María-Jesús García-Murria

Saeid Karkehabadi

Julia Marín-Navarro

Sriram Satagopan

Inger Andersson

Robert J Spreitzer

Joaquín Moreno
email: joaquin.moreno@uv.es

Structural and Functional Consequences of the Substitution of Proximal Residues Cys-172 and Cys-192 in the Large Subunit of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Chlamydomonas reinhardtii †

Keywords: Rubisco, D-ribulose-1,5-bisphosphate carboxylase/oxygenase, crystallographic symmetry, r.m.s.d., root-mean-square deviation Chlamydomonas reinhardtii, chloroplast mutants, Rubisco, specificity factor, proximal cysteines, X-ray crystallography

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Structural and functional consequences of the substitution of proximal residues Cys-172 and Cys-192

in the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii SYNOPSIS: Proximal cysteines 172 and 192 in the large subunit of the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are evolutively conserved among cyanobacteria, algae, and higher plants. Mutation of Cys-172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here we report the effect of the substitution of Cys-172 and Cys-192 by serine on the catalytic properties, thermostability, and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor compared to the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V max for carboxylation was similar to that of wild type Rubisco in the case of the C172S enzyme, but about 30% lower for the C192S Rubisco. In contrast, the K m for CO 2 and O 2 were similar for C192S and wildtype enzymes, but distinctly higher (about double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature about 2 ºC lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 ºC, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side-chains. This may cause the exceptional catalytic features of C172S.

In contrast, the C192S mutation does not produce similar structural perturbations.

INTRODUCTION

The bifunctional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco 1 , EC 4.1.1.39) operates at a metabolic branch point channelling carbon either to the photosynthetic carbon fixation (Calvin-Benson) cycle or to the photorespiratory pathway [START_REF] Spreitzer | Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme[END_REF]. The enzyme converts ribulose 1,5-bisphosphate (RuBP) to a reactive enediolate form, which is prone to attack by either CO 2 or O 2 .

The resulting carboxylated or oxygenated intermediate is hydrolyzed in two further steps (hydration and C 2 -C 3 bond cleavage) producing two molecules of 3phosphoglycerate or one each of 3-phosphoglycerate and 2-phosphoglycolate, respectively, as final products [START_REF] Cleland | Mechanism of Rubisco: The carbamate as general base[END_REF]. Because the carboxylation and oxygenation reactions share the same enediol substrate, the oxygenation reaction may be an unavoidable consequence of the catalytic mechanism of the enzyme [START_REF] Chen | How various factors influence the CO 2 /O 2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Tcherkez | Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized[END_REF], which evolved under anoxic conditions. The intrinsic preference of Rubisco for one or the other reaction is measured by the so called specificity factor (Ω), which is defined as the ratio of the V max for carboxylation (V c ) to the K m for CO 2 (K c ), divided by the ratio of the V max for oxygenation (V o ) to the K m for O 2 (K o ) [START_REF] Laing | Regulation of soybean net photosynthetic CO 2 fixation by the interaction of CO 2 , O 2 and ribulose 1,5diphosphate carboxylase[END_REF]. The value of Ω (V c K o /V o K c ) is known to vary among Rubisco enzymes from different species [START_REF] Jordan | Species variation in kinetic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase[END_REF].

Because of the relevance to plant growth of the photosynthesis/photorespiration ratio, the beneficial manipulation of crop-plant Ω has been an unfulfilled biotechnological goal.

The crystal structures of the active enzyme has been solved for a number of species including prokaryotes, algae, and land plants [START_REF] Andersson | Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. The most common form of Rubisco (type I present in cyanobacteria, green algae, and higher plants) is composed of eight chloroplast-encoded large subunits (ca 55 kDa) and eight nuclear-encoded small subunits (ca 15 kDa). Two large subunits assemble into functional dimers with two active sites at the dimer interface. Catalytic competence requires carbamylation by CO 2 of the ε-amino group of a specific lysine residue in the active site, and subsequent binding of a Mg 2+ ion to the carbamyl group [START_REF] Lorimer | Carbamate formation on the epsilonamino group of a lysyl residue as the basis for the activation of ribulosebisphosphate carboxylase by CO 2 and Mg 2+[END_REF]. Four small subunits cap the top and bottom of four pairs of the large subunits to form the hexadecameric holoenzyme. The knowledge of Rubisco structure has fostered an understanding of central features of the catalytic mechanism and other properties of Rubisco, but the structural determinants of the variations in Ω have remained elusive. Analysis of Rubisco mutants has been helpful in evaluating the functional role of the substituted residues or deleted sequences [START_REF] Spreitzer | Rubisco: Structure, regulatory interactions, and possibilities for a better enzyme[END_REF][START_REF] Spreitzer | Genetic dissection of Rubisco structure and function[END_REF][START_REF] Hartman | Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Kellog | The structure and function of RuBisCO and their implications for systematic studies[END_REF][START_REF] Parry | Manipulation of Rubisco: the amount, activity, function and regulation[END_REF]. Many of these studies have been carried out in Chlamydomonas reinhardtii, a unicellular green alga that allows for straightforward genetic manipulation of both the chloroplastic and nuclear genomes. Because of their singular redox properties, Cys residues are potential candidates for harboring regulatory effects and, therefore, may be considered as primary targets for site-directed mutagenesis. In fact, it is known that the specific oxidative modification of Cys residues by disulfide exchange causes inactivation and increases proteolytic susceptibility of the purified Rubisco in vitro [START_REF] Penarrubia | Increased susceptibility of ribulose-1,5bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments[END_REF][START_REF] Garcia-Ferris | Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis[END_REF][START_REF] Marin-Navarro | Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase[END_REF]. These findings suggest a physiological role of Cys oxidation in Rubisco catabolism.

Whereas the critical Cys residues have not been unambiguously identified, the conserved Cys-172 residue from the Rubisco large subunit has attracted some attention [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Marcus | Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation[END_REF]. Furthermore, specific modification of this Cys residue by an affinity label has been shown to abolish enzymatic activity [START_REF] Schloss | Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate bromoacetylethanolamine phosphate[END_REF], suggesting a yet undefined role for this residue in catalytic performance. Substitution of Cys-172 by Ser in C. reinhardtii yields an active enzyme but eliminates arsenite sensitivity, shifts the critical redox potential that controls proteolytic susceptibility in vitro, and increases resistance to stress-induced degradation in vivo [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. A slower catabolism of Rubisco under stress has been also reported after substitution of the same residue in a prokaryotic Rubisco [START_REF] Marcus | Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation[END_REF], whereas the replacement of the adjacent Cys-192 or other conserved Cys residues (Cys-247) did not delay the stress-induced degradation [START_REF] Marcus | Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation[END_REF].

Analysis of the crystal structures of Rubisco enzymes from different sources reveals that Cys-172 and Cys-192 are close to each other, but, in most cases, not close enough to form a disulfide bond [START_REF] Andersson | Structural framework for catalysis and regulation in ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. In one of the structures of the noncarbamylated enzyme from tobacco [START_REF] Curmi | Crystal structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-Å resolution[END_REF], a disulfide bond is observed, whereas a second structure, obtained with similar conditions, does not feature the 172-192 disulfide bond [START_REF] Duff | The transition between the open and closed states of rubisco is triggered by the inter-phosphate distance of the bound bisphosphate[END_REF]. Structures of activated (carbamylated) Rubisco [START_REF] Schreuder | Crystal structure of activated tobacco rubisco complexed with the reaction-intermediate analogue 2-carboxyarabinitol 1,5-bisphosphate[END_REF][START_REF] Andersson | Large structures at high resolution: the 1.6 A crystal structure of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol bisphosphate[END_REF][START_REF] Taylor | First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii[END_REF] show residues 172 and 192 as free Cys residues. In any case, the proximity of the Cys residues is likely to support redox interactions that may have structural, catalytic, and/or regulatory relevance. In an attempt to define the structural and functional roles of these proximal Cys residues, they have been replaced by site-directed mutagenesis and chloroplast transformation of C. reinhardtii. The C172S and C192S mutant Rubisco enzymes have been analyzed in detail with respect to catalytic function and their X-ray crystal structures have been solved.

