Nadeene Parker

Charles Affourtit

Antonio Vidal-Puig

Martin D Brand

Energisation-dependent endogenous activation of proton conductance in skeletal muscle mitochondria

Keywords: adenine nucleotide translocase, BSA, bovine serum albumin, FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone, TPMP, triphenylmethylphosphonium, UCP, uncoupling protein, KO, knockout Uncoupling protein, adenine nucleotide translocase, UCP3, GDP, carboxyatractylate, proton leak

Leak of protons into the mitochondrial matrix during substrate oxidation partially uncouples electron transport from phosphorylation of ADP, but the functions and source of basal and inducible proton leak in vivo remain controversial. We describe an endogenous activation of proton conductance in mitochondria isolated from rat and mouse skeletal muscle following addition of respiratory substrate. This endogenous activation increased with time, required a high membrane potential and was diminished by high concentrations of serum albumin. Inhibition of this endogenous activation by GDP (classically considered specific for uncoupling proteins, UCPs), carboxyatractylate and bongkrekate (considered specific for the adenine nucleotide translocase) was examined in skeletal muscle mitochondria from wild type and UCP3-knockout mice. Proton conductance through endogenously-activated UCP3 was calculated as the difference in leak between mitochondria from wild type and UCP3-knockout mice, and was found to be inhibited by carboxyatractylate and bongkrekate, but not GDP. Proton conductance in mitochondria from Ucp3 knockout mice was inhibited strongly by carboxyatractylate, bongkrekate and partially by GDP. We conclude the following. (1) at high protonmotive force, an endogenously-generated activator stimulates proton conductance catalysed partly by UCP3 and partly by the adenine nucleotide translocase.

(2) GDP is not a specific inhibitor of UCP3, but also inhibits proton translocation by the adenine nucleotide translocase. (3) The inhibition of UCP3 by carboxyatractylate and bongkrekate is likely to be indirect, acting through the adenine nucleotide translocase.

INTRODUCTION

During substrate oxidation, protons are pumped out of the mitochondrial matrix, creating an electrochemical proton gradient. This protonmotive force is used to drive ATP synthesis. However, substrate oxidation is not fully coupled to ATP synthesis; some of the electrochemical gradient is dissipated by protons leaking back into the matrix through natural proton conductance pathways across the mitochondrial inner membrane [START_REF] Brand | The proton leak across the mitochondrial inner membrane[END_REF][START_REF] Brand | The causes and functions of mitochondrial proton leak[END_REF]. Mitochondrial proton leak may cause up to 25% of resting metabolic rate [START_REF] Rolfe | Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate[END_REF][START_REF] Rolfe | The physiological significance of mitochondrial proton leak in animal cells and tissues[END_REF] and may still be significant when ATP turnover is high [START_REF] Rolfe | Contribution of mitochondrial proton leak to respiration rate in working skeletal muscle and liver and to SMR[END_REF]. The prominence of mitochondrial proton leak suggests that it is physiologically important, and several roles have been suggested [START_REF] Rolfe | The physiological significance of mitochondrial proton leak in animal cells and tissues[END_REF], including protection against mitochondrial free radical production [START_REF] Echtay | Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants[END_REF] and regulation of insulin secretion in pancreatic beta cells [START_REF] Affourtit | Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion[END_REF].

Proton leak occurs through several pathways, some basal and some inducible. Membranes are themselves intrinsically leaky to protons [START_REF] Dilger | The dielectric constant of phospholipid bilayers and the permeability of membranes to ions[END_REF] but proteins embedded in a bilayer can greatly increase permeability [START_REF] Rolfe | The physiological significance of mitochondrial proton leak in animal cells and tissues[END_REF][START_REF] Brookes | The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria[END_REF][START_REF] Brown | On the nature of the mitochondrial proton leak[END_REF]. Half to two-thirds of the basal mitochondrial proton conductance is dependent upon the most abundant carrier protein in the mitochondrial inner membrane, the adenine nucleotide translocase (ANT) [START_REF] Brand | The basal proton conductance of mitochondria depends on adenine nucleotide translocase content[END_REF]. ANTdependent basal proton conductance occurs even if the carrier is inhibited by carboxyatractylate, showing that this leak pathway does not require normal carrier function [START_REF] Brand | The basal proton conductance of mitochondria depends on adenine nucleotide translocase content[END_REF].

Unlike basal proton conductance, specific activators and inhibitors affect inducible proton conductance. Inducible conductance can be catalysed by ANT in the presence of fatty acids [START_REF] Andreyev | The ATP/ADPantiporter is involved in the uncoupling effect of fatty acids on mitochondria[END_REF][START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF], AMP [14] or the lipid peroxidation product hydroxynonenal [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. This pathway is inhibited by carboxyatractylate, bongkrekate and by the ANT substrates, ATP or ADP. It is not known to be sensitive to other nucleotides. Other mitochondrial carriers, such as the glutamate [START_REF] Samartsev | Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria[END_REF] and phosphate carriers [START_REF] Engstova | Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier[END_REF], will catalyse proton conductance in the presence of fatty acids. Specific uncoupling proteins (UCPs) also catalyse inducible proton conductance [START_REF] Echtay | Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage[END_REF][START_REF] Brand | Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3[END_REF][20][START_REF] Brand | Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins[END_REF]. The best understood is UCP1, found primarily in brown adipose tissue and specialised for adaptive thermogenesis. Proton conductance through UCP1 is activated by fatty acids [START_REF] Rafael | Mitochondria from brown adipose tissue: uncoupling of respiratory chain phosphorylation by long fatty acids and recoupling by guanosine triphosphate[END_REF], superoxide [START_REF] Echtay | Superoxide activates mitochondrial uncoupling proteins[END_REF], derivatives such as hydroxynonenal [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]24], and hydroxynonenal analogues (such as retinoic acid [START_REF] Rial | Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2[END_REF]). UCP2, found in a variety of tissues [START_REF] Fleury | Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia[END_REF], and UCP3, found primarily in skeletal muscle [START_REF] Boss | Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression[END_REF]28], can be activated by fatty acids [29,[START_REF] Jaburek | Transport function and regulation of mitochondrial uncoupling proteins 2 and 3[END_REF], superoxide [START_REF] Echtay | Superoxide activates mitochondrial uncoupling proteins[END_REF] and hydroxynonenal and its analogues [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF].

A striking aspect of the UCPs is the inhibition of their proton conductance by a broad range of purine nucleotides. Unlike the proton conductance of the adenine nucleotide carrier, UCPs are strongly inhibited not only by ATP and ADP, but also by GTP and GDP [14]. This difference in specificity has led to widespread use of GDP as a diagnostic inhibitor of UCP function in vitro (e.g. [START_REF] Dejean | Uncoupling protein-3 sensitizes cells to mitochondrial-dependent stimulus of apoptosis[END_REF][START_REF] Nadtochiy | Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection[END_REF][START_REF] Carroll | Identification of a functioning mitochondrial uncoupling protein 1 in thymus[END_REF][START_REF] Ruzicka | Recruitment of mitochondrial uncoupling protein UCP2 after lipopolysaccharide induction[END_REF]), particularly in systems that lack a UCP-ablated control [START_REF] Fridell | Activation of alternative oxidase and uncoupling protein lowers hydrogen peroxide formation in amoeba Acanthamoeba castellanii mitochondria[END_REF][36][START_REF] Talbot | Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins[END_REF][START_REF] Hourton-Cabassa | Alteration of plant mitochondrial proton conductance by free fatty acids. Uncoupling protein involvement[END_REF].

Here we report an endogenous increase in the proton conductance of skeletal muscle mitochondria incubated at high membrane potential. It is catalysed by UCP3 and ANT. Although it is inhibited by GDP and carboxyatractylate, these inhibitors do not show the expected classical specificity.

4

EXPERIMENTAL

Animals

Female Wistar rats (Charles River) and male or female mice were housed at 21 ± 2 o C, 57 ± 5% humidity, 12/12 h light/dark, with standard laboratory chow and water ad libitum, following UK Home Office Guidelines for the Care and Use of Laboratory Animals. Ucp3 knockout (Ucp3KO) [START_REF] Gong | Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3[END_REF] and wild type (WT) sibling paired mice were used at age 12 -15 weeks. Ucp3 ablation was confirmed by PCR analysis of genomic Ucp3 and Western blot in skeletal muscle mitochondria.

Mitochondria

Animals (1 rat or 4 mice per preparation) were killed by stunning and cervical dislocation. Mitochondria were isolated from total hind limb skeletal muscle [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. Mitochondria from WT and Ucp3KO mice were prepared and assayed in parallel.

Proton leak kinetics

Proton leak rates across the inner membrane of isolated mitochondria were measured as the oxygen consumption required to pump the protons back out of the mitochondrial matrix (in the presence of oligomycin to prevent ATP synthesis) [START_REF] Rolfe | Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat[END_REF]. Proton leak kinetics were measured as the dependence of proton leak rate on its driving force, protonmotive force (measured as membrane potential in the presence of nigericin to abolish pH gradients). Respiration rate and membrane potential were measured simultaneously using electrodes sensitive to oxygen or the membrane potential-dependent probe, triphenylmethylphosphonium (TPMP + ). Assay medium contained 0.35 mg/ml mitochondrial protein, 120 mM KCl, 5 mM KH 2 PO 4 , 3 mM Hepes, 1 mM EGTA, 0.3 % w/v defatted bovine serum albumin (unless otherwise stated), 5 µM rotenone, 1 µg/ml oligomycin and 0.11 µM nigericin. The TPMP electrode was calibrated with sequential additions of 0.5 µM TPMP up to 2.5 µM and the mitochondria were energised with 4 mM succinate. Leak kinetics were measured by varying oxygen consumption and membrane potential by sequential additions of malonate (about 0.1 -2.3 mM). After each run, 0.2 µM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to release matrix TPMP, allowing correction for any small baseline drift. The TPMP binding correction was taken to be 0.35 (µl/mg protein) -1 [START_REF] Brand | Measurement of mitochondrial protonmotive force[END_REF].

