not unsurprising therefore, that the kinase is perceived as a potential drug target whose manipulation may prove beneficial in the treatment of metabolic disorders such as diabetes and obesity [START_REF] Zhang | AMPK: an emerging drug target for diabetes and the metabolic syndrome[END_REF]. However, there is growing recognition that AMPK may also confer an anti-inflammatory response based on work showing that metformin and AICAR, two well established AMPK activators, inhibit NFκB activity and expression of NFκB-dependent gene targets in endothelial cells [24,[START_REF] Huang | Metformin inhibits TNF-alpha-induced IkappaB kinase phosphorylation, IkappaB-alpha degradation and IL-6 production in endothelial cells through PI3Kdependent AMPK phosphorylation[END_REF]. Similarly, incubation of primary human skeletal muscle cells from lean and obese individuals with AICAR has been shown to antagonise the palmitate-induced degradation of IκBα [START_REF] Bikman | Lipid-induced insulin resistance is prevented in lean and obese myotubes by AICAR treatment[END_REF]. Although the latter study did not establish the mechanism by which AICAR spares the fatty acid-induced loss of IκBα a very recent study has reported that the ribonucleoside impairs binding of NFκB to DNA without affecting nuclear translocation of NFκB or turnover of IκBα protein in endothelial cells [24]. In contrast, our data suggests that AMPK targets a point considerably more upstream in the inflammatory signalling cascade; specifically, at the level of, or upstream of, MEK/ERK. The finding that A769662 (which is regarded as a more specific AMPK activator than either metformin or AICAR [27] antagonises the palmitateand PMA-induced activation of ERK, and that this results in a concomitant reduction in IKK/NFκB signalling and restrained expression of IL-6 and CINC-1 (two known NFκB target genes expressed in muscle) would support our proposition of a more proximal AMPK effect. What remains unresolved is the precise nature of this upstream AMPK target. There is no evidence in the literature to suggest that MEK or ERK1/2 serve as direct AMPK substrates, although Raf1, which lies upstream of MEK, can be phosphorylated by AMPK on serine 621 in vitro [START_REF] Sprenkle | Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase[END_REF]. It is currently unknown if AMPK phosphorylates Raf1 in vivo (or in intact cells) and whether it is inhibitory for the downstream activation of MEK and ERK. Alternatively, it is possible that AMPK activation enhances palmitate oxidation, which may help limit accumulation of molecules such as DAG and consequently their potential to promote inflammatory signalling via members of the PKC family. Testing these possibilities represent important investigative goals of future work.

Loss of IKKβ or inhibiting nuclear translocation of NFκB has been shown to protect skeletal muscle against lipid-induced insulin resistance in rodent and cell based studies [22,44]. However, our work indicates that inhibiting the NFκB pathway either via activation of AMPK or by expression of the IκBα (S32A/S36A) super-repressor fails to alleviate the repressive effect that palmitate has upon insulin sensitivity of L6 myotubes. There are a number of reasons that may explain why inhibition of NFκB did not prove to be protective in our experimental model. We believe that it is highly unlikely that inhibition of NFκB would have any significant effect upon palmitate-driven synthesis of ceramide or DAG. Our previous work has shown that ceramide added exogenously to cells as a cell permeant analogue or generated in vivo from palmitate causes a targeted loss in PKB-directed insulin signalling [6,41]. This inhibition, as shown in the current study, occurs in the absence of any detectable activation of NFκB by ceramide; a finding that indicates that in L6 myotubes this sphingolipid is unlikely to be a major contributor to fatty acid-induced inflammation. Moreover, whilst suppressing the de novo synthesis of ceramide from palmitate can be beneficial with respect to insulin action, we have found that sustained inhibition of the metabolic pathway that converts palmitate to ceramide induces a dynamic shift in palmitate metabolism towards greater synthesis of DAG [46]. This increase in DAG exerts an insulindesensitising effect upon IRS-directed insulin signalling via activation of DAG-sensitive PKCs [46]. Consequently, if palmitate-driven synthesis of DAG and ceramide remains unabated under circumstances when NFκB signalling is suppressed it is unlikely that one might expect a significant gain in insulin-sensitivity of muscle cells. In addition, AMPKmediated antagonism of NFκB did not prevent palmitate's capacity to activate p38 MAPK and JNK in L6 myotubes. We cannot exclude the possibility that activation of these MAP kinases may also contribute to lipid-induced insulin resistance either directly by targeting proximal components of the insulin signalling cascade or indirectly via modification of mitochondrial function/lipid oxidation as reported in some studies [START_REF] Solinas | Saturated fatty acids inhibit induction of insulin gene transcription by JNK-mediated phosphorylation of insulin-receptor substrates[END_REF][START_REF] Kadotani | Different impacts of saturated and unsaturated free fatty acids on COX-2 expression in C(2)C(12) myotubes[END_REF].

In summary, our work demonstrates that sustained exposure of skeletal muscle cells to palmitate induces NFκB signalling in an ERK-dependent manner and that activation of AMPK serves to counter-regulate this proinflammatory pathway ( NFκB appears not to contribute significantly to the insulin-desensitising effects of palmitate over the incubation periods that we have studied, we suggest that the pathogenic effects that are likely to be associated with enhanced and sustained expression of proinflammatory cytokines (e.g. IL-6) on, for example, gene expression, cell metabolism and cell survival over the long term, and the ability to potentially offset these via activation of AMPK, should not be discounted or over looked. (A) L6 myotubes were treated with palmitate (750 μM) for 16 h or with A769662 (100 μM) for 2 h. Lysates were either immunoprecipitated with a pan-AMPK α1/α2 antibody and used to assay AMPK activity or immunoblotted to assess the phosphorylation status of AMPK and ACC and total protein abundance of IκBα. The asterisk signifies a significant difference from the control (untreated) value (P < 0.05). (B) L6 myotubes were treated with palmitate for 16 h (750 μM) in the absence or presence of either A769662 (100 μM) or phenformin (200 μM) for the indicated periods. Cell lysates were immunoblotted to assess the phosphorylation status of IKKα/β, ERK1/2 and ACC as well as total protein abundance of IκBα, ACC, AMPK and ERK1/2. Analysis of β-actin was used to assess protein loading in gel lanes. The blots are representative of three separate experiments. (C) Changes in IκBα protein levels from (B) were quantified and expressed as a percentage change from untreated control. Values shown are the mean ± S.E.M from three separate experiments. The asterisks in A signify significant differences (P <0.05) from the untreated control, whereas in C the significance is relative to the single palmitate-treated value. of IKKα/β ACC, ERK1/2, p38 MAPK, JNK and MEK1/2 as well as total protein abundance of IκBα. Analysis of β-actin was used to assess protein loading in gel lanes. L6 myotubes were treated with palmitate (750 μM) for 16 h in the absence or presence of (A) A769662 (100 μM) for the final 8 h or (A, C and D) insulin (100 nM) for 10 min or (B) insulin (100 nM) 30 min. (A) Cell lysates were immunoblotted for assessing the phosphorylation status of ACC, PKB and IKKα/β and total protein abundance of IκBα. Equal loading was ascertained by immunoblotting with an antibody against total PKB. (B) Muscle cells were assayed for 2deoxyglucose (2DG) uptake. Values are the mean ± SEM from four separate experiments each performed in triplicate. (C) L6 myoblasts were infected with an empty control adenoviral vector or one expressing an HA-tagged non-phosphorylatable IκBα (S32A/S36A) construct. Infected myoblasts were allowed to differentiate into myotubes prior to treatment with palmitate and insulin as indicated. Cells were used for semi-quantatative PCR analysis of IL-6 mRNA (upper panel). The IL-6 expression data is expressed as mean ± SEM from three separate experiments. The lower panel shows immunoblot analysis of cell lysates assessing the phosphorylation status of IKKα/β and PKB and total protein abundance of IκBα (endogenous and HA-tagged). Gel loading was ascertained by immunoblotting with an antibody against total PKB. The asterisk signifies a significant difference from appropriate control values (P < 0.05).