EXPERIMENTAL

Strains and cell cultures

Chlamydomonas reinhardtii strain C172S, with a Cys-172 to Ser substitution in the Rubisco large subunit, has been already described [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. Strain C192S was created by site-directed mutagenesis and chloroplast transformation as described below. Strain 2137 mt+ is the wild type [START_REF] Spreitzer | Photosynthesis-deficient mutants of Chlamydomonas reinhardtii with associated light-sensitive phenotypes[END_REF]. Non-photosynthetic strain 18-7G, which has a nonsense mutation in the rbcL large-subunit gene, was used as the host for transformation [START_REF] Spreitzer | Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase[END_REF]. All strains were kept on standard solid medium supplemented with acetate [START_REF] Spreitzer | Photosynthesis-deficient mutants of Chlamydomonas reinhardtii with associated light-sensitive phenotypes[END_REF] at 25 ºC in the dark. For Rubisco purification, 1.5 liter flasks of standard liquid culture medium supplemented with acetate were innoculated with the appropriate strains and the cells were grown at 28 ºC in the light (3 x 30W white light tubes at 30 cm, supplying about 10 µE•m -2 •s -1 ) with moderate (120 rpm) shaking up to a density of 10 7 cells/ml. The cells were collected by centrifugation (3000 x g for 5 min) and the pellet was frozen in liquid nitrogen and stored at -80 ºC.

Directed mutagenesis and chloroplast transformation

Plasmid prbclP containing a HpaI fragment (bases -743 to +1927), which encompasses the entire rbcL coding region (bases +1 to +1428), cloned into the SmaI-site of a pBluescript SK-(Stratagene) vector was used as a source of the C. reinhardtii Rubisco large subunit chloroplast gene. Site-directed mutagenesis of Cys-192 to Ser was achieved by changing TGT (Cys192) to AGC, using the QuickChange kit of Stratagene. This substitution created a new HindIII restriction site (between bases +573 and +574) that expedited the screening of transformants.

The mutated plasmid (called pLS-C192S) was used for chloroplast transformation via projectile bombardment of C. reinhardtii strain 18-7G according to published procedures [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Zhu | Directed mutagenesis of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: Loop-6 substitutions complement for structural stability but decrease catalytic efficiency[END_REF]. Transformation of the non-photosynthetic 18-7G mutant with pLS-C192S yielded photosynthetically competent colonies on minimal medium. The transformants became homoplasmic (as checked by HindIII restriction analysis of polymerase chain reaction amplified rbcL) after succesive rounds of single-colony isolation, replating in selective minimal medium, and growth under light. Finally, the entire coding region of rbcL was sequenced to confirm that only the intended mutation was present. The strain thus created was named C192S.

Purification of Rubisco

Rubisco was extracted and purified by 35-60% (w/v) ammonium sulfate precipitation, sucrose gradient centrifugation, and anion exchange chromatography according to a published procedure [START_REF] Garcia-Ferris | Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis[END_REF]. Alternatively, for the determination of kinetic constants, Rubisco was purified following a somewhat different procedure described elsewhere [START_REF] Spreitzer | Heteroplasmic suppression of an amber mutation in the Chlamydomonas chloroplast gene that encodes the large subunit of Ribulosebisphosphate carboxylase/oxygenase[END_REF].

Rubisco activity assays

Rubisco carboxylase activity was routinely determined using a 14 C-fixation method previously described [START_REF] Garcia-Ferris | Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis[END_REF]. Kinetic constants were measured according to a reported procedure [START_REF] Chen | Reduced CO 2 /O 2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas[END_REF], and Ω was independently determined using the dual-labelling method [START_REF] Jordan | A sensitive assay procedure for simultaneous determination of Ribulose-1,5-bisphosphate carboxylase and oxygenase activities[END_REF].

Crystallization and Structure Determination

Crystals of C. reinhardti Rubisco-mutant enzymes were grown using hanging drop vapor diffusion at 20 °C. The drops were prepared by mixing a solution containing 10 mg/ml Rubisco (in 50 mM HEPES pH 7.5) and 1 mM 2-CABP with an equal volume of a well solution containing 50 mM HEPES (pH 7.5), 0.05 -0.2 M NaCl, 7-12% (w/v) PEG 4000, 10 mM NaHCO 3 , 5 mM MgCl 2 . Crystals grew within one week. Prior to data collection, crystals were transferred to a solution containing the well solution with 30% (v/v) ethylene glycol as a cryoprotectant and frozen in liquid nitrogen. Data were collected from single crystals at 100 K on beam line ID14-3 at ESRF, Grenoble, France (C172S), and on beam line I711 at Max-lab, Lund, Sweden (C192S). The data were processed using DENZO and SCALEPACK [START_REF] Otwinowski | Processing of X-ray diffraction data collected in oscillation mode[END_REF] to a resolution of 2.30 Å for C172S, and 2.65 Å for C192S (Table S1, see online supplementary data).

The mutant crystal structures were solved by molecular replacement using the program AMORE [START_REF] Navaza | AMoRe: An automated molecular replacement program package[END_REF][START_REF]The CCP4 suite-programs for protein crystallography[END_REF]. The search model consisted of one set of large and small subunits of wild-type C. reinhardtii Rubisco (PDB code 1GK8). In each case, eight solutions corresponding to eight different orientations of the search unit within one hexadecamer in the asymmetric unit were found.

Refinement was performed using REFMAC v. 5 [START_REF] Murshudov | Refinement of macromolecular structures by the maximum-likelihood method[END_REF]. For cross-validation, 5% of the data was excluded from the refinement for R free calculations. Initial electron density maps calculated after one round of rigid body refinement showed clear density for the substituted residues 172 and 192, respectively. After building the corresponding residues into density, the mutant enzyme structures were further refined using a maximum likelihood target function and overall B-factor refinement. Tight non-crystallographic symmetry (NCS) restraints were imposed for the eight copies of large and small subunits in the asymmetric unit at the beginning of the refinement. Towards the end of refinement, NCS restraints were relaxed or removed for regions of the structure that clearly deviated from NCS. Solvent molecules were added using ARP/wARP [START_REF] Perrakis | wARP: improvement and extension of crystallographic phases by weighted averaging of multiple-refined dummy atomic models[END_REF]. Throughout the refinement, 2mF o -DF c and mF o -DF c sigma A weighted maps [START_REF] Pannu | 668 methods for building protein models in electron density maps and the location of errors in these models[END_REF] were inspected and the models manually adjusted using O [START_REF]The CCP4 suite-programs for protein crystallography[END_REF]. The structures of C172S and C192S were refined to final R cryst /R free values of 0.171/0.204 and 0.205/0.234 respectively. Coordinates and structure factors have been deposited at the Protein Data Bank with accession codes 2vdh (for C172S) and 2vdi (for C192S).

RESULTS

Isolation and growth of the C192S mutant

Chloroplast transformation of the C. reinhardtii 18-7G mutant lacking Rubisco [START_REF] Spreitzer | Nonsense mutations in the Chlamydomonas chloroplast gene that codes for the large subunit of ribulosebisphosphate carboxylase/oxygenase[END_REF] with the pLS-C192S plasmid, containing the rbcL gene with a C192S substitution, yielded transformant colonies that grew on minimal medium in the light. Amplification and sequencing of the rbcL gene confirmed that the transformants carried the single C192S mutation. This showed that the C192S mutant Rubisco was catalytically active. The C192S strain grew at a similar rate as the wild type, both in liquid culture and on solid media, under photoautotrophic, photoheterotrophic, or strictly heterotrophic conditions (data not shown). In general, the growth of the C192S strain presented no particular features when compared to the wild type, except for a somewhat reduced survival of cells in old cultures.