Measurement of ANT content by carboxyatractylate titre

Respiration was measured without oligomycin or nigericin. 1 mM ADP was added to establish the maximum phosphorylating rate, and then respiration was successively inhibited by additions of carboxyatractylate (Calbiochem) until the resting nonphosphorylating rate was re-established. Carboxyatractylate binds ANT very tightly, so ANT content was interpolated as the minimum amount of carboxyatractylate required to abolish phosphorylating respiration [START_REF] Brand | The basal proton conductance of mitochondria depends on adenine nucleotide translocase content[END_REF][START_REF] Streicher-Scott | Use of carboxyatractylate and tight-binding inhibitor theory to determine the concentration of functional mitochondrial adenine nucleotide translocators in a reconstituted system[END_REF].

Statistics

To test for differences in proton conductance by ANOVA or paired Student's t tests, we calculated respiration rates (by linear interpolation between flanking points) at the highest

RESULTS

Time-dependent endogenous activation of proton conductance in energised rat skeletal muscle mitochondria Preliminary investigations of proton leak kinetics in skeletal muscle mitochondria suggested that proton conductance increased as the time of energisation with succinate was extended. Fig. 1 characterises this endogenous activation of proton conductance. Mitochondria were incubated in assay medium for a total of 8 min (Fig. 1A). They were energised with succinate for the last 0.75, 2.5 or 5.0 min, then proton leak kinetics were measured. Fig. 1B shows that respiration driving proton leak increased as incubation with succinate was extended. To allow comparison of proton leak rates at the same driving force, Fig. 1C shows rates interpolated at 175 mV, the highest common membrane potential in the experiment, indicated by the vertical line. The proton leak rate at 175 mV measured after 5 min with succinate was significantly higher than that measured after 0.75 min with succinate. The total time in the assay was always 8 min, therefore endogenous proton conductance increases as the time of incubation with succinate is extended.

Leak kinetics are measured by titrating membrane potential and respiration rate with malonate over three minutes. If time with succinate is important in inducing proton leak, endogenous activation should continue during the malonate titration, causing later points to be more uncoupled than earlier points. To test this, Fig. 1D shows a compilation of leak kinetics measured in five separate experiments, each with a different concentration of malonate added at t = 0. Time with succinate was the same for all titration points (dashed line). This is overlaid on the leak kinetics measured during one standard experiment using five sequential additions of malonate (as in Fig. 1B), where time with succinate differed between titration points (solid line). There was no difference in leak kinetics between the two protocols. Therefore energisation-dependent uncoupling does not increase over the three minutes taken to measure the proton leak kinetics suggesting no further increases after the first malonate addition.

Malonate attenuates the energisation-dependent increase in proton conductance by lowering membrane potential in rat skeletal muscle mitochondria.

To test the ability of malonate to blunt energisation-dependent, endogenous activation of proton leak, 430 µM malonate was added immediately before succinate. At t = 0, after incubation with succinate for 1.5 min, this concentration of malonate lowered the membrane potential by about 12 mV (X in Fig. 2). Succinate was added 1.5 min or 5.0 min before the leak titration, which was completed as normal with sequential additions of malonate (Fig. 2B). Fig. 2A shows the control without malonate pre-addition.

To allow comparison of proton leak rates at the same driving force, Fig. 2C shows the time-dependent increases in rates with succinate, interpolated at the highest common membrane potential, 169 mV. The leak induced between 1.5 and 5 min was significantly Pre-addition of malonate could inhibit energisation-dependent induced leak directly, or by lowering protonmotive force, or by oxidising the electron transport chain. To test these possibilities, the experiment in Fig. 2C was repeated using the complex IV inhibitor, KCN, at a concentration that lowers the membrane potential to the same extent as 430 µM malonate. Leak kinetics were measured in the absence (Fig. 2D) or presence (Fig. 2E) of 43 µM KCN. At the highest common membrane potential, proton conductance induced by succinate between 1.5 and 5 min in the presence of 43 µM KCN (Fig. 2F) was significantly lower than in its absence. Unlike malonate, KCN reduces electron transport chain intermediates. The two inhibitors decreased energisationdependent proton conductance to a similar extent, suggesting that it is high membrane potential, not a particular redox state, that is required for energisation-dependent induction of proton conductance.

The increase in proton conductance between 1.5 and 5 min incubation with succinate was also measured at intermediate concentrations of pre-added malonate or KCN. This produced intermediate drops in membrane potential at t = 0 following 1.5 min with succinate (equivalent to X in Fig. 2B and Fig. 2E). To characterise the ability of decreased membrane potential to blunt energisation-dependent induction of proton conductance, Fig. 2G shows the increase in leak rate (e.g. Fig. 2C and Fig. 2F) plotted against the drop (X) in membrane potential caused by different concentrations of preadded malonate or KCN. There was a linear decrease in activation as membrane potential during the activatory period was decreased. The linear regressions for malonate and KCN overlap suggesting a conserved mechanism of inhibition through the lowering of membrane potential. Extrapolation to zero effect suggests that a decrease of 20 mV is sufficient to prevent activation of proton conductance.

Therefore, the endogenous energisation-dependent activation of proton conductance following addition of succinate requires a high membrane potential. Activation increases over 5 min with succinate but during subsequent titrations with malonate the membrane potential falls by more than 20 mV and further activation is prevented.

What is the activator of energisation-dependent increase in proton conductance in rat skeletal muscle mitochondria?

Bovine serum albumin (BSA) removes putative endogenous activators, including fatty acids [43] and reactive oxygen species [START_REF] Echtay | Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants[END_REF] and their derivatives such as reactive alkenals [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF]. To test whether these endogenous activators were involved in the observed energisation-dependent proton leak, the concentration of BSA was increased in the standard assay medium from 0.3% to 1% w/v (Fig. 3A-C). Proton leak kinetics were measured after energisation with succinate for 1.5 min or 5.0 min in the presence of 0.3% (Fig. 3A) or 1% (Fig. 3B) BSA. At 172 mV, leak induced between 1.5 and 5 min in assay medium containing 1% BSA was significantly lower than in 0.3% BSA (Fig. 3C), showing that BSA blunts energisation-dependent activation of proton conductance and suggesting that an endogenous activator that is quenched by BSA mediates the induced leak. To test the possibility that the increase in energisation-dependent leak is due to lipid peroxidation products, phenylbutylnitrone, a chain-breaking spin trap that quenches carbon-centred radical intermediates, was pre-added to the assay medium at concentrations known to inhibit lipid peroxidation [START_REF] Murphy | Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone[END_REF]. Proton leak kinetics were measured after energisation with succinate for 1.5 or 5.0 min in the absence (Fig. 3D) or presence (Fig. 3E) of phenylbutylnitrone. At 169 mV, there was no significant effect of phenylbutylnitrone on leak induced between 1.5 and 5 min (Fig. 3F), suggesting that pathways that generate lipid peroxidation products through a carbon-centred radical sensitive to phenylbutylnitrone are not involved in the energisation-dependent uncoupling. However, other reactive oxygen species-related activators remain candidates.

Energisation might either generate a pool of activator, or allow activation of uncoupling using a pre-existing activator pool (e.g. through an energisation-dependent change in conformation of an uncoupling protein). To distinguish between these alternatives, we tested the effect of pre-addition of fatty acids in medium containing 1% BSA. Proton leak kinetics were measured in 1% BSA in the absence (Fig. 3G) or presence (Fig. 3H) of 600 µM palmitate after energisation with succinate for 1.5 or 5.0 min. Fig. 3I replicates the observation in Fig. 3C that 1% BSA alone prevented timedependent activation. Pre-addition of palmitate did not overcome the inhibitory effect of 1% BSA, showing that time-dependent activation did not occur by potential-dependent changes in protein conformation that then allowed activation by a pre-existing pool of fatty acids. Fatty acids remain, however, one candidate for the endogenously-generated activator, since proton leak kinetics in the presence of palmitate (Fig. 3H) were the same after 1.5 min incubation with succinate as those seen in 0.3 % BSA after 5 min with succinate (Fig. 3A).

These experiments are consistent with at least three different interpretations. The endogenous activator may be (a) fatty acids, or (b) some other molecule, generated during 5 min incubation at high membrane potential, or (c) some molecule (e.g. hydroxynonenal) that is already present in the mitochondria before addition of succinate, but requires membrane potential to be high for several minutes to allow the interaction that causes uncoupling.

Which proteins catalyse the energisation-dependent increase in proton conductance in rat skeletal muscle mitochondria?