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517

Biochemical

  Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Fig 5A shows that both A769662 and U0126 inhibit the palmitate-induced phosphorylation of IKK and ERK. However, combined treatment of muscle cells with both drugs did not result in any noticeable enhancement in the antagonism of palmitate's effect on IKK or IκBα Biochemical Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  abundance. Of interest, in parallel experiments we assessed whether AMPK activation would also antagonise the palmitate-induced activation of p38 MAPK and JNK. The immunoblot data presented in Fig 5B shows that unlike the ERK pathway, activation of the two other MAP kinase sub-families was not suppressed by A769662. In order to further substantiate the notion that AMPK activation counters MEK/ERK signalling, L6 muscle cells were treated with PMA (a DAG mimetic and a potent activator of ERK signalling) in the absence or presence of A769662. Fig 5C shows PMA treatment induced an increase in the phosphorylation of MEK and ERK and was associated with an increase in IKK phosphorylation. Importantly, incubation of cells with A769662 antagonised the PMAinduced phosphorylation of MEK, ERK and IKK.

  Fig 7 suggests that the insulin-desensitising effect of ceramide in L6 muscle cells is unlikely to involve NFκB activation, our findings do not exclude the possibility that palmitate may act independently of ceramide to suppress insulin signalling via the NFκB pathway. To test this we investigated the effect of suppressing NFκB signalling by two separate approaches and monitored palmitate's effect upon the insulin-dependent activation Biochemical Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  of PKB. Fig 8A shows that palmitate blunted the insulin-dependent phosphorylation of PKB. Under these circumstances palmitate induces IKK phosphorylation and a loss of IκBα and, in line with data presented in earlier figures (Fig 5A), this was antagonised by co-incubation of cells with A769662. However, despite this antagonism in NFκB signalling, A769662 was unable to halt the loss in the insulin activation of PKB elicited by the fatty acid (Fig 8A).

Biochemical

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  Fig 9). Whilst activation of Biochemical Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .Figure 2 .

 12 Figure 1. Effect of palmitate on NFκB and MAP kinase signalling. L6 myotubes were treated with (A) BSA-conjugated palmitate or BSA (control) for 16 h at indicated concentrations or (B) palmitate (750 μM) for the indicated periods. (A and B) Lysates were immunoblotted to assess the phosphorylation status of IKKα/β, ERK1/2, p38 MAPK and JNK and total protein abundance of IκBα. Equal gel loading was ascertained by immunoblotting with an antibody against β-actin. (B) Effects of palmitate upon IκBα protein (filled bars) was quantified and expressed as a percentage change from untreated control cells (open bar). Values shown are the mean ± S.E.M from at least 3 separate experiments

Figure 3 .

 3 Figure3. Effects of AMPK activators on palmitate-induced NFκB and MAP kinase signalling in L6 myotubes. (A) L6 myotubes were treated with palmitate (750 μM) for 16 h or with A769662 (100 μM) for 2 h. Lysates were either immunoprecipitated with a pan-AMPK α1/α2 antibody and used to assay AMPK activity or immunoblotted to assess the phosphorylation status of AMPK and ACC and total protein abundance of IκBα. The asterisk signifies a significant difference from the control (untreated) value (P < 0.05). (B) L6 myotubes were treated with palmitate for 16 h (750 μM) in the absence or presence of either A769662 (100 μM) or phenformin (200 μM) for the indicated periods. Cell lysates were immunoblotted to assess the phosphorylation status of IKKα/β, ERK1/2 and ACC as well as total protein abundance of IκBα, ACC, AMPK and ERK1/2. Analysis of β-actin was used to assess protein loading in gel lanes. The blots are representative of three separate experiments. (C) Changes in IκBα protein levels from (B) were quantified and expressed as a percentage change from untreated control. Values shown are the mean ± S.E.M from three separate experiments. The asterisks in A signify significant differences (P <0.05) from the untreated control, whereas in C the significance is relative to the single palmitate-treated value.

Figure 4 .

 4 Figure 4. Quantitative real-time PCR analysis of IL-6 and CINC-1 mRNA expression in L6 myotubes. Muscle cells were incubated with palmitate (750 μM) for 16 h in the absence or presence of A769662 (100 μM) for the penultimate 8 h of the fatty acid incubation. RNA was extracted from cells and IL-6 and CINC-1 mRNA determined by real time qPCR as described in methods. Values shown are the mean ± S.E.M from three separate determinations. The asterisks signify significant differences (P <0.05) between the indicated bars.

Figure 5 .

 5 Figure 5. Effects of AMPK activation on palmitate and PMA-induced NFκB and MAP kinase signalling in L6 myotubes. Muscle cells were treated with (A and B) palmitate (750 μM) for 16 h or (C) PMA (100 nM) for 2 h in the absence or presence of A769662 (100 μM) for 8 h (A and B) or 2 h (C).In some experiments, L6 myotubes were also treated with U0126 (10 μM) for 8 h. Cell lysates were immunoblotted to assess the phosphorylation status

Figure 6 .