Thermal stability of the mutant enzymes

The C172S Rubisco has been already shown to be thermosensitive [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. In order to compare the thermal stability of the C192S mutant protein with the C172S and the wild-type enzymes, purified Rubisco from all three strains was incubated at different temperatures (in the 48 to 62 ºC range) for 30 min (Figure 1A), and at 55 ºC for different times (Figure 1B), and the carboxylase activity was subsequently determined. The C172S enzyme showed a critical denaturation temperature (54 ºC) about 2 ºC lower than the wild-type (Figure 1A) and a distinctly higher denaturation rate at 55ºC (Figure 1B). In contrast, the denaturation temperature of the C192S Rubisco (near 55.5 ºC, Figure 1A) was only slightly (about 0.5 ºC) lower than the wild-type, and C192S denatured at a similar rate as the wild type at 55 ºC (Figure 1B). In a previous report [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF], it was suggested that the lower stability of the C172S mutant protein could result from the inability of the mutant to form a Cys-172 to Cys-192 disulfide bond, which might be present in wild-type Rubisco. Because the C192S enzyme is relatively stable, it can now be concluded that the thermosensitivity of the C172S mutant Rubisco is not caused by the absence of a putative Cys-172 to Cys-192 disulfide bridge, but reveals instead a significant structural role played by the Cys-172 residue, which cannot be fully complemented by Ser at the same position. In contrast, the replacement of Cys-192 by Ser seems to have a lesser effect on structural stability.

Catalytic performance of the mutant enzymes

Table 1 displays the mean and standard deviation values of the kinetic constants and Ω values determined for wild-type and mutant Rubisco enzymes. V c was similar to that of the wild-type enzyme in the case of C172S, but somewhat lower (about 30%) for C192S Rubisco. In contrast, K c and K o were similar for C192S and wild-type Rubisco, but distinctly higher (about double) for the C172S mutant enzyme. This indicates that the lower carboxylation specific activity reported for C172S Rubisco previously [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF] was likely due to the increased K c value (Table 1).

Whereas the Ω value of C192S Rubisco was identical to that of wild-type Rubisco (Ω = 61), the C172S mutant enzyme showed a significantly (P>0.95) higher value of 68 (Table 1). With respect to the C192S enzyme, a lower V c was compensated by a lower K c resulting in an unchanged Ω value. However, in the case of C172S Rubisco, an even greater increase in K o relative to an increased K c is apparently responsible for an 11% increase in Ω (Table 1).

X-ray Crystal Structures of the mutant enzymes

To determine the molecular basis for their altered properties, the purified mutant enzymes were crystallized and their structures were determined. The crystallographic data collection and refinement statistics for the mutant-enzyme crystal structures are summarised in Table S1 (see online supplementary data). The final electron density maps showed density for all 140 residues of the small subunit. The amino-terminus of the large subunit displayed disorder that varied in extent from subunit to subunit.

Density was observed for residues 11-475 (from a total of 475 residues) of the large subunit. In several of the subunits, an additional 2-4 amino-terminal residues could be built into density. Clear density was observed for the substituted residues in each case. However, because the substituted residue is isosteric, it cannot be verified by inspection of difference Fourier maps in this case.

In general, no dramatic differences in large-subunit Cα backbone atoms were observed among the wild-type, C172S, and C192S structures. A superposition of the wild-type large subunit with the large subunits of C172S and C192S using the algorithms encoded in O [36] gave root-mean-square deviations of 0.198 and 0.200 Å for all Cα atoms, respectively. This is close to the coordinate errors of the respective structures. Inspection of the region around residue 172 in C172S Rubisco reveals that the main chain atoms of residues 170-174 are displaced significantly (ca. 0.4 Å) compared to wild-type Rubisco (Figure 2A,B) resulting in a shift of β-strand 1 of the α/β-barrel. This is a larger displacement than the average for this β-strand and also for the average of all strands of the α/β-barrel (mean displacement 0.2 Å).

Cys-192 is located in α-helix 6 of the α/β-barrel facing Cys-172. Substitution of Cys-192 with Ser did not cause a similar displacement of the backbone atoms of αhelix 6 or β-strand 6. In the wild-type enzyme, the distance between the sulfur atoms of residues 172 and 192 is 3.9 Å, which is too long for establishing a disulfide bond.

In the C172S and C192S mutant enzymes, the oxygen-to-sulfur distances are even somewhat longer, 4.1 Å. In contrast, the distance between the sulfur atoms of Cys-449 and Cys-459 (2.0 Å) and their conformations in the wild-type enzyme are ideal for disulfide formation [START_REF] Taylor | First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii[END_REF]. In C172S, the sulfur to sulfur distance between Cys-449 and Cys-459 was similar to that of wild-type Rubisco (2.1 Å). In C192S this distance was lengthened to 2.9 Å (Figure 3), resulting in a shift of the α-helix harboring Cys-449 (residues 438-452), breaking the disulfide bond.

A superposition of the wild-type small subunit with the small subunits of C172S and C192S gave root-mean-square deviations of 0.235 and 0.224 Å for Cα atoms 1-126, respectively. This is close to the coordinate errors of the respective structures, indicating that the C172S and C192S substitutions in the large subunit had no noticeable effect on the small-subunit structure.

Alterations in the Active Site

Residue 172 is located in β-strand 1 of the α/β barrel and is close to the active site.

The most immediate effect of the C172S substitution is to shift the main-chain atoms of β-strand 1 by 0.4 Å relative to wild-type. This shifted residue 172 by 0.4 Å and resulted in an increase (by 0.2 Å) in the distance between the side chains of residues 172 and 192. It also resulted in a significant increase in the distance (by more than 0.3 Å) between Thr-173 and CABP relative to wild type Rubisco (Figure 2). All other shifts of active-site residues were close to coordinate error. geometry required for a disulfide bond. This implies that, in order for a disulfide to form under oxidative conditions, the main chain backbone around the residues would have to move considerably. Nevertheless, disulfide bonding between Cys-172 and Cys-192 appears to be possible if Rubisco becomes decarbamylated, as observed in the unactivated tobacco enzyme [START_REF] Curmi | Crystal structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-Å resolution[END_REF]. On the other hand, conformational changes are known to be induced in Rubisco by oxidative modification of Cys residues [START_REF] Marin-Navarro | Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase[END_REF]. Therefore, Cys-172 and Cys-192 offer, in principle, a potential for redox modulation of Rubisco through structural changes induced by oxidation.

DISCUSSION

We have addressed the structural and functional role of the conserved Cys residues by substitution with Ser via site-directed mutagenesis and chloroplast transformation of C. reinhardtii. As a result of this single-atom substitution (O for S) the redox sensitivity of the residue is abolished. We have shown that the substitution of Cys-172 with Ser increases Rubisco thermosensitivity (Figure 1), perturbs the structure near the substitution site (Figure 2), and alters the kinetic constants of the enzyme (Table 1). Because the symmetric substitution of Cys-192 does not induce the same changes, it appears that the described effects are not directly related to the presence/absence of a disulfide bond between the proximal Cys residues. Thus, it seems that Cys-172 plays by itself a remarkable role in the structure and catalytic properties of Rubisco.

The substitution of Cys-172 by Ser increases the K m for both gaseous substrates (Table 1). Because CO 2 and O 2 do not bind to the enzyme before reacting with the enediolate form of RuBP, the increased K m values might result from a structural change that causes a restricted accessibility of the bound enediolate RuBP to CO 2 and O 2 . However, because the Ω value of the C172S enzyme is also altered (Table 1), it is more likely that conformational changes occur that affect the stabilities of the carboxylation and oxygenation transition states differently [START_REF] Chen | How various factors influence the CO 2 /O 2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. Cys-172 is situated in β-strand 1 of the α/β-barrel, which forms the scaffold for the active site [START_REF] Andersson | Structure of the active site of ribulose-bisphosphate carboxylase[END_REF]. Thus, replacement of this residue in the barrel may affect active-site residues. Such perturbations, however subtle, may have dramatic consequences for the kinetics of the reaction. Indeed, substitution of either of the flanking residues, Gly-171 or Thr-173, in C. reinhardtii Rubisco eliminates carboxylase activity [START_REF] Spreitzer | Genetic dissection of Rubisco structure and function[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

backbone atoms of β-strand 1 of the α/β-barrel. This shift could disturb the geometry of the active site as well as the position of individual residues in the active site. In particular, the interactions of the proximal Thr-173, hydrogen bonding to the C2hydroxyl of the CABP transition-state analogue [START_REF] Knight | Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site[END_REF], and perhaps Lys-175, which is thought to participate as a secondary proton acceptor in generating the reactive enediolate [START_REF] Cleland | Mechanism of Rubisco: The carbamate as general base[END_REF], are likely to be strained by the displacement of the β-strand. In contrast, the C192S mutation, which produces relatively minor alterations of enzymatic properties (Table 1), does not involve structural changes around the catalytic site.