Uncoupling through the adenine nucleotide translocase is inhibited by carboxyatractylate [START_REF] Andreyev | Carboxyatractylate inhibits the uncoupling effect of free fatty acids[END_REF], whereas uncoupling through UCPs is classically inhibited by GDP [14,[START_REF] Nicholls | Hamster brown-adipose-tissue mitochondria. Purine nucleotide control of the ion conductance of the inner membrane, the nature of the nucleotide binding site[END_REF]. Proton leak kinetics were measured after energisation with succinate for 2.5 min in the absence and presence of carboxyatractylate and GDP to determine whether ANT and UCP3 were involved (Fig. 4A). Fig. 4B shows that at the highest common membrane potential, 180 mV, GDP and carboxyatractylate significantly inhibited proton conductance separately and in combination. The extent of the carboxyatractylate inhibition of proton conductance suggests that the majority is attributable to ANT. As observed previously [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF], inhibition of proton conductance by GDP and carboxyatractylate was non-additive; characterisation of the relative contributions of ANT and UCP3 therefore required further experiments. The contribution of UCP3 to energisation-dependent proton conductance was assessed using skeletal muscle mitochondria prepared from WT and Ucp3KO mice. Proton leak kinetics were measured after energisation with succinate for 1.5 or 5.0 min. Fig. 5A shows that skeletal muscle mitochondria from WT mice showed a similar time-dependent increase in proton leak after addition of succinate to mitochondria from rat (Fig. 1B). Fig. 5B shows the same experiment performed using skeletal muscle mitochondria from Ucp3KO mice. There was still a difference between the curves, but it was smaller than in WT. The difference is quantified in Fig. 5C, which shows proton leak through UCP3, expressed as the difference between rates in skeletal muscle mitochondria from WT and Ucp3KO mice at 169 mV. UCP3-associated proton conductance was significantly greater after incubation with succinate for 5.0 min than for 1.5 min. In the presence of UCP3, the increase in respiration rate between 1.5 and 5 min was 75 nmol/min/mg protein while in the absence of UCP3 it was 62 nmol/min/mg protein. Thus about 17% of the energisation-dependent proton leak induced between 1.5 and 5 min incubation with succinate requires the presence of UCP3.

UCP3 and ANT each catalyse part of the energisation-dependent increase in proton conductance in mouse skeletal muscle mitochondria

The contribution of ANT to the proton conductance observed after 5 min incubation with succinate was assessed as the carboxyatractylate-sensitive effect in skeletal muscle mitochondria from Ucp3KO mice. Fig. 6B shows that carboxyatractylate substantially inhibited energisation-dependent proton conductance in the absence of UCP3. The effect is quantified in Fig. 6D, which shows UCP3-independent leak at 161 mV. Carboxyatractylate lowered the rate from 90 to 30 nmol O/min/mg protein, equivalent to about 50-55% of the total effect in mitochondria from WT mice in Fig. 6A. Together, these results show that the energisation-dependent proton conductance in mouse skeletal muscle mitochondria is catalysed about 15-20% by UCP3, 50-55% by ANT, and 25-30% by other pathways not sensitive to GDP or carboxyatractylate (e.g. the dicarboxylate carrier).

Specificity and lack of additivity of inhibition by GDP and carboxyatractylate in mouse skeletal muscle mitochondria

To investigate the lack of additivity between the inhibitors GDP and carboxyatractylate in rat skeletal muscle (Fig. 4), and also seen in other studies [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF], inhibition was measured in skeletal muscle mitochondria from WT and Ucp3KO mice. The same lack of additivity was seen in skeletal muscle mitochondria from WT mice (Fig. 6A) as in rat: GDP partially inhibited proton conductance in the absence of carboxyatractylate but caused no further inhibition in the presence of carboxyatractylate. Fig. 6B shows the same effect in mitochondria from Ucp3KO mice.

Figs 6C and 6D dissect out the effects of the inhibitors. Fig. 6C represents the contribution of UCP3 (Fig. 6A minus 6B) at the highest common membrane potential, 161 mV, after 5 min incubation with succinate. Surprisingly (since GDP is the classical inhibitor of UCPs), the proton conductance through UCP3 under these conditions was insensitive to GDP. More surprisingly (since carboxyatractylate is thought to be a highly specific inhibitor of ANT), it was almost fully sensitive to carboxyatractylate. This sensitivity of UCP3 to carboxyatractylate partially explains the lack of additivity of the two inhibitors: once UCP3 is inhibited by carboxyatractylate, any inhibition by GDP would have no further effect. Fig. 6D represents the contribution of ANT and other pathways (data from Fig. 6B). The energisation-dependent proton conductance of mitochondria from Ucp3KO mice was significantly lower in the presence of GDP, indicating that an endogenously activated pathway other than UCP3 was inhibited by GDP. Since this conductance was also strongly inhibited by carboxyatractylate, it was catalysed by ANT. This experiment shows that GDP is not specific for UCPs, but can also inhibit proton conductance through other pathways, specifically ANT. It completes the explanation of the lack of additivity of GDP and carboxyatractylate: once ANT is inhibited by carboxyatractylate, inhibition by GDP would have no further effect. Together, these results highlight potential promiscuity of the inhibitors GDP and carboxyatractylate: neither is specific for its classical target in our experiments.

A possible alternative explanation of the sensitivity of mitochondria from Ucp3KO mice to GDP is that the Ucp3KO targeting vector upregulates Ucp2 expression, due to proximity of the Ucp2 and Ucp3 genes [START_REF] Solanes | The human uncoupling protein-3 gene. Genomic structure, chromosomal localization, and genetic basis for short and long form transcripts[END_REF], and it is this UCP2 that is inhibited by GDP. We detected Ucp2 transcript in total hind limb skeletal muscle using real-time PCR, but at levels 30 times lower than in spleen or lung, with no significant difference between WT and Ucp3KO (data not shown). UCP2 was undetectable by Western blot using skeletal muscle mitochondria (20 µg protein) from either WT or Ucp3KO mice. The antibody can detect 125 pg affinity-purified recombinant UCP2, so UCP2 content is below 6 ng/mg mitochondrial protein, less than 5% of the UCP3 content of 140 ng/mg protein in mouse skeletal muscle mitochondria [START_REF] Harper | Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria[END_REF]. UCP1 is not expressed in skeletal muscle of WT or Ucp3KO mice [48]. Therefore, the putative presence of UCP1 or UCP2 does not explain the sensitivity of proton conductance in skeletal muscle mitochondria from Ucp3KO mice to GDP.

A possible alternative explanation of the decreased effect of carboxyatractylate in mitochondria from Ucp3KO mice is that ablation of UCP3 somehow decreased ANT content. We tested this possibility by titrating adenine nucleotide translocase activity with carboxyatractylate in skeletal muscle mitochondria from WT and Ucp3KO mice. UCP3 (4 pmol/mg mitochondrial protein [START_REF] Harper | Artifactual uncoupling by uncoupling protein 3 in yeast mitochondria at the concentrations found in mouse and rat skeletal-muscle mitochondria[END_REF]) is about 0.2% as abundant as ANT (2 nmol/mg [START_REF] Brand | The basal proton conductance of mitochondria depends on adenine nucleotide translocase content[END_REF]), so any sequestering of carboxyatractylate by UCP3 would be negligible. ANT content was not significantly different (WT, 1.52; Ucp3KO, 1.56 nmol/mg protein, Fig. 6E), eliminating this explanation.

Inhibition of UCP3 by carboxyatractylate could be direct or through ANT. To examine this, we tested the effects of an alternative ANT inhibitor, bongkrekate. Unlike carboxyatractylate, which binds to the C conformation of the carrier, bongkrekate binds to the M conformation [START_REF] Kramer | Reconstitution of adenine nucleotide transport from beef heart mitochondria[END_REF]. BSA was omitted from the assay medium because it binds bongkrekate. Proton leak kinetics were measured in skeletal muscle mitochondria isolated from WT (Fig. 7A) and Ucp3KO (Fig. 7B) mice after energisation with succinate for 5.0 min in the absence or presence of bongkrekate or carboxyatractylate. Bongkrekate partially inhibited the energisation-dependent proton conductance in both cases. Fig. 7C shows the effects that were dependent on UCP3 (Fig. 7A minus Fig. 7B). At the highest common membrane potential, 158 mV, both carboxyatractylate and bongkrekate inhibited proton conductance through UCP3. It is unlikely that UCP3 and ANT share two different high-affinity inhibitor sites, therefore both carboxyatractylate and bongkrekate act on the UCP3-associated leak indirectly through primary effects on ANT. . Whatever the activator and mechanism, energisation-dependent uncoupling of skeletal muscle mitochondria may be important in limiting membrane potential during rest, to limit reactive oxygen species production [START_REF] Brand | Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins[END_REF] or prevent dielectric breakdown of the membrane [START_REF] Rolfe | The physiological significance of mitochondrial proton leak in animal cells and tissues[END_REF]. Two catalysts of energisation-dependent proton conductance were found to be UCP3 and ANT. In WT mouse skeletal muscle mitochondria after 5 min incubation with succinate, the relative contributions of UCP3, ANT and other pathways to proton conductance were 15-20%, 50-55% and 25-30% respectively. Since UCP3 content is only about 0.2% of ANT content (see above), this result demonstrates that the specific proton flux through UCP3 is 100-200 times greater than that through ANT. The greater specific activity of UCP3 may allow finer control of high membrane potential in tissues that contain this protein in addition to ANT.

We found that GDP partially inhibited the contribution of ANT to energisationdependent proton conductance. Khailova et al [START_REF] Khailova | Participation of ATP/ADP antiporter in oleateand oleate hydroperoxide-induced uncoupling suppressed by GDP and carboxyatractylate[END_REF] also concluded that GDP inhibits proton translocation by ANT, although they did not use Ucp knockout controls, which provide our most compelling evidence that GDP can inhibit proton conductance in the absence of UCP3. GDP and GTP bind weakly to ANT [START_REF] Huber | Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy[END_REF], but compete poorly with ADP and ATP, so they are not regarded as inhibitors of adenine nucleotide exchange. However, adenine nucleotides are absent in assays of proton conductance, which may allow guanine nucleotides to bind and partially inhibit ANT-catalysed proton leak.