 6 Figure 6. Expression of a dominant negative AMPK in L6 myotubes antagonises the repressive effect of A769662 on palmitate-induced NFκB and ERK signalling. L6 myoblasts were infected with a dominant negative AMPK adenoviral construct or mockinfected with virus lacking the AMPK construct and allowed to differentiate into myotubes. Cells were treated with palmitate (750 μM) for 16 h in the absence or presence of A769662 (100 μM) for the final 8 h of treatment. (A) Cell lysates were immunoblotted to assess the phosphorylation status of ACC and ERK1/2 and cellular levels of IκBα, AMPKα1/α2 and myc. Immunoblotting for β-actin provided a means for assessing protein loading in gel lanes. (B) Quantification of IκBα abundance and ERK1/2 phosphorylation, values shown are the mean ± S.E.M for between 3-5 separate experiments.Asterisks signify significant differences from the appropriate mock-infected untreated control, the hash signifies a significant difference from the palmitate treatment value (P < 0.05).

Figure 7 .

 7 Figure 7. Effects of C2-ceramide on activation of the NF-κB proinflammatory and MAP kinase pathways. Representative immunoblots showing the effect of incubating L6 myotubes with either palmitate (Pal, 750 μM) for 16 h or C2-ceramide for 2 h (Cer, 100 μM)or 16 h (Cer, 10 μM) in the absence or presence of insulin (100 nM) for 10 min. Lysates from control (Con) and treated cells were immunoblotted for the phosphorylation status of PKB, ERK, IKK and total protein amount of IκBα. Equal loading was ascertained by immunoblotting with an antibody against total PKB.

Figure 8 .

 8 Figure8. Effects of antagonising NFκB signalling on palmitate-induced inhibition of insulin-stimulated PKB activation and glucose uptake in L6 myotubes. L6 myotubes were treated with palmitate (750 μM) for 16 h in the absence or presence of (A) A769662 (100 μM) for the final 8 h or (A, C and D) insulin (100 nM) for 10 min or (B) insulin (100 nM) 30 min. (A) Cell lysates were immunoblotted for assessing the phosphorylation status of ACC, PKB and IKKα/β and total protein abundance of IκBα. Equal loading was ascertained by immunoblotting with an antibody against total PKB. (B) Muscle cells were assayed for 2deoxyglucose (2DG) uptake. Values are the mean ± SEM from four separate experiments each performed in triplicate. (C) L6 myoblasts were infected with an empty control adenoviral vector or one expressing an HA-tagged non-phosphorylatable IκBα (S32A/S36A) construct. Infected myoblasts were allowed to differentiate into myotubes prior to treatment with palmitate and insulin as indicated. Cells were used for semi-quantatative PCR analysis of IL-6 mRNA (upper panel). The IL-6 expression data is expressed as mean ± SEM from three separate experiments. The lower panel shows immunoblot analysis of cell lysates assessing the phosphorylation status of IKKα/β and PKB and total protein abundance of IκBα (endogenous and HA-tagged). Gel loading was ascertained by immunoblotting with an antibody against total PKB. The asterisk signifies a significant difference from appropriate control values (P < 0.05).

Figure 9 .

 9 Figure 9. Schematic illustrating the effects of palmitate and AMPK activation upon the IKK/NFκB signalling axis in L6 muscle cells. (A) Sustained exposure of L6 myotubes to palmitate induces phosphorylation and activation of MAP kinase family members. Activation of ERK promotes downstream phosphorylation/activation of IKK causing a concomitant phosphorylation and proteosomal mediated loss of IκBα thereby promoting an increase in the expression of NFκB target genes.

Figure 4

 4 Figure1

  Biochemical Journal Immediate Publication. Published on 17 Feb 2011 as manuscript BJ20101517 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101517

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGMENTS:

We thank Dr Chris Lipina and Dr Clare Stretton for providing helpful comments on the manuscript and are grateful to the Division of Signal Transduction and Therapy for providing some of the reagents used in this study. We are also grateful to Pascal Ferre and Fabienne Foufelle for providing us with the adenovirus encoding dominant negative AMPK. This work was supported by the UK Medical Research Council, the EXGENESIS project of the European Commission (LSHM-CT-20004-005272), Dundee District Diabetes UK volunteer group and Diabetes UK.


Nadja Schröder-Kress

Eva Dütting

Heinrich Sticht

Dr Ralf Enz
email: ralf.enz@biochem.uni-erlangen.de

Nadja Schröder-Kreß

Sumo E3 ligases are expressed in the retina and regulate sumoylation of the metabotropic glutamate receptor 8b

Keywords: bp -base pairs, CNS -central nervous system, GCL -ganglion cell layer, GST -glutathione-S-transferase, INL G-protein coupled receptor, GPCR, metabotropic glutamate receptor, mGlu receptor, Pias1, retina Biochemical

Sumo E3 ligases are expressed in the retina and regulate sumoylation of the metabotropic glutamate receptor 8b

INTRODUCTION

The correct function of the central nervous system (CNS) requires a highly ordered and tightly controlled arrangement of neurotransmitter receptors at subcellular specializations. Thus, the regulation of neurotransmitter receptors at synapses represents an important mechanism to adjust neuronal excitability. Excitatory neurotransmission is mainly mediated by the neurotransmitter glutamate that binds to ion channel-associated (ionotropic) and Gprotein-coupled (metabotropic) glutamate receptors. Metabotropic glutamate receptors regulate intracellular second messenger systems that take part in a variety of physiological and pathophysiological processes, including development, learning and memory, pain, stroke, epileptic seizures and schizophrenia, as well as in chronic neurodegenerative diseases such as Alzheimer´s, Huntington´s and Parkinson´s disease [START_REF] Bordi | Group I metabotropic glutamate receptors: implications for brain diseases[END_REF][START_REF] Schoepp | Unveiling the functions of presynaptic metabotropic glutamate receptors in the central nervous system[END_REF].

The eight known metabotropic glutamate receptors (mGluR1-8) are subdivided into three groups, based on sequence homology, agonist specificity and associated second messenger systems [START_REF] Ferraguti | Metabotropic glutamate receptors[END_REF]. Group III contains mGluR4 and mGluR6-8 that negatively couple to adenylyl cyclase. With the exception of mGluR6, group III mGluRs are found pre-synaptically where they are suggested to operate as auto-receptors [START_REF] Shigemoto | Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus[END_REF].