Because the effect of C172S on enzyme kinetics appears to be structural and indirect rather than resulting from the elimination of the redox active sulfhydryl group, the particular impairment imposed on the catalytic site by the substitution might be expected to depend on the volume and nature of the substituting residue.

Accordingly, the replacement of Cys-172 by Ala in a prokaryotic Rubisco produced only a limited increase of K c but a significant reduction of V c [START_REF] Marcus | Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation[END_REF]. In this regard, the modification of Cys-172 with the bulky N-bromoacetylethanolamine phosphate affinity label is likely to cause a stronger structural distortion resulting in activity loss [START_REF] Schloss | Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate bromoacetylethanolamine phosphate[END_REF].

Another pair of proximal Cys residues, Cys-449 and Cys-459, are located close to the surface and about 20 Å away from the 172/192 pair. A survey of all available C. reinhardtii Rubisco structures to date [START_REF] Taylor | First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii[END_REF][START_REF] Mizohata | Crystal structure of activated ribulose-1,5-bisphosphate carboxylase/oxygenase from green alga Chlamydomonas reinhardtii complexed with 2-carboxyarabinitol-1,5bisphosphate[END_REF][START_REF] Karkehabadi | Altered intersubunit interactions in crystal structures of catalytically compromised ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Karkehabadi | Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF] revealed that whereas the sulfur to sulfur distance of the 172/192 pair remains fairly constant, the distance of the 449/459 pair varies considerably. This variation is also illustrated by the present structures. In C172S, the sulfur to sulfur distance of the 449/459 pair was similar to that of wild-type, but in C192S this distance was considerably lengthened and there was no sign of a covalent bond in the electron density maps. This variation at a location close to the molecular surface may suggest a role for the 449/459 pair as a redox sensor. It is not clear at this stage if a long-distance structural connection exists between both redox centers or if some of the redox effects associated with the C172S substitution [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF] might in fact result from the perturbation of the redox properties of Cys-449 and Cys-459. However, the in vitro and in vivo redox properties of mutant enzymes in which Cys-449 and Cys-459 have been replaced by Ser are very different from those of C172S [START_REF] Marin-Navarro | Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress[END_REF]. Therefore, a physiologically relevant effect resulting from the cooperation between the two Cys pairs seems unlikely.

It is noteworthy that the C172S substitution produces a modest but significant increase in Ω (Table 1). Changes in Ω have been proposed to result from differences in the differential stabilization of the carboxylation and oxygenation transition states [START_REF] Chen | How various factors influence the CO 2 /O 2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Chen | Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii[END_REF]. It has been recently hypothesized by Tcherkez and co-authors [START_REF] Tcherkez | Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized[END_REF] that an increase in Ω should result in a decreased V c , and that a negative correlation between Ω and K c may also exist [START_REF] Tcherkez | Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized[END_REF]. These predictions are, however, not fulfilled in the case of the C172S mutant enzyme, which displays an increased Ω value without alteration of V c but with a significant increase of K c (Table 1).

Considering that Cys-172 is a critical residue that renders Rubisco inactive when oxidatively modified by a number of agents [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF][START_REF] Marcus | Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation[END_REF][START_REF] Schloss | Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate bromoacetylethanolamine phosphate[END_REF], one may wonder why it is so highly conserved and why it has not been substituted by Ser during evolution for the gain of substrate specificity and oxidative resistance. Moreover, the only apparent disadvantages of the C172S substitution would be a slight structural destabilization that does not seem to be relevant at physiological temperatures (Figure 1) and increases in K c and K o (Table 1). Despite the increased Ω value, the increase in K c would likely cause a decrease in net CO 2 fixation, which is determined by the difference in the velocities of carboxylation and oxygenation at physiological concentrations of CO 2 and O 2 [START_REF] Laing | Regulation of soybean net photosynthetic CO 2 fixation by the interaction of CO 2 , O 2 and ribulose 1,5diphosphate carboxylase[END_REF]. Nevertheless, the C. reinhardtii C172S mutant has been shown to grow at the same rate as the wild type under photoautotrophic conditions [START_REF] Moreno | C172S substitution in the chloroplastencoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase[END_REF]. There may be, however, additional reasons for the conservation of Cys-172. Perhaps it is precisely its ability to act as an oxidation-activated switch, as a part of a wider design of redox control of CO

  Cys residues 172 and 192 of the large subunit are highly conserved among both eukaryotic and cyanobacterial Rubiscos. Their structural vicinity, potentially allowing for a disulfide bond to form between them, could be a possible source of redox regulatory effects on the enzyme. Analysis of the high-resolution (1.4 Å) structure of the wild-type C. reinhardtii enzyme [23] clearly shows the presence of free Cys-172 and Cys-192 residues in the electron density maps. The distance and geometries of the two residues in activated Rubisco are also not compatible with the S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071422 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071422 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Our X-ray diffraction analysis of the C172S mutant Rubisco indicates that the substitution to Ser at position 172 causes a significant shift of the main chain S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071422 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071422

Figure 1 .

 1 Figure 1. Thermal inactivation of C172S ( ), C192S ( ), and wild type ( ) Rubiscos. Purified Rubisco (0.2 mg/ml) was incubated at the given temperatures for 30 min (A), or at 55 ºC for the indicated times (B). Afterwards, the Rubisco samples were chilled on ice for 5 min, and assayed for RuBP carboxylase activity at 30 ºC. Activity values are given as a percentage of the activity at 30 ºC (1.29, 1.27 and 1.97 µmol of CO 2 fixed per minute and mg of enzyme, respectively for C172S, C192S and wild type Rubiscos), which was taken as the initial value in B. Error bars indicate ± standard deviation of independent triplicates. Spline interpolated curves have been included in A.

Figure 2 .

 2 Figure 2. Displacement of β-strand 1 in the C172S mutant enzyme relative to wild-type Rubisco. (A) Cα trace showing β-strands in the α/β-barrel. (B) Structural differences around residues 172 and 192 and part of the active site in the C172S mutant enzyme relative to wild type. In the wild-type structure, carbon atoms are colored blue, and in C172S carbon atoms are colored yellow. In 2CABP, in the lower right of (B), phosphorus atoms are colored magenta.

Figure 3 .Figure 2 23S

 32 Figure 3. Region around residues Cys449 and Cys459 in the C172S and the C192S mutant enzymes. In C172S, carbon atoms are colored yellow. In C192S, carbon atoms are coloured gray.

Figure 3 S

 3 Figure 3 S t a g e 2 ( a ) P O S T -P R I N T

Table 1 Kinetic properties of Rubisco from wild-type C. reinhardtii, C172S and C192S mutants.

 1 2 fixation and Rubisco turnover, what has prevented the substitution of this residue during evolution.

	carboxylase/oxygenase	modified	by	the	affinity	label	N-

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by Grants from the Spanish MCYT (BMC2003-03209 and BFU2006-07783), Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (FORMAS), the European Union (No. QLK3-CT-2002-01945), and U. S. Department of Agriculture National Research Initiative (No. 2006-35318-17376).