We found that carboxyatractylate inhibited the contribution of UCP3 to proton leak. Carboxyatractylate is a high-affinity inhibitor of ANT, and has not been shown previously to act on other proteins. However, most other studies have not looked for carboxyatractylate inhibition of UCP3 function. The observation that bongkrekate also inhibits UCP3-dependent proton conductance suggests that inhibition of UCP3 is indirect, via primary effects on ANT. It is unclear what these indirect effects might be, but they might involve protein-protein interactions between ANT and UCP3, or interactions through some other protein, lipid or small molecule. More exploration of the interaction is required to uncover the mechanism. However, the (indirect) inhibition of UCP3 by carboxyatractylate fully explains the hitherto puzzling lack of additivity between inhibition of proton conductance by GDP and carboxyatractylate.

Since inhibition by GDP and carboxyatractylate is also non-additive in mitochondria from kidney [START_REF] Echtay | A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling[END_REF][START_REF] Murphy | Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone[END_REF], carboxyatractylate may also inhibit UCP2 by a However, it appears that carboxyatractylate does not inhibit potato UCP [START_REF] Smith | Activation and function of mitochondrial uncoupling protein in plants[END_REF] or mammalian UCP1, since guanine nucleotides are still inhibitory in the presence of carboxyatractylate in mitochondria from potato or mammalian brown adipose tissue. Thus caution should be shown when using sensitivity to GDP or carboxyatractylate as diagnostic of the involvement of a UCP or ANT in proton conductance. Observations of GDP-or carboxyatractylate-sensitivity should be verified by independent assays of the involvement of UCPs and ANT, such as sensitivity to genetic ablation or over-expression.

13

Energisation-dependent proton conductance of rat skeletal muscle mitochondria Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at t = -8 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between t = 0 and t = 3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2. 1A). Proton leak kinetics were measured between t = 0 and t = 3 min after no treatment (A, D) or addition of 430 µM malonate (B) or 43 µM KCN (E) immediately prior to addition of succinate. (C, F), inhibition of energisation-dependent proton leak by malonate and KCN. The increase in proton conductance at t = 0 caused by incubation with succinate between -5.0 min and -1.5 min was interpolated at the highest common membrane potential, 169 mV (solid line (A,B)) in (C) and 170 mV (solid line (D,E)) in (F). 'X' indicates the drop in membrane potential measured at t = 0 caused by addition of malonate or KCN prior to addition of succinate at t = -1.5. (G), effect of this drop in membrane potential on energisation-dependent increase in proton conductance. The increases in proton leak rate from (C) and additional experiments (not shown) using 140 µM and 290 µM malonate (circles), and from (F) and additional experiments (not shown) using or 14 µM and 29 µM KCN (triangles) are plotted against the drop ('X') in membrane potential at t = 0 after succinate addition at t = -1. 1A). Proton leak kinetics were measured between t = 0 and t = 3 min in medium containing 0.3% (A,D) or 1% w/v BSA (B,G,H), after no pre-treatment or pre-treatment with 0.1 mM (triangles) or 1 mM phenylbutylnitrone (diamonds) (E) or 600 µM palmitate (H). (C,F,I), effect on energisation-dependent proton leak of 1% BSA, phenylbutylnitrone or palmitate. The increase in proton conductance at t = 0 caused by incubation with succinate between -5 and -1.

Figure 4

Inhibition of proton conductance in rat skeletal muscle mitochondria by GDP and carboxyatractylate (A) Proton leak kinetics measured between t = 0 and t = 3 min following addition of inhibitors (where present) and mitochondria at t = -5.5 min. 4 mM succinate was added at t = -2.5 min (see Fig. 1A) in the presence of no additions (squares), 500 µM GDP (triangles), 2.5 µM carboxyatractylate (diamonds) or GDP and carboxyatractylate (circles).

Biochemical

  Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N T membrane potential common to all conditions on a given day. Means for each condition (with S.E.M.) are plotted in bar graphs, with the mean value of the highest common potential indicated.

Biochemical

  Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N T lower when 430 µM malonate was present, therefore malonate does indeed attenuate the energisation-dependent increase in proton conductance.

  Biochemical Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N TDISCUSSIONWe describe an endogenous activation of the proton conductance of skeletal muscle mitochondria isolated from both rats and mice that increases with time after energisation. This effect is not dependent upon redox state but requires a high membrane potential upon succinate addition. The effect of membrane potential may be to increase the production of endogenous fatty acids or other molecules that progressively activate uncoupling, or to sensitise target proteins to the effects of generated or pre-existing activators such as products of reactive oxygen species. The mechanism remains unclear. Membrane potential might change the conformation of membrane lipids to facilitate access by phospholipases, or might change the conformations of UCP3 and ANT to expose activator-binding sites. Mobilisation of fatty acids could explain the lack of GDP sensitivity of the UCP3-dependent proton conductance, by analogy with UCP1, where fatty acids functionally compete with inhibitory nucleotides [50]

Biochemical

  Journal Immediate Publication. Published on 06 Feb 2008 as manuscript BJ20080006 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080006 S t a g e 2 ( a ) P O S T -P R I N T similar indirect mechanism.

  Figure 1Energisation-dependent proton conductance of rat skeletal muscle mitochondria Proton leak kinetics were measured in rat skeletal muscle mitochondria as described in the experimental section. (A) Timings of additions to the incubation chamber. Mitochondria plus inhibitors (M) were added at t = -8 min, 4 mM succinate (S) was added at the times indicated, then leak kinetics (LK) were measured between t = 0 and t = 3 min by titration of membrane potential and oxygen consumption rate with malonate from 0.14 to 2.3 mM. (B) Kinetic response of mitochondrial proton leak rate (measured as oxygen consumption rate) to membrane potential measured after energisation with 4 mM succinate for 0.75 (open squares), 2.5 (grey squares) or 5.0 min (black squares). (C) Proton leak rate at 175 mV (solid line (B)); the average highest protonmotive force common to all curves generated in the experiment. (D) Comparison of proton leak curves generated either in a single experiment using sequential malonate additions over 3 min (S at t = -5, leak kinetics measured between t = 0 and t = 3 min; squares) or in five separate experiments each with a different concentration of malonate added at t = 0 (S at t = -5; 0.14 to 2.3 mM malonate added at t = 0, leak kinetics measured between t = 0 and t = 0.5 min; triangles). Data are means ± S.E.M. of duplicate experiments performed on four (B-C) or three (D) separate preparations. One-way ANOVA was performed for (C); p = 0.0034, with Dunnett's multiple comparison test used posthoc comparing other bars to 0.75 min: **significantly different: p < 0.01.

Figure 2

 2 Figure 2Sensitivity of energisation-dependent proton conductance in rat skeletal muscle mitochondria to membrane potential (A-C), effect of malonate. (D-F), effect of KCN. (A,B,D,E), proton leak kinetics. 4 mM succinate was added at t = -1.5 min (open symbols) or t = -5 min (closed symbols) (see Fig.1A). Proton leak kinetics were measured between t = 0 and t = 3 min after no treatment (A, D) or addition of 430 µM malonate (B) or 43 µM KCN (E) immediately prior to addition of succinate. (C, F), inhibition of energisation-dependent proton leak by malonate and KCN. The increase in proton conductance at t = 0 caused by incubation with succinate between -5.0 min and -1.5 min was interpolated at the highest common membrane potential, 169 mV (solid line (A,B)) in (C) and 170 mV (solid line (D,E)) in (F). 'X' indicates the drop in membrane potential measured at t = 0 caused by addition of malonate or KCN prior to addition of succinate at t = -1.5. (G), effect of this drop in membrane potential on energisation-dependent increase in proton conductance. The increases in proton leak rate from (C) and additional experiments (not shown) using 140 µM and 290 µM malonate (circles), and from (F) and additional experiments (not shown) using or 14 µM and 29 µM KCN (triangles) are plotted against the drop ('X') in membrane potential at t = 0 after succinate addition at t = -1.5 min. Data are means ± S.E.M. of 5 (A-C) or 6 (D-F) experiments each performed in duplicate. *Significantly different by paired Student's t test: p < 0.03. Lines in (G) are linear regressions.

  Figure 2Sensitivity of energisation-dependent proton conductance in rat skeletal muscle mitochondria to membrane potential (A-C), effect of malonate. (D-F), effect of KCN. (A,B,D,E), proton leak kinetics. 4 mM succinate was added at t = -1.5 min (open symbols) or t = -5 min (closed symbols) (see Fig.1A). Proton leak kinetics were measured between t = 0 and t = 3 min after no treatment (A, D) or addition of 430 µM malonate (B) or 43 µM KCN (E) immediately prior to addition of succinate. (C, F), inhibition of energisation-dependent proton leak by malonate and KCN. The increase in proton conductance at t = 0 caused by incubation with succinate between -5.0 min and -1.5 min was interpolated at the highest common membrane potential, 169 mV (solid line (A,B)) in (C) and 170 mV (solid line (D,E)) in (F). 'X' indicates the drop in membrane potential measured at t = 0 caused by addition of malonate or KCN prior to addition of succinate at t = -1.5. (G), effect of this drop in membrane potential on energisation-dependent increase in proton conductance. The increases in proton leak rate from (C) and additional experiments (not shown) using 140 µM and 290 µM malonate (circles), and from (F) and additional experiments (not shown) using or 14 µM and 29 µM KCN (triangles) are plotted against the drop ('X') in membrane potential at t = 0 after succinate addition at t = -1.5 min. Data are means ± S.E.M. of 5 (A-C) or 6 (D-F) experiments each performed in duplicate. *Significantly different by paired Student's t test: p < 0.03. Lines in (G) are linear regressions.