MGluR mediated neurotransmission is dynamically regulated by associated molecules, e.g. enzymes and scaffold proteins. This is achieved by the formation of macromolecular signalling complexes that contain functionally related proteins, including neurotransmitter receptors, kinases and phosphatases. The intracellular mGluR C-termini play a key role in the association and regulation of these networks. Alternative splicing within these C-termini generates a large variety of different intracellular domains accessible for cytosolic binding partners and indeed, various protein interactions of these domains are described [START_REF] Enz | The trick of the tail: protein-protein interactions of metabotropic glutamate receptors[END_REF]. Importantly, mutations in mGluR binding proteins can lead to neuropsychiatric disorders including addiction, depression, epilepsy and schizophrenia [START_REF] Szumlinski | Homer proteins: implications for neuropsychiatric disorders[END_REF].

Protein-protein interactions are regulated by post-translational modifications, such as phosphorylation or sumoylation that determine the structure of binding surfaces. The covalent attachment of Sumo isoforms (Sumo1-4 in mammals) to target proteins is guided by the sequential action of three enzymes [START_REF] Gareau | The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition[END_REF]. First, Sumo proteins are activated by a heterodimeric E1-activating enzyme and subsequently transferred to the E2-conjugating enzyme Ubc9. Finally, Ubc9 recognizes specific sumoylation consensus sequences in target proteins and attaches Sumo to the ε-amino group of lysine residues. Often, this enzymatic process is facilitated by E3-ligases, e.g. members of the PIAS protein family. The covalent conjugation of Sumo isoforms to substrate proteins may prevent, enable or modify proteinprotein interactions or protein activities. Thereby, sumoylation regulates a variety of cellular functions, including gene transcription, protein trafficking and synaptic excitability. In recent years, many Sumo targets were identified in neurons and an involvement of Sumo proteins in CNS disorders was proposed [START_REF] Wilkinson | Targets and Consequences of Protein SUMOylation in Neurons[END_REF].

In order to identify new regulatory mechanisms of mGluR mediated signal transduction, we performed yeast two-hybrid screens against the intracellular C-termini of two mGluR8 isoforms (mGluR8a, mGluR8b) and identified several proteins for post-translational modification by sumoylation and neddylation. A previous study reported sumoylation of mGluR8a [START_REF] Tang | Pias1 interaction and sumoylation of metabotropic glutamate receptor 8[END_REF]. However, while this study used the isolated C-terminus of mGluR8a, here we show that the complete mGluR8b protein inserted in the plasma membrane of mammalian cells can be sumoylated in-vivo. MGluR8b contains a bona-fide sumoylation motif that is identical between mGluR8a and mGluR8b, and a non-canonical sumoylation site located in the mGluR8b isoform-specific C-terminus. We found that lysine residues present in both motifs can be sumoylated and observed that addition of the Sumo E3-ligase Pias1 enhanced this enzymatic process.

EXPERIMENTAL Yeast two-hybrid techniques

Proteins interacting with the C-terminal domains of mGluR8a or mGluR8b were searched in a rat brain cDNA-library as described [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF]. In brief, yeast AH109 cells were sequentially transformed with mGluR8 C-termini and library containing vectors and incubated for 5 days at 30 °C on selection plates. Library plasmids were isolated and sequenced as in [START_REF] Rose | Band 4.1 proteins are expressed in the retina and interact with both isoforms of the metabotropic glutamate receptor type 8[END_REF].

GST pull-down assays

Plasmids encoding Pias1-4 in pET28 [START_REF] Azuma | PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes[END_REF] and Pc2, Ubc9 and Sumo1 in pET21 were used for in-vitro expression in the presence of [S35]methionine (MP Biomedicals, Eschwege, Germany) using the TnT Quick Coupled Transcription/Translation System (Promega, Mannheim, Germany). MGluR C-termini were ligated to the coding sequence of GST in pET-41 (Novagen, Madison, WI), expressed in E. coli, purified and immobilized under native conditions on glutathione Sepharose using the BugBuster GST-Bind-Purification Kit (Novagen). Coated glutathione Sepharose was incubated with in-vitro translated proteins in the presence of 0.1% Triton X-100 and 0.1% bovine serum albumin. Bound proteins were eluted by boiling in SDS sample buffer and analyzed on protein gels as described [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF].

RT-PCR

Total RNA was extracted from adult rat retina, reverse transcribed and amplified for 35 cycles as reported [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF] using primer pairs specific for Pias1 (sense nt1299-1318; antisense nt1961-1939), Pias2 (sense nt1519-1541; antisense nt1961-1941), Pias3L (sense nt1299-1318; antisense nt1893-1873), Pias4 (sense nt1256-1274; antisense nt1533-1516), Pc2 (sense nt1201-1219; antisense nt1780-1759), Sumo1 (sense nt117-133; antisense nt422-404) and Ubc9 (sense nt1-20; antisense nt477-456). Amplifications without cDNA served as negative controls. All PCR products were identified by DNA sequencing.

Immunocytochemistry

Retinal cryostat sections of adult mice were prepared as described [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF] and incubated with primary antibodies recognizing Pias1 (1:25; ZP008, Zymed Laboratories, South San Francisco, CA), Pias3L (1:300; AB1395 [START_REF] Duval | The 'PINIT' motif, of a newly identified conserved domain of the PIAS protein family, is essential for nuclear retention of PIAS3L[END_REF]), Ubc9 (1:20; AB-1, Oncogene Research Products, San Diego, CA), Sumo1 (1:100; FL-101, Santa Cruz Biotechnology, Santa Cruz, CA) or mGluR8b (1:1000; [START_REF] Ferraguti | Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus[END_REF]). In addition, retina sections were co-stained with DAPI (1:5000; Sigma-Aldrich, Munich, Germany). Binding sites of primary antibodies were revealed by the secondary antibodies Alexa TM 594 and Alexa TM 488 (1:500; Molecular Probes, Eugene, OR). In controls, the primary antibodies were omitted, resulting in a complete loss of immunereactivity. Labelled proteins were examined by a Zeiss Axio Imager Z1 equipped with an ApoTome (Zeiss, Oberkochen, Germany).