Dr Katarina Luciakova
email: katarina.luciakova@savba.sk

Gabriel Kollarovic

Peter Barath

Buck Dean

GROWTH-DEPENDENT REPRESSION OF HUMAN ADENINE NUCLEOTIDE TRANSLOCASE-2 (ANT2) TRANSCRIPTION: EVIDENCE FOR THE PARTICIPATION OF SMAD AND SP FAMILY PROTEINS IN THE NF1-DEPENDENT REPRESSOR COMPLEX

Keywords: adenine nucleotide translocator-2, NF1, repression, transforming growth factor-1, human diploid fibroblasts, protein interaction

Growth-dependent repression of human adenine

nucleotide translocase-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex

INTRODUCTION

The Nuclear factor 1 (NF1) transcription factors are encoded in four genes (Nf1-a, Nf1-b, Nf1-c and Nf1-x) [START_REF] Gronostajski | Roles of the NFI/CTF gene family in transcription and development[END_REF] that are expressed as a large number of splice variants [START_REF] Santoro | A family of human CCAAT/box/binding proteins active in transcription and DNA replication> cloning and expression of multiple cDNAs[END_REF][START_REF] Rupp | Chicken NF1/TGGCA proteins are encoded by at least three independent genes> NFI-A, NFI-B, and NFI-C with homologues in mammalian genomes[END_REF][START_REF] Kruse | The genes for transcription factor nuclear factor I give rise to corresponding splice variants between vertebrate species[END_REF].

The expressed forms of NF1 bind to DNA as homo-or hetero-dimers [START_REF] Kruse | Transcription factor nuclear factor I proteins form stable homo-and heterodimers[END_REF][START_REF] Gounari | Amino-terminal domain of NF1 binds to DNA as a dimer and activates adenovirus DNA replication[END_REF], thus creating the potential for a large and varied transcriptional network in which NF1 family members activate or repress transcription depending on promoter and cellular context [START_REF] Gronostajski | Roles of the NFI/CTF gene family in transcription and development[END_REF]. Many genes are dependent upon NF1 for proper and optimal expression, and NF1 targets the promoters of genes with widely varying functions (reviewed in [START_REF] Gronostajski | Roles of the NFI/CTF gene family in transcription and development[END_REF]. NF1 has also been implicated in several growth [START_REF] Nakamura | Nuclear factor 1 is a negative regulator of gadd153 gene expression in vascular smooth muscle cells[END_REF][START_REF] Furlong | YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity[END_REF] and developmental [START_REF] Das Neves | Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum[END_REF][10][START_REF] Steele-Perkins | Essential role for NFI-C/CTF transcription-replication factor in tooth root development[END_REF] processes, but never in a central regulatory capacity.

We recently described a unique role for NF1 as an active repressor of gene expression in serum-starved cells [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF] using the human adenine nucleotide translocator-2 (ANT2) gene as a model [START_REF] Luciakova | Differential regulation of the transcript levels of some nuclear-and mitochondrial-encoded respiratory chain components in response to growth activation[END_REF]. ANT proteins, which play an important role in maintaining cellular energy homeostasis by catalyzing the exchange of cytosolic ADP for mitochondrial ATP, are encoded in four genes [START_REF] Dolce | A fourth ADP/ATP carrier isoform in man: identification, bacterial expression, functional characterization and tissue distribution[END_REF]. The ANTs have also been implicated as both positive and negative regulators of apoptosis [START_REF] Bras | Chemosensitization by knockdown of adenine nucleotide translocase-2[END_REF]. ANT2 differs from the other ANT isoforms in that its expression is regulated by the growth-and differentiation-states of cells. In general, ANT2 expression is repressed in growth inhibited [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF][START_REF] Barath | The growth-dependent expression of the adenine nucleotide translocase-2 (ANT2) gene is regulated at the level of transcription and is a marker of cell proliferation[END_REF][START_REF] Battini | The multiple ADP/ATP translocase genes are differentially expressed during human muscle development[END_REF] or differentiating [START_REF] Battini | The multiple ADP/ATP translocase genes are differentially expressed during human muscle development[END_REF][18][START_REF] Stepien | Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation[END_REF] cells. Furthermore, reactivation of growth-inhibited cells activates ANT2 expression on the same time scale as the 3 immediate-early genes [START_REF] Battini | The multiple ADP/ATP translocase genes are differentially expressed during human muscle development[END_REF], suggesting a functional role in the early events of cell growth. In spite of this, the importance of growth-modulated ANT2 expression remains unclear.

Growth-arrest repression of ANT2 is mediated through NF1 bound to two upstream repressor elements (Go-1 and Go-2) [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF][START_REF] Barath | The growth-dependent expression of the adenine nucleotide translocase-2 (ANT2) gene is regulated at the level of transcription and is a marker of cell proliferation[END_REF]. The Go-1 and-2 repressor elements are occupied by NF1 only in the growth-arrested state, and are required and sufficient for NF1-mediated transcriptional repression [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF]. Since ANT2 expression is maintained by Sp1 bound to two juxtaposed Sp1 activation-elements (AB boxes) in the proximal promoter [START_REF] Li | Sp1 activates and inhibits transcription from separate elements in the proximal promoter of the human adenine nucleotide translocase 2 (ANT2) gene[END_REF], NF1 either directly or indirectly regulates the activity of Sp1.

In the present work, we demonstrate that, when acting as a growth-dependent repressor, NF1 is a component of a protein complex that includes members of the Smadand Sp-family proteins. This repressor complex contacts both the upstream NF1-binding repressor elements and the proximal promoter Sp1-dependent activation element. In growth-activated cells, NF1 leaves the promoter and the multimeric repressor complex dissociates. The presence of Smad proteins in the NF1 repressor complex, together with our observations that NF1 and Smad proteins interact physically, and that transforming growth factor-beta (TGF-β) represses ANT2 transcription in normally growing cells, suggest a functional collaboration between NF1 and Smad that has not previously been observed.

EXPERIMENTAL

Cell culture. Human primary diploid foreskin fibroblasts in passages 7-20 were grown as described [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF]. To arrest cell growth, cells were washed twice with phosphatebuffered saline and incubated for 48 h in serum free media. For growth-activation, serum starved cells were grown an additional 24 h in the presence of 10% serum.

Expression plasmids. The GST-Sp1, GST-Smad2, -Smad3, -Smad4 and the GST- As a template the appropriate full length Smad in pGEX was used. Amplified MH domains were cloned into the pGEX-6P-1 (Amersham Biosciences) and verified by sequencing. cDNAs coding the NF1-A1, -B2, -C2 and -X1 were provided by Dr. M.

Imagawa (Nagoya City University, Japan). For in vitro transcription/translation, inserts containing the whole coding region of individual NF1s were recloned into the expression vector pETM11 (EMBL, Heidelberg).

Treatment of cells with TGF-β. Cells in complete medium were treated with 1 ng/ml of TGF-β for 48 h [START_REF] Poncelet | Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human glomerular mesangial cells[END_REF].

Preparation of nuclear extract. Cells from starved and serum-induced human diploid fibroblasts were harvested, nuclei were prepared [22] and nuclear proteins were extracted as described in [START_REF] Barath | Identification of NF1 as a silencer protein of the human adenine nucleotide translocase-2 gene[END_REF]. The protein was measured using the Bio-Rad Protein Assay (Bio-Rad). Nuclear extracts were stored at -70 0 C.

Immunoprecipitation.

Antibodies were covalently linked to Protein A-Sepharose using the Seize X Protein Immunoprecipitation Kit (Pierce). Binding, antigen immunoprecipitation and elution of the antigen was performed according to the manufacturer's manual.

SDS-PAGE and Western blot analysis.

Eluted immunoprecipitated samples were precipitated for 20 min on ice in 10% trichloroacetic acid, followed by a 15-min centrifugation at 10 000 x g and two washes with ice-cold acetone. Samples were airdried, dissolved in sample buffer and separated by 10% SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] After electrophoresis, samples were electroblotted onto the Hybond ECL membrane (Amersham Biosciences). Membranes were incubated with appropriate antibodies and developed with SuperSignal West Pico Chemiluminiscent Substrate (Pierce).

Chromatin immunoprecipitation.

Chromatin immunoprecipitation from growtharrested and growth-induced human diploid cells was performed as in [START_REF] Weinmann | Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation[END_REF], except that 100 µl of Protein A-Sepharose was used instead of Staphylococcus aureus cells.