Figure 3

 3 Figure 3Modulation of energisation-dependent leak in rat skeletal muscle mitochondria by BSA, phenylbutylnitrone and palmitate

  5 min was interpolated at the highest common membrane potential, 172 mV (solid line (A,B,D,E)) in (C) and (I) and 167 mV (solid line (G,H)) in (F). Data are means ± S.E.M. of 4 (A-C), 3 (D-F) or 3 (G-H) experiments, each performed in duplicate. *Significantly different by paired Student's t test: p < 0.01.

  Figure 4Inhibition of proton conductance in rat skeletal muscle mitochondria by GDP and carboxyatractylate (A) Proton leak kinetics measured between t = 0 and t = 3 min following addition of inhibitors (where present) and mitochondria at t = -5.5 min. 4 mM succinate was added at t = -2.5 min (see Fig.1A) in the presence of no additions (squares), 500 µM GDP (triangles), 2.5 µM carboxyatractylate (diamonds) or GDP and carboxyatractylate (circles). (B) Absolute respiration rate at 180 mV (solid line (A)) in each condition. Data are means ± S.E.M. of 15 experiments, each performed in duplicate. One-way ANOVA was performed for (B); p = 0.0001, with Dunnett's multiple comparison test used posthoc comparing other bars to control: **significantly different: p < 0.01.

Figure 5

 5 Figure 5 Contribution of UCP3 and ANT to energisation-dependent leak in mouse skeletal muscle mitochondria (A, B) proton leak kinetics. (A) mitochondria from WT mice. (B), mitochondria from UCP3KO mice. 4 mM succinate was added at t = -1.5 min (open symbols) or t = -5 min (closed symbols) (see Fig. 1A). (C), proton leak due to UCP3 measured at 169 mV (solid line (A,B)) as (A) minus (B). Data are means ± S.E.M. of 7 experiments, each performed in duplicate. **Significantly different by paired Student's t test: p < 0.01.

Figure 6 Figure 7 3 Dx

 673 Figure 6Inhibition of proton conductance in skeletal muscle mitochondria from Ucp3WT and Ucp3KO mice by GDP and carboxyatractylate (A-B), proton leak kinetics. (A) mitochondria from WT mice. (B), mitochondria from UCP3KO mice. 4 mM succinate was added at t = -5 min (see Fig.1A). Proton leak kinetics were measured after 5 min incubation with 4 mM succinate in the absence (closed symbols) or presence of 500 µM GDP (dark grey symbols), 2.5 µM carboxyatractylate (light grey symbols) or GDP and carboxyatractylate (open symbols). (C), proton leak due to UCP3 measured at 161 mV (solid line (A,B)) as (A) minus (B). (D), proton leak not due to UCP3 measured at 161 mV, data from (B). (E), carboxyatractylate titration of ANT content in mitochondria from WT mice (squares) and Ucp3KO mice (circles). Data are means ± S.E.M. of 10 (A-D) and 4 (E) experiments, each performed in duplicate. One-way ANOVA was performed for (C) and (D); p = 0.0001, with Dunnett's multiple comparison test used posthoc comparing other bars to control: *significantly different: p < 0.05; **, p < 0.01.

BFigure 7

 7 Figure 3

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We thank Dr. Mary-Ellen Harper (Ottawa) for Ucp3KO mice and Julie Buckingham and Helen Boysen for technical assistance. This work was supported by the Medical Research Council and the Wellcome Trust (grants 065326/Z/01/Z and 066750/B/01/Z).


Debipriya Das

Rebecca A Randall

Caroline S Hill
email: caroline.hill@cancer.org.uk

An N-terminally truncated Smad2 protein can partially compensate for loss of full-length Smad2

Keywords: ARE, Activin-responsive element, DE, distal element, MEF, mouse embryo fibroblast

TGF-β superfamily signalling is critical both for early embryonic development and later for tissue homeostasis in adult organisms. The use of gene disruption techniques in mice has been essential to understand the functional roles of components of the pathways downstream of TGF-β superfamily ligands, in particular the receptors and the Smads that transduce the signals from the plasma membrane to the nucleus. Smad2 functions downstream of TGF-β, Activin and Nodal, and a number of Smad2 mutant mice have been generated by different laboratories. Although in all cases these Smad2-deficient mice were embryonic lethal, those created by deletion of the first coding exon survived longer than those generated by replacing part of the Mad homology (MH) 1 domain or deleting all or part of the MH2 domain. Moreover, they displayed a less severe phenotype, as they were capable of transiently inducing mesoderm. Here we show that embryonic fibroblasts taken from the Smad2 mutant mice created by deletion of the first coding exon express a small amount of an Nterminally truncated Smad2 protein. We show this protein results from internal initiation at methionine 241 and encodes the entire MH2 domain with the C-terminal part of the linker. We demonstrate that this protein is incorporated into Smad heteromeric complexes, can interact with DNA-binding transcription factors, and thereby mediate TGF-β-induced transcriptional activation from a number of TGF-βresponsive elements. We propose that this functional truncated Smad2 protein can partially compensate for the loss of full-length Smad2, thereby providing an explanation for the differing phenotypes of Smad2 mutant mice.

INTRODUCTION

Ligands of the TGF-β superfamily have a wide variety of functional roles including embryonic patterning and organogenesis, wound healing, tissue homeostasis, and cell migration, proliferation and differentiation. The effects that the TGF-β superfamily ligands exert depend on the cellular context; the cell type, the age of the cell, and the competence of the cell to respond to signal [START_REF] Massague | How cells read TGF-β signals[END_REF]. Additionally strength and duration of signal have important roles in the specificity of the biological consequences of this signalling pathway [START_REF] Schmierer | TGFβ-SMAD signal transduction: molecular specificity and functional flexibility[END_REF]. Members of this superfamily of ligands signal to the nucleus via complexes of activated Smads. These complexes are composed of phosphorylated receptor-regulated Smads (R-Smads), specifically Smad2 and Smad3 in response to TGF-β/Activin/Nodal signalling, in combination with the common mediator Smad (Co-Smad), Smad4 [START_REF] Feng | Specificity and versatility in TGF-β signaling through Smads[END_REF].

Sequence alignment reveals that the R-and Co-Smad proteins have two highlyconserved domains, the N-and C-terminal Mad Homology (MH) 1 and 2 domains, which are separated by a less well-conserved proline-rich linker. The MH1 domain is responsible for the auto-inhibition of the MH2 domain in the basal unphosphorylated state, and additionally, in the case of Smad3 and Smad4 but not Smad2, has some weak intrinsic DNA-binding activity. In contrast, the MH2 domain is commonly considered to be the 'effector' domain of the Smad, mediating multiple proteinprotein interactions, including those with other Smads, and with the receptors, transcription factor partners, coactivators and corepressors [START_REF] Feng | Specificity and versatility in TGF-β signaling through Smads[END_REF].

Owing to their poor affinity for DNA, Smad complexes are primarily recruited to TGF-β-responsive promoter elements via interactions with site-specific transcription factors. During the development of the early Xenopus embryo, Smad2 interacts with a number of transcription factors, such as those of the Mix family, namely Mixer, Milk and Bix3 [START_REF] Germain | Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif[END_REF][START_REF] Randall | Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif[END_REF], and of the Fast/FoxH1 family, XFoxH1a and XFoxH1b [START_REF] Chen | A transcriptional partner for MAD proteins in TGF-β signalling[END_REF][START_REF] Howell | A novel Xenopus Smadinteracting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements[END_REF], resulting in transcriptional activation of, for example, the goosecoid gene via the DE (distal element), and the Mix.2 gene via the ARE (activin-responsive element), respectively. Here the Smad-transcription factor interaction is mediated by the highly conserved proline-rich Smad interaction motif (SIM) present in the C-termini of these transcription factors, which binds to a hydrophobic pocket in the Smad2 MH2 domain [START_REF] Randall | Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif[END_REF]. Additionally, the FoxH1s contain a Fast/FoxH1 motif (FM), which allows them to specifically interact with phosphorylated Smad2-containing activated Smad complexes [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF].

Targeted gene disruption in mice has hugely advanced our understanding of how and when the Smads function. Unlike Smad3 mutant mice that are viable and survive to term [START_REF] Datto | Targeted disruption of Smad3 reveals an essential role in transforming growth factor β-mediated signal transduction[END_REF][START_REF] Yang | Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β[END_REF][START_REF] Zhu | Smad3 mutant mice develop metastatic colorectal cancer[END_REF], Smad2-deficient mice that were generated by replacing part of the MH1 domain or inserting a LacZ reporter in the MH2 domain [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF], or by deleting part of the MH2 domain [START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF] are embryonic lethal (E7.5-8.5) and exhibit severe gastrulation defects. These mice fail to form an egg cylinder and lack all mesoderm, as evidenced by the complete lack of Brachyury expression [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF][START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF]. A proportion of Smad2 heterozygous embryos also exhibit gastrulation defects, and lack mandibles and eyes later in development, indicating that signalling through Smad2 may function in a dose-dependent fashion [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF][START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF], confirming previous studies in Xenopus [START_REF] Baker | A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway[END_REF][START_REF] Graff | Xenopus Mad proteins transduce distinct subsets of signals for the TGF-β superfamily[END_REF]. Interestingly, Smad2 mutant mice generated by two further laboratories by disruption of the first coding exon displayed a transient induction of mesoderm [16,[START_REF] Waldrip | Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo[END_REF], suggesting that Smad2 was dispensible for mesoderm induction. We previously demonstrated that mouse embryonic fibroblasts (MEFs) taken from such homozygous mice [16,18] express a low level of truncated Smad2 protein that is phosphorylated in response to TGF-β [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF]. Since the phenotype of the mice expressing this N-terminally truncated Smad2 [16,[START_REF] Waldrip | Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo[END_REF] was less severe than that of the other Smad2 mutant mice [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF][START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF], we wanted to fully characterise this truncated protein to determine if it is functional. Here we show that this truncated Smad2 protein results from internal initiation from methionine 241 and encodes the entire MH2 domain as well as the Cterminal region of the linker. We demonstrate that, as for wild-type Smad2, this protein forms heteromeric complexes with Smad4 in response to TGF-β that are able to mediate ligand-induced transcriptional activation. We go on to show that truncated Smad2 is incorporated into stable DNA-binding complexes with FoxH1a/b and Smad4 or FoxH1a/b, Smad3 and Smad4. Therefore, the truncated Smad2 protein expressed in these MEFs is functional and we propose that it could partially compensate for loss of full-length Smad2 in the Smad2-deficient mice, thereby accounting for the observed differences in phenotype of the previously described Smad2 mutant mice. 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.