Sumo1 conjugation

Sumoylation sites in mGluR8b were predicted with SUMOPlot (Abgent, San Diego, CA). The mGluR8b coding sequence was N-terminally fused to a c-myc epitope in the eukaryotic expression vector pRK7 and mutations were introduced by PCR cloning techniques. Coding sequences of Pias1, Ubc9 and Sumo1 were fused to N-terminal T7-tags and cloned in the eukaryotic expression vector pRK5. Human embryonic kidney (HEK-293) cells were transfected in 10 cm dishes with 8 μg DNA (2 μg mGluR8b constructs + 6 μg containing equal amounts of various combinations of Sumo1, PIAS1, Ubc9) and 15 μl jetPEI (Biomol, Hamburg, Germany). After 48 h, cells were homogenized as described [START_REF] Rose | Band 4.1 proteins are expressed in the retina and interact with both isoforms of the metabotropic glutamate receptor type 8[END_REF] in the presence of 20 mM N-ethylmaleimide (Sigma-Aldrich). Solubilized proteins were incubated for 18 h with 50 μl of a 50% (v/v) suspension of protein A-Agarose (Amersham, Braunschweig, Germany) that was pre-incubated with c-myc specific antibodies (5 μl; 9E10, Santa Cruz). Thereafter, beads were washed 3 times in 500 μl washing buffer (20 mM Tris-HCl, 0.16 mM NaCl, pH=7.4) and the c-myc epitope of precipitated mGluR8b was detected by Western Blotting (1:10000, ab9106, Abcam, Cambridge, UK). Sumoylation of mGluR8b was analyzed using Sumo1 specific antibodies (1:200; Santa Cruz). Surface expression of mGluR8b constructs was analyzed in HEK-293 cells that were grown on poly(L-lysine) coated glass coverslips and transfected with 1 μg DNA as above. The extracellular c-myc tag of glutamate receptors was labelled with c-myc specific antibodies (1:1000, Abcam) under non-permeabilizing conditions and resulting fluorescence was analyzed with a confocal microscope (TCS SL; Leica Microsystems, Wetzlar, Germany).

Structural and energetic analysis

Position 1 and 4 of the LKSE binding motif of the Ubc9-RanGAP1 crystal structure (PDB code: 1KPS; [START_REF] Bernier-Villamor | Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitinconjugating enzyme Ubc9 and RanGAP1[END_REF]) were mutated to V and G, respectively, using Sybyl7.3 (Tripos Inc.). Energetic analyses were performed with Fold-X [START_REF] Guerois | Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations[END_REF] and Concoord/PBSA [START_REF] Benedix | Predicting free energy changes using structural ensembles[END_REF] with standard settings. DS Visualizer 2.5 (Accelrys Inc.) was used for structural analysis and visualization.

RESULTS

Proteins of the sumoylation machinery interact with mGluR C-termini

Previously, we used yeast two-hybrid screens to search for binding partners of the intracellular mGluR8a and mGluR8b C-termini (Fig. 1A) in the CNS. These studies identified several proteins involved in sumoylation and neddylation (Fig. 1B; [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF]). Here, we focused on the role of proteins belonging to the sumoylation cascade, namely the Sumo E2-conjugating enzyme Ubc9 and several Sumo E3-ligases (Pc2, Pias1 and Pias3L). Pias3 exists in two isoforms (Pias3 and Pias3L) that differ in 35 amino acids inserted in Pias3L. The long Pias3 variant is highly expressed in brain [START_REF] Duval | The 'PINIT' motif, of a newly identified conserved domain of the PIAS protein family, is essential for nuclear retention of PIAS3L[END_REF] and consistently, screening a rat brain cDNA library we identified exclusively Pias3L, also designated K + -channel associated protein (KChAP, [18]). Interestingly, the identified Pias1/3L and Ubc9 interacted exclusively with the mGluR8b isoform, but not with the mGluR8a counterpart.

To verify the protein-protein interactions observed in yeast cells, glutathione Sepharose beads were coated with mGluR8a/b C-termini. Because C-terminal domains of group III mGluRs are highly homologous, sequences of mGluR4, mGluR6 and mGluR7a/b were included in this study. Immobilized mGluR C-termini were then incubated with in-vitro translated proteins of the sumoylation machinery. The sumoylation cascade covalently attaches Sumo proteins to a lysine residue of the target protein. Therefore, we included Sumo1 and other related proteins not identified in the screens (Pias2 and Pias4) in our study. Analyzing interacting proteins on a Western Blot verified the interaction between Pias1 and Pias3L with the mGluR8b C-terminus, while the detected signal intensity for mGluR8a was extremely low (Fig. 2), consistent with the binding profile observed in yeast cells. Besides binding to the mGluR8b C-terminus, Pias1 and Pias3L also interacted with C-termini of mGluR7a/b and to a lower extend of mGluR4 and mGluR6. Unlike Pias1/3L, Pias2 and Pias4 did not bind to any of the mGluR C-termini tested (Fig. 2), consistent with the fact that these two E3-ligases were not detected in the initial yeast two-hybrid screens. Finally, Pc2 showed a relative strong interaction with mGluR7b, while binding intensities to C-termini of other group III mGluRs were much lower.

Compared to Pias1 and Pias3L, Ubc9 interacted only weakly with the mGluR8b C-terminus (Fig. 2). Indeed, often Ubc9 needs a Sumo E3-ligase for target recognition that localizes the Sumo1-Ubc9 dimer in vicinity to the target protein [START_REF] Wilkinson | Targets and Consequences of Protein SUMOylation in Neurons[END_REF]. Subsequently, Sumo1 is enzymatically transferred from Ubc9 onto a lysine residue of the target. This mechanism explains the observed weak binding of Ubc9 and the lack of interaction between Sumo1 and mGluR Ctermini in the absence of E2-and E3-enzymes (Fig. 2). Besides being covalently linked to target proteins, also non-covalent Sumo interaction motifs (SIMs) were reported [START_REF] Martin | Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction[END_REF]. However, none of the intracellular domains of group III mGluRs fulfil SIM consensus sequences, consistent with the lack of interaction between Sumo1 and mGluRs C-termini in our binding assays.

MGluR8b and proteins of the sumoylation machinery are co-expressed in the mammalian retina

A pre-requisite for protein-protein interactions under physiological conditions is their coexpression in the same cell. The retina is widely used as a model for the CNS because of its clear organization in distinct horizontal layers. The expression of mGluR8 isoforms in the mammalian retina has been shown previously [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF][START_REF] Koulen | Pre-and Postsynaptic Sites of Action of mGluR8a in the mammalian retina[END_REF][START_REF] Koulen | Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor[END_REF]. Therefore, we first analyzed which of the proteins tested in our binding assay are present in the retina. The E3-ligases Pias1-4 were clearly expressed in the retina, as well as Ubc9 and Sumo1 (Fig. 3A). In contrast, the Pc2 specific signal was extremely faint and thus this protein was excluded from further studies. In addition, we excluded Pias2 and Pias4 because they showed no interaction with the mGluR C-termini (see Fig. 2).