Immunoprecipitation was performed with either 2 µl of antiserum against NF1 (#8199, kindly provided by Dr. Tanese), or 2 µg of antiserum against Smad4 (sc-7154, Santa In vitro and bacterial expression of proteins. In vitro expression and radiolabeling of proteins was performed using the Promega coupled transcription/translation system (TNT). Plasmids were transcribed using the T7 RNA polymerase according to the manufacturer's manual. The quality of translated proteins was verified by SDS-PAGE.

Glutathione S-transferase (GST) fusion proteins were expressed in E. coli strain BL21, and purified as described in [26]. Solubilization and purity of the expressed proteins were monitored by SDS-PAGE.

Pull-down experiments. 1μl of the 35 GGGTCAAGCTGCTGCTGCAGG-3'), respectively. The 3'-primer of ANT2 covered nts +509/+532 (5'-CGGAATTCCCTTTCAGCTCCAGC-3'), and the 3'-primer for ANT3 covered nts +443/+464 (5'-CCGGCCGCACCGCCGGAGGC-3'). PCR reaction was performed for 28 cycles with 1 min of denaturation at 94 0 C, 30 s annealing at 63 0 C and extension for 1 min at 72 0 C. PCR products were separated on 2% agarose and stained with ethidium bromide.

RESULTS

The ANT2 promoter contains 3 repressor elements that modulate the activity of Sp1 p300 interacts directly with Smad proteins [START_REF] Pardali | Role of Smad proteins and transcription factor Sp1 in p21(Waf1/Cip1) regulation by transforming growth factor-beta[END_REF][START_REF] Park | p300 cooperates with Smad3/4 and Runx3 in TGFbeta1-induced IgA isotype expression[END_REF] and functions as a co-activator.

We therefore tested if p300 acts as a co-activator on the ANT2 promoter. Antibodies to p300 co-precipitate Smad4 from nuclear extracts of serum-activated cells, but not from serum-starved cells (Fig 6A). Thus, formation of p300/Smad4 complexes is also growth- The inclusion of Smad proteins in the Go-repression complex suggests that TGF-β might be a signaling ligand in ANT2 repression. To test this we measured ANT2 transcripts in normally growing diploid fibroblast treated with TGF-β. The results (Fig 8)

show that TGF-β prevents ANT2 expression in growing cells, and that the magnitude of the TGF-β inhibitory effect is similar to that obtained by serum removal (Fig 8, ref. [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF].

As expected [START_REF] Dörner | Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans[END_REF], serum starvation does not effect the expression of ANT3 (Fig 8). grown to confluence [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF][START_REF] Barath | The growth-dependent expression of the adenine nucleotide translocase-2 (ANT2) gene is regulated at the level of transcription and is a marker of cell proliferation[END_REF]. We earlier mapped and identified two upstream promoter elements (Go-1 and Go-2) required for repression [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF]. These elements bind NF1, which acts as an active repressor. To gain insight into the mechanism by which NF1 represses Sp1-dependent activation, we studied the proteins associated with the Go and AB box elements in growth-repressed and growth-activated human skin fibroblasts. We show that NF1 bound to the Go elements in growth-repressed cells is part of a larger repressor complex that includes both Smad and Sp-family proteins. Figure 9 Whatever the nature of the complexes, growth-activation results in an extensive reorganization on the promoter in which NF1 and the associated Smad and Sp proteins leave the Go element. These findings are in accord with our earlier failure to detect protein binding to the Go elements in growth-activated cells using in vivo foot printing [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF]. Release of the repressor complex from the Go elements in growth-activated cells is also accompanied by reorganization around the AB elements (Fig 9). Sp1 remains on the AB boxes, but is no longer associated with NF1, Smad2/3 or Sp3. Sp1 is, however, associated with Smad4. Reorganization of Sp1 bound to the AB elements in growthactivated cells is also suggested by a shift in the specific nucleotides contacted by Sp1 [START_REF] Luciakova | In vivo mapping of the human adenine nucleotide translocator-2 (ANT2) promoter provides support for regulation by a pair of proximal Sp1-activating sites and an upstream silencer element[END_REF].

NF1 binding to the Go element in the growth-repressed state, and its release in the growth-activated state [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF], suggest a central role for this protein in ANT2 repression.

However, the mechanism of NF1 recruitment to the promoter and the order in which the components of the repressor complex are assembled is difficult to assess since Sp1 only one of many possible mechanisms by which assembly of a repressor complex might be regulated, especially as the cytoplasmic/nuclear distribution of Smad proteins is a dynamic process, and some of these proteins are present in the nucleus even in nonstimulated cells [START_REF] Schmierer | Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads[END_REF][START_REF] Nicolas | Endogenous TGF-beta activity is modified during cellular aging: effects on metalloproteinase and TIMP-1 expression[END_REF]. Furthermore, all three proteins can be modified by various combinations of phosphorylation, acetylation or Sumoylation, and are, thus, potential targets for signaling pathways that initiate repressor assembly/disassembly. Despite these complications, our observation that TGF-β induces repression of the ANT2 promoter in proliferating human diploid fibroblasts implies involvement of a Smad-dependent pathway. Furthermore, since TGF-β is released from serum-starved human diploid fibroblasts [39] and is responsible for initiating a senescence response [39], it is a prime candidate as the ligand that initiates ANT2 repression in the absence of serum.

A mechanism of NF1-dependent repression similar to that described here and in our previous studies [START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF] on the ANT2 promoter was recently reported for the p21Waf1/Cip1

promoter in human skin fibroblasts [START_REF] Ouellet | Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells[END_REF]. With both promoters, NF1 binding represses transcription, whereas release of NF1 is associated with transcriptional activation.

Furthermore, Sp3 is associated with NF1 on both promoters in the repressed state, but not in the activated state (discussed below). However, regulation of the two genes by NF1 differs in that repression of ANT2 takes place in growth-arrested human skin fibroblasts, whereas p21 repression occurs in proliferating cells. Thus, NF1, acting as a direct repressor, is able to regulate genes with vastly different growth-related functions in skin fibroblasts.

NF1 has been implicated in a variety of growth-related processes, even though a central role for NF1 in growth regulation has not been established. NF1 represses gadd 153 [START_REF] Nakamura | Nuclear factor 1 is a negative regulator of gadd153 gene expression in vascular smooth muscle cells[END_REF] and activates p53 [START_REF] Furlong | YY1 and NF1 both activate the human p53 promoter by alternatively binding to a composite element, and YY1 and E1A cooperate to amplify p53 promoter activity[END_REF] expression, both of which have key roles in growth arrest of damaged or stressed cells. Furthermore, NF1-X, together with c-myc and mdm2, were the only genes identified in a screen performed to identify complementary DNAs that prevent TGF-β-induced growth arrest of mink lung epithelial cells [28]. Finally, expression of NF1 in chick embryo fibroblasts has also been reported to prevent transformation induced by nuclear, but not cytosolic, oncogene products [START_REF] Schuur | Nuclear factor I interferes with transformation induced by nuclear oncogenes[END_REF]. These NF1-mediated repression of ANT2 described here is reminiscent of the TGFdependent repression of the myc and Id1 genes which are mediated through Smads cooperating with a transcription factor acting as a co-repressor. Myc repression is mediated through the p107 repressor protein which is recruited to a Smad/E2F/DP1 complex [START_REF] Chen | E2F4/5 and p107 as Smad cofactors linking the TGFbeta receptor to c-myc repression[END_REF], and Id1 repression is mediated via ATF3 in cooperation with Smad3 [START_REF] Kang | A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells[END_REF].

In the case of ANT2, assembly of the NF1/Smad complexes on the promoter is also associated with recruitment of a known repressor protein, Sp3. Sp3 is associated with a NF1-repressor complex on both the Go and the AB box elements in growth-arrested cells, and is released from the promoter in growth-activated cells. Similarly, Sp3, but not Sp1, is bound to the NF1-repressed p21Waf1/Cip1 promoter in proliferating skin fibroblasts [START_REF] Ouellet | Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells[END_REF]. These results suggest that Sp3 could be a specific component of the NF1-dependent repression machinery. Sp3, unlike Sp1, is a strong transcriptional repressor [START_REF] Hagen | Sp1-mediated transcriptional activation is repressed by Sp3[END_REF].