MATERIALS AND METHODS Plasmids:

The following plasmids have been described: pEF-HA Mixer [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF]; XFoxH1a and XFoxH1b in pEF-HA and pEF-Flag [START_REF] Germain | Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif[END_REF][START_REF] Howell | A novel Xenopus Smadinteracting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements[END_REF]; pEF-HA Smad3 [START_REF] Inman | Stoichiometry of active Smad-transcription factor complexes on DNA[END_REF]; (DE) 4 -Luc, (ARE) 3 -Luc and pEF-LacZ [START_REF] Pierreux | Transforming growth factor β-independent shuttling of Smad4 between the cytoplasm and nucleus[END_REF]; CAGA . pEF and pEF-HA Smad2(241-467) were constructed by PCR. Oligos were designed to amplify the region by PCR of mouse Smad2 corresponding to amino acids 241 to 467 and the resulting sequence was cloned into pEF-plink [START_REF] Hill | The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF[END_REF] and pEF-HA. The M241A point mutation was made in the context of pEF-Smad2(241-467) using PCR. All constructs were verified by sequencing.

Transfections, reporter assays and RT-PCR:

Maintenance of NIH-3T3 cells, transfection and reporter assays were as described [START_REF] Pierreux | Transforming growth factor β-independent shuttling of Smad4 between the cytoplasm and nucleus[END_REF]. HEK-293T cells and MEFs (wild-type and S2 KO) were cultured in Dulbecco's modified Eagle's medium (DMEM)/10% fetal bovine serum and were transfected using jetPEI (Polyplus-transfection) or Lipofectamine 2000 in combination with Plus Reagent (Invitrogen), respectively, both according to the manufacturers' instructions. siRNA transfections in S2 KO MEFs were performed in 6-well plates. Cells were seeded at 1 x 10 5 cells per well on the day prior to transfection. Each well was transfected with 8 nM final concentration of siRNA using Interferin (Polyplustransfection). Cells were incubated for 72 hrs and then treated with TGF-β. When cells were treated with both siRNA and plasmid DNA, cells were seeded at 2.5 x 10 4 cells per well in 12-well plates. Cells were transfected first with siRNA for 48 hours, and then transfected for an additional 24 hours with plasmid DNA prior to being treated ± TGF-β. siRNAs were purchased from Dharmacon and had the following catalogue numbers: Smad2 (D-040707-04), Smad3 (M-040706-00). TGF-β1 (Peprotech Ltd) was used at 2 ng/ml, and Activin at 40 ng/ml (R&D Systems), and inductions were for 1 hour, except in reporter assays when cells were treated with ligand for 8 hours. In the case of HEK-293Ts, cells were treated overnight with 10 µM SBI (SB-431542; Tocris) to inhibit autocrine TGF-β/Activin/Nodal signalling prior to washing the cells with fresh medium and induction with Activin. We use Activin in this case instead of TGF-β as HEK-293T cells respond better to Activin [START_REF] Levy | Arkadia activates Smad3/Smad4-dependent transcription by triggering signalinduced SnoN degradation[END_REF]. RT-PCR was performed as previously described [START_REF] Levy | Smad4 dependency defines two classes of transforming growth factor β (TGF-β) target genes and distinguishes TGF-β-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses[END_REF]. Primers used have the following sequences: GAPDH-Fwd, 5'-ACCACAGTCCATGCCATCAC-3'; GAPDH-Rev, 5'-TCCACCACCCTGTTGCTGTA-3'; mSmad2-Fwd, 5'-GCTTAGGCTTGCTCTCCAAC-3'; mSmad2-Rev, 5'-ATTCTTATGGTGCACATT CGG-3'.

Immunoprecipitation, Western blotting and bandshift assays

Nuclear, cytoplasmic and whole-cell extracts were prepared as previously described [START_REF] Howell | A novel Xenopus Smadinteracting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements[END_REF][START_REF] Marais | The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain[END_REF]. Immunoprecipitations were carried out as previously described [START_REF] Pierreux | Transforming growth factor β-independent shuttling of Smad4 between the cytoplasm and nucleus[END_REF]. Bandshifts probes corresponding to the ARE were synthesised as described [START_REF] Inman | Stoichiometry of active Smad-transcription factor complexes on DNA[END_REF], and bandshifts were performed as described [START_REF] Germain | Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif[END_REF] except that 50 µg whole-cell extract was used per lane. Antibodies used in Western blots, immunoprecipitations and bandshifts were anti-phospho-Smad2 (Cell Signalling Technology); anti-Smad4, (B8; Santa Cruz); anti-Flag, M2 (Sigma); anti-Smad2/3 and anti-Grb2 (BD Biosciences); anti-Smad3 (Zymed); anti-PARP, anti-HA and anti-HA-HRP (Roche).

RESULTS

Smad2-null mouse embryonic fibroblasts express a truncated form of Smad2 that is nuclear and can be phosphorylated in response to TGF-

In response to TGF-, Smad2 is phosphorylated on the last two serines of the Cterminal SSXS motif and, in complex with Smad4, accumulates in the nucleus where it regulates gene expression. The truncated Smad2 protein expressed by the Smad2null MEFs (hereafter called S2 KO MEFs [18]) is phosphorylated when cells are stimulated with TGF- [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF]. In order to test whether this protein acts like full-length Smad2, we compared its behaviour to Smad2 protein from wild-type MEFs in a number of assays. Nuclear and cytoplasmic extracts were prepared from wild-type and S2 KO MEFs cultured in the presence or absence of TGF-and were Western blotted for the phosphorylated form of Smad2. As expected, wild-type MEFs express Smad2, and this is phosphorylated and accumulates in the nucleus upon TGFaddition (Figure 1). As previously demonstrated [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF], the S2 KO MEFs express a small quantity of truncated Smad2 protein that can be phosphorylated upon TGF-addition. This protein was nuclear when the cells were stimulated with ligand (Figure 1). As antibodies specific for unphosphorylated Smad2 cannot recognise this truncated Smad2 protein it is not possible to determine its cellular localisation in the absence of signal. Antibodies against PARP and Grb2 were used as loading controls for nuclear and cytoplasmic extracts, respectively.

Internal initiation of translation at methionine 241 gives rise to the truncated Smad2 protein

We next determined what amino acid residues the truncated Smad2 corresponded to. It was previously shown that mice deleted in the first coding exon of Smad2 expressed an RNA transcript 300 base pairs shorter than wild type [START_REF] Tremblay | Formation of the definitive endoderm in mouse is a Smad2-dependent process[END_REF]. This transcript arises by splicing around the PGKneomycin cassette replacing the first coding exon. There are only three in-frame methionines from which the truncated Smad2 protein could start: M241, M327 and M411. Moreover, we know it contains the extreme C-terminus of Smad2 as it is phosphorylated in response to TGF-. From its size, the truncated Smad2 protein, which runs at approximately 28 kDa, would be predicted to start at the first internal in-frame methionine, Met241, and would therefore encode the entire MH2 domain along with the most C-terminal 33 amino acids of the linker (Figure 2A). To test this, a version of mouse Smad2 beginning at Met241, Smad2(241-467), was constructed. This plasmid was transfected into NIH-3T3 cells and whole-cell extracts from cells treated with or without TGF-were compared with extracts from wild-type and S2 KO MEFs by Western blot using the antibody against phosphorylated Smad2. The endogenous truncated Smad2 protein migrated on SDS-PAGE identically to the transfected Smad2 construct (Figure 2B). Full-length endogenous Smad2 is present and is phosphorylated upon TGFstimulation in both the NIH-3T3s and the wild-type MEFs. To confirm that Met241 was indeed the initiation codon, we made a construct in which it was mutated to alanine. This plasmid was transfected, along with wild-type Smad2(241-467) expression plasmid into human HEK-293T cells, so as to be able to distinguish mouse and human Smad2 transcripts. Mutation of Met241 to alanine resulted in complete loss of the truncated Smad2 protein (Figure 2C) despite significant mRNA levels being detected (Figure 2D). This confirms that the protein expressed in S2 KO MEFs is the result of internal initiation at Met241. A c c e p t e d M a n u s c r i p t Truncated Smad2 forms a heteromeric complex with Smad4 in response to TGFβ Upon TGF-β stimulation, Smad oligomerisation occurs in order to form transcriptionally active R-Smad-Smad4 heterodimers and heterotrimers [START_REF] Inman | Stoichiometry of active Smad-transcription factor complexes on DNA[END_REF][START_REF] Chacko | The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization[END_REF][START_REF] Shi | A structural basis for mutational inactivation by the tumour suppressor Smad4[END_REF][START_REF] Wu | Formation of a Stable Heterodimer between Smad2 and Smad4[END_REF][START_REF] Wu | Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGFβ signaling[END_REF]. These complexes are the result of extensive protein-protein interactions between the MH2 domains of the Smads, and are stabilised by binding of the phosphorylated Cterminus of the R-Smad with the L3 loop/B8 β-strand of the adjacent monomer [START_REF] Chacko | The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization[END_REF]. Since the Smad2 protein expressed by the S2 KO MEFs is N-terminally truncated and therefore has an intact MH2 domain, we wanted to determine whether it could form heteromeric complexes with Smad4. Whole-cell extracts from uninduced or TGF-βinduced wild-type and S2 KO MEFs were immunoprecipitated with the anti-Smad4 antibody and then Western blotted to detect endogenous phosphorylated Smad2. Smad4 immunoprecipitated full-length phospho-Smad2 from wild-type MEFs, and truncated phospho-Smad2 from S2 KO MEFs (Figure 3). Thus, internal initiation from Met241 produces a Smad2 protein that is appropriately folded and capable of forming heteromeric complexes with Smad4.