Next, we compared the localization of mGluR8b with the distribution of Pias1, Pias3L, Ubc9 and Sumo1 in the mammalian retina. MGluR8b specific antibodies detected a prominent expression of the glutamate receptor in somata located in the ganglion cell layer (GCL), as reported previously (Fig. 3B, asterisks; [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF]). In addition, cell bodies of the inner nuclear layer (INL) were labelled with lower intensity. MGluR8b specific signals visible as one horizontal strata in the upper half of the inner plexiform layer (IPL) have been shown before to colocalize with processes of cholinergic amacrine cells, mGluR2 and RanBPM [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF].

Staining retinal sections with immunesera specifically recognizing Pias1, Pias3L, Ubc9 and Sumo1 showed strong expression of the four proteins in cell bodies of the GCL (Fig. 3C, asterisks). This can be better seen in the enlargements of the boxed insets that were superimposed with the DAPI stains to label somata of retinal cells (Fig. 3C, right panels). Based on our data, we conclude that mGluR8b interacts with Pias1, Pias3L and Ubc9 and that all these proteins potentially involved in the sumoylation of mGluR8b are co-expressed in somata of the ganglion cell layer in the mammalian retina.

Sumoylation of mGluR8b at specific lysine residues is regulated by Pias1

In a next step we tested if mGluR8b is indeed sumoylated in-vivo. To analyze the function of PIAS proteins in this process, we concentrated our studies on the first member of this protein family, PIAS1. The complete coding sequence of mGluR8b was expressed in HEK-293 cells together with different combinations of T7-tagged Sumo1, Ubc9 and Pias1. First, we ensured that the transfected cDNAs for the T7-tagged constructs generated the desired proteins of the sumoylation machinery (Fig. 4B). Next, mGluR8b was purified from cell lysates by immuneprecipitation using an N-terminally fused c-myc tag. Subsequently, precipitated mGluR8b was analyzed on a Western Blot for the presence of receptor-bound Sumo1 using Sumo1 specific antibodies. Co-expression of mGluR8b with an irrelevant sequence revealed no detectable sumoylation of mGluR8b (Fig. 4C, Mock). In contrast, co-expressing mGluR8b together with Sumo1 resulted in a clear sumoylation of mGluR8b, indicating that the used HEK-293 cells contain endogenous enzymes needed for successful sumoylation of target proteins. Nevertheless, addition of Pias1 and Sumo1 to mGluR8b in a triple-transfection enhanced the sumoylation efficiency of mGluR8b, being consistent with the observed protein-protein interaction between Pias1 and mGluR8b (Fig. 4C). Further addition of Ubc9 did not significantly change mGluR8b sumoylation levels.

Protein sumoylation occurs preferably at lysine residues in target proteins being located in the consensus sequence ψ-K-X-[D/E] (ψ -large hydrophobic amino acid, X -any amino acid). Intracellular domains of mGluR8 contain several lysine residues, but only K882 in the mGluR8b C-terminus is located in a bona-fide consensus sequence for sumoylation (VKSE, Fig. 4A). Upon mutation of K882 into an arginine, mGluR8b sumoylation was drastically reduced, but not completely abolished (Fig. 4D). Therefore, we co-mutated a second lysine (K903) located in a motif highly similar to the sumoylation consensus sequence (VKSG, Fig. 4A). This motif is present in the distal isoform-specific region of the mGluR8b C-terminus and is not present in the mGluR8a variant. In the generated double-mutant the attachment of Sumo1 to the glutamate receptor was largely prevented (Fig. 4E). Importantly, as the wildtype mGluR8b, also both mutant receptor constructs were targeted to the cell surface of HEK-293 cells (Fig. 4F).

Structural analysis of Ubc9 in complex with mGluR8b

As shown above, the mGluR8b C-terminus uses two consensus sites for sumoylation -VKSE and VKSG. The VKSE site shares a high sequence similarity with the LKSE sumoylation site present in the Ran GTPase activating protein 1 (RanGAP1). In the RanGAP1-Ubc9 complex crystal structure [START_REF] Bernier-Villamor | Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitinconjugating enzyme Ubc9 and RanGAP1[END_REF], L525 interacts with a hydrophobic surface patch formed by residues P128, A129 and A131 of Ubc9 (Fig. 5A). Molecular modelling reveals that the valine present in mGluR8b can form similar hydrophobic interactions at the respective site and an energetic analysis suggests that the effect of this mutation on binding affinity is negligible. Thus, the VKSE site of mGlu8Rb represents a strong sumoylation site, consistent with our data.

The VKSG site of mGlu8Rb is characterized by an E to G replacement at the C-terminal end of the motif. In the RanGAP1-Ubc9 crystal structure the C-terminal glutamate forms two hydrogen bonds to S89 and T91 of Ubc9 (Fig. 5A). These interactions cannot be formed by a glycine (Fig. 5B). Two independent energetic analyses indicate that the E to G replacement will reduce the free binding energy by 1.2 to 1.6 kcal/mol, corresponding to an 8-to 15-fold weaker binding affinity.

DISCUSSION

The modulation of neurotransmitter receptors by post-translational modification represents a key mechanism in the regulation of synaptic signal transduction. In this study, we describe new protein-protein interactions between mGluR types and Sumo ligases. Sumoylation is an important cellular mechanism controlling a variety of cellular functions, most prominently gene transcription in the nucleus. Outside the nucleus, sumoylation regulates different cell functions including the transport of mRNA and proteins, synaptic excitability and proteinprotein interactions [START_REF] Wilkinson | Targets and Consequences of Protein SUMOylation in Neurons[END_REF]. These features define Sumo proteins as important players in neuronal function. In support of this view, a connection between sumoylation and neurodegeneration has been proposed recently [START_REF] Wilkinson | Targets and Consequences of Protein SUMOylation in Neurons[END_REF].

Besides the proteins involved in sumoylation and neddylation listed in Fig. 1B, our yeast twohybrid screen identified additional proteins [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF]. Of these, RanBPM (Ran binding protein in the microtubule-organizing centre) functions as an E3-ligase and sumoylates the immediateearly transcription factor Rta of the Epstein-Barr virus [START_REF] Chang | Enhancement of transactivation activity of Rta of Epstein-Barr virus by RanBPM[END_REF]. Filamin-A (FLNA) was described previously as an interactor of mGluR8a, but not of mGluR8b [START_REF] Enz | The actin-binding protein Filamin-A interacts with the metabotropic glutamate receptor type 7[END_REF]. Consistently, we identified FLNA 3 times in our screen against the mGluR8a C-terminal domain, but never as mGluR8b interactor.