Silencing is dependent on the lysine residue in an IKEE repressor motif, which, when mutated, converts Sp3 into a transcription activator [START_REF] Braun | Transcription factor Sp3 is regulated by acetylation[END_REF]. In vivo acetylation of Sp3 activates expression of the TGF-β type II receptor [START_REF] Ammanamanchi | Sp3 is a transcriptional repressor of transforming growth factor-beta receptors[END_REF] and the SOCS [START_REF] Ehlting | Sp3 is involved in the regulation of SOCS3 gene expression[END_REF] genes, suggesting a possible signaling mechanism for the Sp3 repressor/activator switch.

However, the IKEE motif is also Sumoylated, which, like acetylation, eliminates Sp3 silencing and promotes Sp3 activation [START_REF] Ross | SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization[END_REF][START_REF] Sapetschnig | Transcription factor Sp3 is silenced through SUMO modification by PIAS1[END_REF][START_REF] Sapetschnig | Complexity of translationally controlled transcription factor Sp3 isoform expression[END_REF]. Thus, the specific role of Sp3 in ANT2 repression remains to be sorted out.

To our knowledge, no structural or functional associations between members of Smad and NF1 families have been reported previously, even though, as discussed above, several reports suggest interfacing between the NF1 proteins and TGF-mediated processes. However, the present study demonstrates clearly: a) that Smad and NF1 proteins interact physically, as shown by in vitro pull-down assays, and b) that nuclear complexes of Smad/NF1 are formed and disassembled in human skin fibroblasts in a growth-state dependent manner. These findings, coupled to our observation from ChIP analysis that Smad proteins are components of a NF1 complex that acts to repress ANT2 A representative picture of three independent experiments is shown.

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T

Smad4

  MH1 plasmids were generously provided by Dr. A. Moustakas (Ludwig Institute for Cancer Research, Uppsala, Sweden). The Smad2 MH1 domain was prepared by amplifying the domain from amino acid 1 to 251, and the MH2 domain was prepared by Biochemical Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 4 amplifying the domain from amino acid 251 to 467. Similarly, the Smad3 MH1 (amino acid 1 to amino acid 199) and MH2 (amino acid 199 to amino acid 425) domains were prepared; and the Smad4 MH2 domain (amino acid 321 to amino acid 552) was prepared.

Biochemical 5 Cruz

 5 Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T Biotechnology), Smad 1/2/3 (sc-7960, Santa Cruz Biotechnology), Sp1 (sc-59, Santa Cruz Biotechnology), Sp3 (sc-644, Santa Cruz Biotechnology), Smad 2 (sc-6200, Santa Cruz Biotechnology) and p300 (sc-585, Santa Cruz Biotechnology). Amplification of immunoprecipitated DNA fragments (2 µl) was performed using the primers -931 (5'-AGTTCTTAACCTTCCTAAGCCTC-3') and -666 (5covering the AB boxes. PCR was performed for 26-30 cycles, with 45 s of denaturation at 94 0 C, followed by 45 s of annealing at 60 0 C and 45 s of extension at 72 0 C. The last step included extension for 10 min at 72 0 C.

  on the AB box (Fig1). However, repression of ANT2 transcription in growth-arrested cells (either by serum starvation or by growth to confluence) is mediated solely through NF1-binding elements (Go-1 and Go-2) in the GoR[START_REF] Luciakova | Repression of the human adenine nucleotide translocase-2 gene in growtharrested human diploid cells: the role of nuclear factor-1[END_REF][START_REF] Barath | The growth-dependent expression of the adenine nucleotide translocase-2 (ANT2) gene is regulated at the level of transcription and is a marker of cell proliferation[END_REF]. The ANT2 promoter also contains a number of putative Smad binding elements (5'-GTCT-3', or 5'-AGAC-3') that are distributed around and between the Go-1 and Go-2 elements, between the two Silencer elements, and immediately upstream of the Sp1 activating elements (Fig1). This distribution, together with data suggesting a link between NF1 and the TGF-β signaling system [28], raised the question if Smad proteins could play a role in NF1dependent repression of ANT2 in serum-starved cells.To address this issue, we first carried out a series of co-immunoprecipitation experiments to determine if NF1 and Smad proteins interact, either directly or as components of a common multimeric complex. Experiments were done on nuclear extracts from serum-starved (growth-arrested) and serum re-fed (growth-activated) diploid fibroblasts (Fig2). The existence of Smad/NF1 nuclear complexes could be demonstrated by co-immunoprecipitation with antibodies to both Smad4 (Fig.2, left panel) and NF1 (Fig 2, right panel). Unexpectedly, NF1/Smad4 complexes were detected in nuclear extracts predominantly, if not exclusively, in serum-starved cells (Fig 2, lane S vs. I). Thus, formation of nuclear complexes containing NF1 and Smad protein appears to be growth-state dependent. To determine if Smad/NF1 nuclear complexes are associated with the ANT2 promoter, chromatin immunoprecipitation (ChIP) analysis was performed on chromatin Biochemical Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 7 isolated from serum-starved and serum re-fed cells. The results show that Smad4 (Fig 3A, upper panel, compare lanes 4 and 5) and NF1 (Fig 3B, lanes 4) are both associated with the Go-2/Go-1 elements in serum-starved cells. By contrast, neither protein is associated with the ANT2 promoter in growth-activated cells, suggesting that release of NF1 is accompanied by release of Smad4. However, Smad4 is detected on the AB box in re-fed cells (Fig 3A, lower panel, lane 5). The growth-activated loss of NF1 and Smad4 from the Go repressor and the appearance of Smad4 associated to the AB activation boxes suggest a re-organization of the repressor complex in growth activated cells. We also tested for the presence of R-Smads (Smad2/3) [29] on the GoR and AB box elements. ChIP analysis using antibodies to Smad2/3 or Smad2 show that Smad2, and perhaps also Smad3, are associated with both the GoR and the AB box regulatory elements in serum-starved cells (Fig. 4A, lanes 4), but to neither of these elements in growth-activated cells (Fig 4A, lanes 5). Thus, Smad2/3 is associated with both the Go and AB elements, but only in the repressed state. However, co-immunoprecipitation of nuclear extracts (Fig 4B) show that NF1/Smad2/3 complexes are present in the nucleus under all conditions of growth, suggesting that they may not dissociate after leaving the Go repressor element. By contrast, nuclear complexes containing both Smad4 and Smad2/3 appear to decrease in serum-activated cells (Fig 4B, lane I), suggesting that the Smads might dissociate upon growth-activation. Since co-precipitation experiments probe the total population of nuclear complexes, not just the subset of those bound to the ANT2 promoter, identification of Smad/NF1 complexes by this method provides reason to believe that such complexess participate in the regulation of additional genes. The association of NF1/Smad complexes to the AB box is most probably mediated through the Sp proteins. We, therefore, conducted ChIP analysis of Sp1 and Sp3 binding to the ANT2 promoter in serum-starved and serum-activated cells (Fig 5). Sp3 was included in the analysis since it binds the same element as Sp1, but is the only member of the Sp family that acts as a repressor [30]. Sp1 (Fig 5A, lane 4) and Sp3 (Fig 5B, lane 4) are associated with both the Go element and the AB box elements in serum-starved cells, suggesting that these elements might be linked through a larger repressor complex. Upon serum-reactivation, Sp3 is no longer detected (Fig 5B, lanes 5) and appears to leave the Biochemical Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 8 promoter, while Sp1 remains bound only to the AB boxes (Fig 5A, lower panel, lane 5). Thus, it would appear as though Sp1 and Sp3 are anchored to the Go repressor and the AB activation boxes together with NF1 and Smad2/3 in growth-arrested cells. However, upon re-feeding with serum Sp3 leaves the promoter while Sp1 remains anchored to the AB activation element (Fig. 5, lane 5, lower panels). To gain further insight into the repressor complex, we investigated the association between the Sp and Smad proteins under different growth states. Sp1 antibodies coprecipitate Smad4 from nuclear extracts prepared from both serum-starved and re-fed cells (Fig 6A), indicating the presence of Sp1/Smad4 nuclear complexes under all conditions of growth. However, a second Smad protein is also co-precipitated by Sp1 antibodies, but only from serum-starved cells (Fig 6A, asteriks). The growth-state dependency of this latter Smad/Sp1 complex suggests a possible role in G0 repressor assembly/disassembly. However, the identity and functional significance of this Smad protein remains unknown. In contrast to the presence of nuclear Sp1/Smad4 complexes in both serum-starved and serum-refed cells, Sp3/Smad4 complexes are detected by co-immunoprecipitation only in nuclear extracts of serum-starved cells (Fig 6C). Thus, the dissociation of nuclear Sp3/Smad4 complexes appears to be related to the growth repression/activation transition in diploid fibroblasts. This conclusion is strengthened by ChIP analysis showing that Sp3 leaves the AB boxes in growth-activated cells (Fig 5B), resulting in the Sp1-dependent activation of ANT2 expression [20]. Finally, Sp1 antibodies co-precipitate NF1 from nuclear extracts prepared from growth-arrested cells, but not from growth-activated cells (Fig 6B). Thus, as with the Sp3/Smad4 complexes, formation of nuclear Sp1/NF1 complexes in human diploid fibroblasts is growth-state dependent. This conclusion is also consistent with ChIP experiments showing that Sp1/NF1 complexes bind to the ANT2 promoter only in serumstarved, growth-inhibited cells (Fig 3)