TGF-β-induced transcriptional activation is supported by truncated Smad2

Smad2 cannot bind DNA directly and thus forms transcriptionally active complexes through its interaction with a number of different transcription factors. The best characterised Smad2-interacting transcription factors are found in the early Xenopus embryo. Here, Smad2-Smad4 complexes interact with paired-like homeodomain transcription factors of the Mix family, such as Mixer, Milk and Bix3, in order to be recruited to the distal element (DE) of the goosecoid promoter, and with the forkhead/winged-helix transcription factors FoxH1a and FoxH1b at the Mix.2 ARE [START_REF] Germain | Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif[END_REF][START_REF] Randall | Different Smad2 partners bind a common hydrophobic pocket in Smad2 via a defined proline-rich motif[END_REF][START_REF] Chen | A transcriptional partner for MAD proteins in TGF-β signalling[END_REF][START_REF] Howell | A novel Xenopus Smadinteracting forkhead transcription factor (XFast-3) cooperates with XFast-1 in regulating gastrulation movements[END_REF]. Since the Smad complex interacts with these transcription factors through the Smad2 MH2 domain [START_REF] Germain | Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif[END_REF], and we have demonstrated that the truncated Smad2 protein can form Smad2-Smad4 complexes (Figure 3), it is conceivable that this protein can also interact with its transcription factor partners and thereby mediate TGF-β-induced transcriptional activation. Indeed, it has been demonstrated in in vitro transcription assays from chromatinised templates that this portion of Smad2 has transcriptional activity [START_REF] Ross | Smads orchestrate specific histone modifications and chromatin remodeling to activate transcription[END_REF]. In order to test whether the truncated Smad2 protein can form active Smad-transcription factor complexes and is therefore functional, wild-type and S2 KO MEFs were transfected with Mixer and the (DE) 4 -Luc reporter, or with FoxH1a or FoxH1b and the (ARE) 3 -Luc reporter construct, and then treated with or without TGF-β. Additionally, the two cell lines were transfected with the Smad3-dependent reporter CAGA 12 -Luc, which acted as a TGF-β-responsive, but Smad2-independent, control. The results indicate that there is no significant difference in the ability of fulllength or truncated Smad2 to mediate TGF-β-induced transcriptional activation. In the wild-type MEFs, treatment with TGF-β resulted in an average 3.1-fold induction of (DE) 4 -Luc when Mixer was also transfected (Figure 4A and Table 1) and, in the same way, this reporter was induced 3.8-fold in the S2 KO MEFs. Similarly, (ARE) 3 -Luc was activated comparably in both cell types when FoxH1a/FoxH1b and TGF-β were present. As expected for a Smad3-dependent reporter, induction of the CAGA 12 -Luc reporter was not significantly different in the S2 KO MEFs when compared to the wild-type cells, being induced 12.3-fold and 10.1-fold, respectively (Figure 4A and Table 1).

Since the S2 KO MEFs contain levels of Smad3 comparable with those in wild-type MEFs, it remained a possibility that Smad3 could be mediating the TGF-β-induced overexpressed, the lower two complexes of the native trimer were enhanced at the expense of the upper complex as would be expected if they contain the truncated Smad2, and these complexes shifted with the addition of the anti-HA antibody (Figure 5B, compare lanes 8 and 12). In contrast, exogenous HA-tagged Smad3 increased the amount of the top two complexes, supporting the theory that these contain Smad3-Smad4 and truncated Smad2-Smad3-Smad4 heterotrimers, respectively. These complexes were shifted with a Smad3 antibody and also with an anti-HA antibody, (Figure 5B, compare lanes 14, 16 and 18). Extracts were Western blotted to check levels of expression of the HA-tagged Smads (Figure 5C).

Thus, taking all our results together we conclude that truncated Smad2 can form stable complexes with either Smad4 and FoxH1 or with Smad3

DISCUSSION

Here we show that mouse embryonic fibroblasts lacking expression of full-length Smad2 [18] express a small amount of a truncated form of Smad2 that starts at Met241 and encodes the entire MH2 domain and the most C-terminal end of the linker. This protein is functional in terms of its ability to be phosphorylated by the receptor, to form complexes with transcription factors and other Smads, and consequently it mediates ligand-induced transcriptional activation of a number of TGF-β-responsive reporter constructs. In fact, further N-terminal deletion of Smad2 to residue 245 results in loss of the ability to be phosphorylated by the receptor [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF], indicating that this truncated Smad2 is the minimal fragment that can be activated by TGF-β. The mice from which these embryonic fibroblasts were taken were created by disruption of the first coding exon of Smad2 [16]. Mice created using this strategy [16,[START_REF] Waldrip | Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo[END_REF] showed a less severe phenotype than Smad2-null mice generated differently by two further labs [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF][START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF], displaying a transient induction of mesoderm as well as surviving for an additional day. We propose that this is the result of the incomplete loss of all functional Smad2 protein, and that there is sufficient Smad2 activity in the form of this N-terminally truncated Smad2 to account for the less severe phenotype.

Studies in Xenopus demonstrate that Activin, Nodal and Smad2 all act in a dosedependent manner to induce mesoderm [START_REF] Baker | A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway[END_REF][START_REF] Graff | Xenopus Mad proteins transduce distinct subsets of signals for the TGF-β superfamily[END_REF][START_REF] Green | Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm[END_REF][START_REF] Jones | Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation[END_REF], the result of different transcriptional activation thresholds of specific Smad2-responsive genes. Since the genetic response of a cell to TGF-β superfamily ligands is a consequence of the strength and duration of the signal, and this itself is a function of the concentration of active Smad complexes in the nucleus, decreased cellular Smad levels would be expected to considerably affect TGF-β/Activin/Nodal-responsive transcriptional regulation.

A previous study investigated the expression of a number of TGF-β-dependent genes in Smad2-and Smad3-deficient fibroblasts [18]. Here the authors identified a number of Smad3-dependent genes, including c-fos, Smad7 and TGF-β1, whereas loss of Smad2 alone had no significant effect on the expression of all but one of the genes analysed (MMP-2). However, the Smad2-null MEFs used in this study were the same ones as those described here and therefore express truncated, functional Smad2 protein. The small number of Smad2-dependent target genes identified in this study is thus expected to be artificially low. It is therefore also likely that a number of the 'Smad3-dependent' genes identified are also additionally regulated by Smad2, and that these two R-Smads have considerable overlapping but non-redundant roles in TGF-β-mediated gene expression.

The ability of Smad3 to interact with FoxH1 family members and thus compensate for loss of Smad2 has been previously investigated. Experiments using overexpressed or recombinant purified Smad3 have supported the proposed interaction between these proteins [START_REF] Labbe | Smad2 and Smad3 positively and negatively regulate TGF β-dependent transcription through the forkhead DNA-binding protein FAST2[END_REF][START_REF] Nagarajan | Regulation of Smad7 promoter by direct association with Smad3 and Smad4[END_REF][START_REF] Yagi | Alternatively spliced variant of Smad2 lacking exon 3. Comparison with wild-type Smad2 and Smad3[END_REF][START_REF] Yeo | The role of FAST-1 and Smads in transcriptional regulation by activin during early Xenopus embryogenesis[END_REF]. However, unless Smad3 is overexpressed, an ARF1 complex formed in HaCaT cells does not contain Smad3 when assayed by bandshift [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF]. This suggests that Smad2 is preferentially recruited into ARF complexes over Smad3. FoxH1a and FoxH1b are thought to interact with a Smad heterotrimer composed of two Smad2s and one Smad4 through their two Smad-interaction motifs; the SIM contacts one Smad2 subunit, and the FM contacts the second [START_REF] Randall | Recognition of phosphorylated-Smad2-containing complexes by a novel Smad interaction motif[END_REF]. The FM allows FoxH1a/FoxH1b to discriminate between Smad2/Smad4 heterotrimeric and Smad3/Smad4 heterodimeric complexes. This has been attributed to the fact that the FM recognises the R-Smad-R-Smad interface in the Smad heteromer and, since the Smad3/Smad4 complex is dimeric, at least when bound to the c-Jun Smad binding region [START_REF] Inman | Stoichiometry of active Smad-transcription factor complexes on DNA[END_REF], it therefore lacks this binding groove. However, in vitro purified activated Smad3 can form heterotrimeric complexes in association with Smad4, with a stoichiometry of two Smad3s and one Smad4 [START_REF] Chacko | The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization[END_REF][START_REF] Correia | Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo-and heterotrimerization[END_REF][START_REF] Qin | Smad3 allostery links TGF-β receptor kinase activation to transcriptional control[END_REF]].