In this study, we describe Pias1, Pias3L and Ubc9 as binding partners of mGluR8b. Transfection of mGluR8b without Sumo1 did not result in detectable mGluR8b sumoylation levels, consistent with a previous report [START_REF] Tang | Pias1 interaction and sumoylation of metabotropic glutamate receptor 8[END_REF] and indicating that the used HEK-293 cells contain only a small amount of unconjugated endogenous Sumo1. Nevertheless, the used cells contain a functional sumoylation machinery, because the sole addition of Sumo1 was sufficient for mGluR8b sumoylation. Still, sumoylation efficiency could be significantly increased upon co-expression of Pias1, supporting a functional interaction between both proteins. Within the mGluR8b C-terminus, lysines K882 and K903 were highly scored to be located in functional sumoylation motifs (VKSE: 0.93; VKSG: 0.76) and indeed, mutations into arginines drastically reduced the mGluR8b sumoylation efficiency. We predicted that the E to G replacement at position +4 of the sumoylation motif VKSG decreases the binding affinity of about one order of magnitude, which is moderate when compared to the divergence of three orders of magnitude observed in naturally occurring sumoylation sites [START_REF] Macauley | Beads-on-a-string, characterization of ETS-1 sumoylated within its flexible N-terminal sequence[END_REF]. Enzymatic sumoylation can occur at lysine residues present in non-canonical sumoylation motifs and the presence of a glycine at position +4 has been reported in several proteins (see supplementary Tab. 1 in [START_REF] Xue | SUMOsp: a web server for sumoylation site prediction[END_REF]). A c c e p t e d M a n u s c r i p t 8 K882 is located in the proximal part of the mGluR8b C-terminus, which is identical between both mGluR8 isoforms and highly conserved in mGluR7a and mGluR7b. While the observed binding of Pias1, Pias3 and Pc2 to mGluR7a/b in our pull-down assays is consistent with a potential sumoylation of these glutamate receptors, mGluR8a interaction with E3-ligases was extremely weak (Pias1, Pc2) or undetectable (Pias2, Pias3L, Pias4). Weak binding of Pias1 to mGluR8a, relative to mGluR8b, was described previously [START_REF] Tang | Pias1 interaction and sumoylation of metabotropic glutamate receptor 8[END_REF]. The study demonstrated sumoylation of K882 in the isolated C-terminus of mGluR8a. Although this indicates that faint signals observed in Fig. 2 might represent biological functions, it is not known if the complete mGluR8a protein inserted into the plasma membrane is sumoylated as well. However, because (i) the K882 containing sumoylation motif is identical between mGluR8a and mGluR8b, and because (ii) we observed Sumo1 conjugation to K882 in the context of the complete mGluR8b protein expressed in mammalian cells, we propose that both mGluR8 variants can be sumoylated in-vivo.

Other studies identified glutamate receptors as targets for Pias1. In an bacterial sumoylation assay, intracellular domains of ionotropic and metabotropic glutamate receptors were modified by Sumo1 [START_REF] Wilkinson | Analysis of SUMO-1 modification of neuronal proteins containing consensus SUMOylation motifs[END_REF]. The authors report sumoylation of isolated C-terminal domains of mGluR2, 4, 6, 7a/b and 8a in the absence of E3-ligases. While the mGluR8b C-terminus was not tested, the sumoylation profile of the other mGluR types is comparable to the mGluR binding profile for Pias1 and Pias3L shown in Fig. 2. Other transmembrane proteins reported to be sumoylated include the kainate receptor GluR6 [START_REF] Martin | SUMOylation regulates kainate-receptor-mediated synaptic transmission[END_REF], the cannabinoid receptor CB1 [START_REF] Gowran | Delta(9)-tetrahydrocannabinol regulates the p53 post-translational modifiers Murine double minute 2 and the Small Ubiquitin MOdifier protein in the rat brain[END_REF], the voltage-gated potassium channel Kv1.5 [START_REF] Benson | SUMO modification regulates inactivation of the voltage-gated potassium channel Kv1.5[END_REF] and the TGF-β receptor type I [START_REF] Kang | The type I TGF-beta receptor is covalently modified and regulated by sumoylation[END_REF].

In Pias1 knockout mice no neurological phenotype was reported, probably due to compensatory mechanisms by other Pias proteins [START_REF] Liu | PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity[END_REF]. Thus, exact functions of Pias1 interacting with and sumoylating mGluR8b remain elusive. Possible physiological consequences include internalization and/or degradation of the glutamate receptor, as interaction of group I mGluRs with the ubiquitin E3-ligase Siah1a leads to receptor degradation [START_REF] Moriyoshi | Seven in absentia homolog 1A mediates ubiquitination and degradation of group 1 metabotropic glutamate receptors[END_REF]. Intracellular C-termini of group III mGluR types do not contain preformed folds, but are rather unstructured and use short linear motifs containing three to ten successive residues for protein-protein interactions [START_REF] Seebahn | Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors[END_REF]. The accessibility of these short linear motifs might be modified or eliminated by sumoylation of the mGluR8b C-terminus which could influence mGluR8b coupled signal pathways. Indeed, examples for the generation of new binding surfaces in G-protein signalling of μ-opioid receptors and phosducin were reported [START_REF] Rodriguez-Munoz | Sumoylated RGS-Rz proteins act as scaffolds for Mu-opioid receptors and G-protein complexes in mouse brain[END_REF][START_REF] Klenk | SUMO-1 controls the protein stability and the biological function of phosducin[END_REF].