Here we have demonstrated that when endogenous levels of Smad2 protein are low, as in the wild-type MEFs, or when Smad2 is truncated and expressed at low level, as in the S2 KO MEFs, Smad3 can become incorporated into ARF complexes and mediate ligand-induced activation of the (ARE) 3 -Luc reporter. In the S2 KO MEFs TGF-β-induced transcriptional activation is thus likely to be mediated by complexes containing Smad4 and truncated Smad2, either alone or in combination with Smad3. Indeed, our knockdown experiment (Figure 4B) confirms that both truncated Smad2 and Smad3 contribute to transcriptional activation via the ARE. Thus the truncated Smad2 protein has transcriptional activity and can clearly partially compensate for the loss of full-length Smad2, since the mice that express this protein [16,[START_REF] Waldrip | Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo[END_REF] have a less severe phenotype than those generated by methods other than deletion of the first coding exon [START_REF] Nomura | Smad2 role in mesoderm formation, left-right patterning and craniofacial development[END_REF][START_REF] Weinstein | Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2[END_REF]. However, it is important to note that since the mice expressing truncated Smad2 still fail to develop normally and make it to term, this truncated Smad2 cannot fully compensate for the loss of the full-length protein. This could either be the result of the reduced levels of expression of the truncated protein, which mean it is simply not at a high enough threshold to activate certain developmental genes, or might indicate an as yet unidentified role for the MH1 domain of Smad2. This second possibility seems likely in the light of recent work showing that directed expression of Smad2(Δexon3), but not Smad2(full length) can rescue the ability of Smad2-deficient ES cells to contribute to descendants of definitive endoderm in wildtype host embryos [START_REF] Dunn | Mice exclusively expressing the short isoform of Smad2 develop normally and are viable and fertile[END_REF]. In addition, mice that express exclusively Smad2(Δexon3) develop normally and are viable and fertile. The difference in ability of Smad2(Δexon3) versus Smad2(full length) to rescue the loss of Smad2 suggests a critical function of the Smad2 MH1 domain which lacks exon 3. Since the truncated Smad2 we study here has no MH1 domain it cannot therefore fully rescue Smad2 null embryos. overexpressing Smad2(241-467), were assayed by Western blotting with the antiphosphorylated Smad2 antibody. (C) HEK-293T cells were transfected with plasmids expressing Smad2(241-467) or the mutant Smad2(M241A-467) or remained untransfected. Cells were then treated with the ALK5 inhibitor SB-431542 to inhibit autocrine signalling (lanes marked -). After 15 hours, the SB-431542 was washed out in half the samples and these were treated with Activin for 1 hour (lanes marked +). Whole-cell extracts were assayed by Western blot for phospho-Smad2, and Smad2/3 and Grb2 as loading controls. Note that although the 15-hour treatment with SB-431542 was sufficient to cause dephosphorylation of full-length Smad2 in the HEK-293 cells, it was not sufficient to cause dephosphorylation of the truncated Smad2. (D) Total RNA was isolated from the HEK-293Ts (untransfected), or those overexpressing the wild-type or M241A truncated Smad2 constructs and RT-PCR was performed using primers specific for mouse Smad2, and GAPDH as a control (+RT panels). To ensure plasmid DNA was not being detected, RT-PCR reactions were also performed in the absence of reverse transcriptase (-RT panels).

Figure 3

Smad4 can form a complex with the truncated Smad2 protein. Whole-cell extracts were prepared from wild-type or S2 KO MEFs that had been incubated with or without TGF-β for 1 hour. Extracts were then assayed by immunoprecipitation of complexes with anti-Smad4 antibody followed by Western blotting with antiphosphorylated-Smad2 antibody (top). Western blotting of whole-cell extracts (inputs) with anti-phosphorylated-Smad2 (middle) and anti-Smad4 (bottom) antibodies acted as loading controls. A non-specific background band is marked by an asterisk.

Figure 4

Truncated Smad2 can mediate TGF-β-induced transcriptional activation via the DE and ARE, comparable with wild-type full-length Smad2. (A) Wild-type (top) and S2 KO MEFs (bottom) were transfected with the (DE) 4 -luciferase, (ARE) 3 -luciferase or CAGA 12 -luciferase reporters along with plasmids expressing Mixer, FoxH1a or FoxH1b, as indicated. Cells were incubated in the presence or absence of TGF-β for 8 hours, and luciferase was quantitated relative to the β-Gal internal control. Data are from one representative experiment performed in triplicate. Refer to Table 1 for the mean and standard deviation of four independent experiments. (B) S2 KO MEFs were transfected with the control non-target siRNA SMARTpool or with an siRNA single duplex targeting mouse Smad2 (mS2) or an siRNA SMARTpool targeting mouse Smad3 (mS3), followed by transfection with (ARE) 3 -Luciferase with or without a plasmid expressing FoxH1b and treatment with or without TGF-β, as for (A). Data are the mean and standard deviation of one representative experiment performed in triplicate. (C) Western blot to show specificity of knock down of mouse Smad2 and mouse Smad3 siRNAs used in (B), blotted with anti-Smad2/3, anti-phosphorylated Smad2, and anti-Grb2 as a control.

Figure 5

The truncated Smad2 protein can form stable complexes on the ARE with Smad3 and Smad4, and either FoxH1a or FoxH1b. (A) Wild-type and S2 KO MEFs were transfected with plasmids expressing Flag-tagged FoxH1a (left panel) or FoxH1b (right panel). Cells were either untreated or treated with TGF-β for 1 hour. Whole-cell extracts were then analysed by bandshift using the ARE probe. The ARF1/2 complexes were shifted with the indicated antibodies to show that they contained FoxH1a/FoxH1b, Smad2, Smad3 and Smad4. Asterisks represent the three ARF1/2 complexes formed in the S2 KO MEFs. (B) Whole-cell extracts from wild-type and S2 KO MEFs treated with or without TGF-β and transfected with Flag-tagged FoxH1a, either alone or with plasmids expressing HA-tagged Smad2 or Smad3, were assayed by bandshift using the ARE probe. Complexes were incubated with antibodies to demonstrate that the three ARF1 bands (as indicated by asterisks) contained varying heterotrimers of Smad3 and truncated Smad2, with Smad4 and FoxH1a. (C) Western blot of whole-cell extracts from (B) probed with anti-HA-HRP, anti-phosphorylated Smad2 and anti-Grb2 antibodies to show levels of transfected plasmids. Grb2 acts as a loading control.

  Biochemical Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20080014 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080014

Biochemical

  Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20080014 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080014A 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.

LEGENDS Figure 1

 1 Truncated Smad2 is phosphorylated and nuclear in response to TGF-β signalling. Cytoplasmic (Cyt) and nuclear (Nuc) extracts were prepared from wild-type and S2 KO MEFs that had been treated with or without TGF-β for 1 hour. Extracts were then analysed by Western blot, probing with an anti-phosphorylated Smad2 antibody, along with anti-PARP and anti-Grb2 antibodies that acted as controls for nuclear and cytoplasmic proteins, respectively. The positions of the molecular weight markers are shown on the left of the top panel.

Figure 2

 2 Figure 2The truncated Smad2 protein expressed by S2 KO MEFs is the result of an internal initiation at methionine 241. (A) Schematic of Smad2 showing the position of Met241, and the truncated Smad2(241-467) protein. (B) Whole-cell extracts prepared from uninduced or TGF-β-induced wild-type and S2 KO MEFs, and from NIH-3T3s overexpressing Smad2(241-467), were assayed by Western blotting with the antiphosphorylated Smad2 antibody. (C) HEK-293T cells were transfected with plasmids expressing Smad2(241-467) or the mutant Smad2(M241A-467) or remained untransfected. Cells were then treated with the ALK5 inhibitor SB-431542 to inhibit autocrine signalling (lanes marked -). After 15 hours, the SB-431542 was washed out in half the samples and these were treated with Activin for 1 hour (lanes marked +). Whole-cell extracts were assayed by Western blot for phospho-Smad2, and Smad2/3 and Grb2 as loading controls. Note that although the 15-hour treatment with SB-431542 was sufficient to cause dephosphorylation of full-length Smad2 in the HEK-293 cells, it was not sufficient to cause dephosphorylation of the truncated Smad2. (D) Total RNA was isolated from the HEK-293Ts (untransfected), or those overexpressing the wild-type or M241A truncated Smad2 constructs and RT-PCR was performed using primers specific for mouse Smad2, and GAPDH as a control (+RT panels). To ensure plasmid DNA was not being detected, RT-PCR reactions were also performed in the absence of reverse transcriptase (-RT panels).

  Figure 3

Figure 5 HBiochemical

 5 Figure 5

  Biochemical Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20080014 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080014

Table 1

 1 Average TGF-β-induced transcriptional activation of the (DE) 4 -, (ARE) 3 -and CAGA 12 -luciferase reporters in wild-type and S2 KO MEFs. In all instances n=4, except for CAGA 12 -Luc where n=2. Biochemical Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20080014 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080014 A c c e p t e d M a n u s c r i p t

				Wild-type MEFs		S2 KO MEFs
	Reporter +		TGF-β	Average fold	± Standard	Average fold	± Standard
	transcription factor			induction	deviation	induction	deviation
				relative to
				uninduced
	(DE) 4 -Luc + Mixer		+	3.1	0.2	3.8	1.4
	(ARE) 3 -Luc	+	+	2.7	0.6	2.3	1.3
	FoxH1a
	(ARE) 3 -Luc	+	+	4.5	0.9	2.8	0.6
	FoxH1b
	CAGA 12 -Luc (n=2)		+	12.3	1.0	10.1	1.0

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 03 Sep 2008 as manuscript BJ20080014 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080014 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. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

We would like to thank Liz Robertson and Liz Bikoff and members of the Hill lab for useful discussions, and Laurence Levy for comments on the manuscript. The work was supported by Cancer Research UK.

TGF-β α-P-Smad2