In the retina, mGluR8 isoforms were described at synapses of photoreceptors, bipolar and horizontal cells, as well as extra-synaptically on cell bodies of amacrine, ganglion and horizontal cells [START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF][START_REF] Koulen | Pre-and Postsynaptic Sites of Action of mGluR8a in the mammalian retina[END_REF]. In photoreceptor terminals, the pre-synaptically localized mGluR8a is part of a negative feed-back loop that regulates glutamate release of these neurons [START_REF] Koulen | Modulation of the intracellular calcium concentration in photoreceptor terminals by a presynaptic metabotropic glutamate receptor[END_REF]. Recently, it has been shown that sumoylation of pre-synaptic proteins can regulate glutamate release of neurons, although the identity of the sumoylated target proteins was not specified [START_REF] Feligioni | Protein SUMOylation modulates calcium influx and glutamate release from presynaptic terminals[END_REF]. One of these targets might be the C-terminal binding protein CtBP1. CtBP1 can be sumoylated and is present in pre-synaptic ribbons of photoreceptors [START_REF] Chinnadurai | Transcriptional regulation by C-terminal binding proteins[END_REF][START_REF] Tom Dieck | Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex[END_REF]. These organelles mediate the release of glutamate from photoreceptor terminals, a process that is regulated by mGluR8a. The sub-cellular localization of mGluR8b in photoreceptors has not been analyzed yet. Thus, a function of mGluR8b in glutamate release from photoreceptors is currently speculative. Alternatively, mechanisms similar to those described above might be present in neurons of the ganglion cell layer. Clearly, pre-synaptic proteins involved in neurotransmitter release are sumoylated, pointing towards a function of the sumoylation machinery in neuronal signal transduction. In addition, recent studies describe a Pias3 regulated sumoylation of transcription factors in photoreceptors, demonstrating additional functions for Sumo proteins in the CNS, such as the development of rods [START_REF] Onishi | Pias3dependent SUMOylation directs rod photoreceptor development[END_REF]. (B) T7 tagged proteins of the sumoylation machinery were expressed in HEK-293 cells and detected on Western Blots (arrowheads). (C) C-myc tagged mGluR8b was co-expressed in HEK-293 cells with different proteins combinations of the sumoylation machinery, as indicated. Immuneprecipitation was performed with c-myc specific antibodies and receptorbound Sumo1 was detected on Western Blots with an immuneserum against Sumo1 (upper panel: IP). Expression efficiencies of mGluR8b was compared in the lower panel (Input) using c-myc antibodies. MGluR8b appears as monomer and dimer (arrowheads), as reported before [START_REF] Ferraguti | Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus[END_REF]. (D, E) C-myc tagged mutant receptors were expressed and analyzed as in (C). (F) To ensure a comparable surface expression of wild-type and mutant receptors, their extracellular c-myc tag was labelled under non-permeabilizing conditions in transfected HEK-293 cells.

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854

BiochemicalFig. 1 :

 1 FIGURE LEGENDS Fig. 1: Proteins of the sumoylation machinery interact with mGluR C-termini. (A)Membrane topology of mGluRs. A grey box represents the membrane and transmembrane regions 1-7 are indicated by black rectangles. The intracellular C-terminus (bold) is alternatively spliced (triangle). (B) A subset of proteins identified in yeast two-hybrid screens against mGluR8a and mGluR8b C-termini as reported in[START_REF] Seebahn | RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors[END_REF].

Fig. 2 :

 2 Fig. 2: The Sumo E3-ligases Pias1 and Pias3L show high affinity for mGluR8b, but not for the mGluR8a isoform. GST or GST fused to C-terminal domains of group III mGluRs were immobilized on glutathione Sepharose and incubated with in-vitro translated Pias1-4, Pc2, Ubc9 or Sumo1. Protein-protein interactions were detected by radiography. Protein concentrations of coated beads are shown on Coomassie stained SDS-PAGE (bottom panel).

Fig. 3 :

 3 Fig. 3: Co-expression of mGluR8b and proteins of the sumoylation machinery in the retina. (A) Agarose gels showing PCR products amplified from reverse transcribed mRNA isolated from the rat retina. Calculated sizes are: Pias1: 662 bp, Pias2: 442 bp, Pias3L: 594 bp, Pias4: 277 bp, Pc2: 579 bp, Sumo1: 305 bp, Ubc9: 477 bp. (B) A vertical cryostat section of the retina stained with specific antibodies for mGluR8b shows prominent expression of the receptor in cell bodies of the GCL (red, asterisks). Retinal layers are indicated by labelling nuclei of cell bodies with DAPI (blue). Scale bar: 40 μm. (C) Retinal sections incubated with immunesera specific for Pias1, Pias3L, Ubc9 or Sumo1 (red) and DAPI (blue). Prominent expression of proteins in cell bodies of the GCL (asterisks) is evident from the enlargement of the boxed regions and their superimposure with the DAPI stainings (right panels).

Fig. 4 :

 4 Fig. 4: Pias1 enhances sumoylation of specific lysine residues in the mGluR8b Cterminus. (A)The C-terminal 39 amino acids of mGluR8b contain four lysine residues (asterisks) and a splice-site (triangle) that separates the proximal region being identical between mGluR8a and mGluR8b from the distal mGluR8b specific sequence. Sumoylation consensus sequences as identified by SUMOPlot are underlined, calculated sumoylation probabilities are shown by numbers and the two mutations analyzed are indicated by arrows. (B) T7 tagged proteins of the sumoylation machinery were expressed in HEK-293 cells and detected on Western Blots (arrowheads). (C) C-myc tagged mGluR8b was co-expressed in HEK-293 cells with different proteins combinations of the sumoylation machinery, as indicated. Immuneprecipitation was performed with c-myc specific antibodies and receptorbound Sumo1 was detected on Western Blots with an immuneserum against Sumo1 (upper panel: IP). Expression efficiencies of mGluR8b was compared in the lower panel (Input) using c-myc antibodies. MGluR8b appears as monomer and dimer (arrowheads), as reported before[START_REF] Ferraguti | Metabotropic glutamate receptor 8-expressing nerve terminals target subsets of GABAergic neurons in the hippocampus[END_REF]. (D, E) C-myc tagged mutant receptors were expressed and analyzed as in (C). (F) To ensure a comparable surface expression of wild-type and mutant receptors, their extracellular c-myc tag was labelled under non-permeabilizing conditions in transfected HEK-293 cells.

Fig. 5 :

 5 Fig. 5: Structural analysis of sumoylation sites in the mGluR8b C-terminus. Enzymesubstrate interactions formed by Ubc9 with the LKSE site present in RanGAP1 (A) and with the VKSG site of mGlu8Rb (B). The backbone of Ubc9 is indicated by a black tube. Interacting hydrophobic residues (P128, A129, A131) are shown as space-filled presentation, hydrogen bonded residues (S89, T91) are shown in stick presentation and hydrogen bonds between E528 and S89/T91are indicated as straight dotted lines. The residues of the Sumo1 conjugation site in the substrate (V902 -G905) are shown in stick presentation.

  Figure 3

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© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Mary Dasso for Pias plasmids, Francesco Ferraguti for the mGluR8b expression plasmid, Helene Boeuf and Ryuichi Shigemoto for Pias3L and mGluR8b specific antibodies and Johann Helmut Brandstätter for retinal cryostat sections.

FUNDING

This work was supported by the Deutsche Forschungsgemeinschaft (SFB539, SFB796, EN349/5-1 and 5-2). Biochemical Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101854 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101854

AUTHOR CONTRIBUTION

Nadja Schröder-Kreß performed pull-down assays, RT-PCR and retinal stainings, Eva Dütting performed sumoylation assays, Heinrich Sticht analyzed the mGluR8b/Ubc9 binding interface and Ralf Enz performed the initial yeast two-hybrid screen, designed the research and wrote the paper.