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 9 state dependent in diploid fibroblasts, favoring the growth-activated state. However, even though nuclear p300/Smad4 complexes are formed in growth-activated fibroblasts (Fig 6A), a significant increase in p300 binding to the AB activation boxes could not be detected by ChIP analysis (Fig 5A, lower panel). Thus, p300 may not act as a coactivator on the ANT2 promoter. Antibodies to p300 co-precipitated little or no NF1 from nuclear extracts of either growth-arrested or growth-activated cells (Fig 6B), suggesting that the population of nuclear complexes containing both p300 and NF1 is relatively small, if existent, in human diploid fibroblasts. This result supports our conclusions from ChIP analysis that p300 is not associated with the G0 repressor complex (Fig 5A). The question remains if nuclear NF1/Smad complexes are formed through direct or indirect physical contacts. Smad [31, 33] and NF1 [34] interact directly with Sp1, providing a possible assembly pathway for NF1/Smad-containing complexes. To test for physical interactions, GST fusion proteins (full length Smads or their MH1 or MH2 domains) were used to pull-down in vitro translated, radiolabeled full length NF1s. Smad3 and Smad4 interact relatively strongly with the NF1-A and -C (Fig 7, left panel) through their MH1 domain (Fig 7, right panel). Smad3 and Smad4 appear to interact more weakly with full length NF1-B and NF1-X (left panel). However, pull down with the MH domains (right panel) suggests that Smad3 and Smad4 might interact with NF1-X through the MH1 domain. Interactions of Smad2 are less clear. Though it appears to interact as strongly as Smad3 and 4 with full length NF1-A (Fig 7, left panel), this was not confirmed using the MH-domain fusion proteins (Fig. 7, right panel). Taken together, results in Fig. 7 show for the first time a direct physical interaction between the transcription factor NF1 and Smad2, Smad3 and Smad4.

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 10 DISCUSSION Expression of the human ANT2 gene is repressed in serum starved cells and in cells

  summarizes the proteins found by ChIP analysis to be associated with the Go and AB box elements in growth-activated and growth-repressed cells. Complexes containing NF1, Smads2/3, Smad4, Sp1 and Sp3 are associated with both the Go and AB Box elements in serumstarved cells. These data could be explained by the binding of separate and distinct complexes to each set of elements, as depicted in Fig 9. However, it is equally feasible that NF1 and Sp proteins anchored to the Go and AB box elements, respectively, are linked in a repressor complex that includes Smad proteins.

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 11 interacts directly with the Smads [31, 33] and NF1 [34], and, as demonstrated in the present study, NF1 also interacts with Smads. The dynamic redistribution of Smads is

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 12 studies, together with the present results demonstrating the existence of NF1/Smad nuclear complexes (Figs. 2 and 4B), suggest that NF1 may have a more general role in regulating growth-related processes than previously appreciated.

Biochemical

  Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T 13 expression, raise the possibility that NF1/Smad may have a broader function in the regulation of gene expression than heretofore appreciated. region; Go-1 and Go-2, Go NF1-binding repressor elements 1 and 2; TGF-β, transforming growth factor-β; GST, glutathione S-transferase; ChIP, chromatin immunoprecipitation. Biochemical Journal Immediate Publication. Published on 21 Jan 2008 as manuscript BJ20071440 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071440 S t a g e 2 ( a ) P O S T -P R I N T

Fig. 1 .

 1 Fig. 1. The human ANT2 promoter. Positions of known regulatory regions are placed relative to the transcription start (arrow). Empty boxes represent the Sp1 ABactivation elements. Three repressor regions, the GoR, Silencer and the C box are marked as grey boxes. Black ovals represent putative Smad-binding elements. The Go repressor and the Silencer bind NF1, whereas the C-box binds Sp1.

Fig. 2 .

 2 Fig. 2. NF1 and Smad4 co-immunoprecipitate in growth-arrested cells. To arrest cell growth, cells were washed twice with phosphate-buffered saline and incubated in serum free media for 48 h. To reactivate growth, serum starved cells were grown an additional 24 h in the presence of 10% serum. Nuclear extracts (500 μg) from serumstarved (S), and serum-induced (I) cells were isolated and immunoprecipitated (IP) with antibodies against Smad4 or NF1. Protein eluted from the ProteinA-Sepharose were resolved by SDS-PAGE, transferred to nitrocellulose membranes and probed with specific antibodies (WB for western blots). Arrows indicate the MW standards.A representative picture of four independent experiments is shown.

Fig. 3 . 20 Fig. 4 .

 3204 Fig. 3. NF1/Smad4 repressor complex binds to the GoR only in growtharrested human diploid cells. Chromatin immunoprecipitation was carried out on formaldehyde-crosslinked cells deprived of serum for 48 h (lanes 4), or serum-deprived and subsequently grown in the presence of serum (lanes 5). Chromatin was isolated and immunoprecipitated with: A) anti-Smad4 or B) anti-NF1 antibodies. DNA was amplifiedwith primers covering the Go-2/Go-1 repressor elements (nts -931/-666) and the AB box Sp1-activation elements (nts -151/+39). Primer amplification was also carried out on: a negative control containing no template (lanes 1), template isolated from chromatin which was not immunoprecipitated (lanes 2), chromatin immunoprecipitated with an unrelated antibody, anti-F1-ATPase (lanes 3), or total chromatin (lanes 6). A 100-bp gene ruler (MBI Fermentas) marker is also shown (M). A representative picture of four independent experiments is shown.

Fig. 5 .Fig 6 .Fig. 7 .Fig. 8 .

 5678 Fig. 5. Sp1 and Sp3 are associated with the Go repressor. A) Chromatin immunoprecipitation was carried out on serum-starved (lane 4 and 6) and serum-induced (lane 5 and 7) diploid fibroblasts using antibodies against Sp1 or p300. B) Chromatin immunoprecipitation was carried out on starved (lanes 4) and induced (lanes 5) cells using the antibodies against Sp3. DNA was amplified with primers covering the GoR (Go-2/Go-1) and the AB box (AB box). Lanes 1-3 are as described in Fig 3, lanes 8 (A) and 6 (B) is total chromatin. A representative picture of three independent experiments is shown.

Fig. 9 .Fig. 1 .

 91 Fig. 9. The role of NF1, Smad and Sp proteins in growth repression/activation of ANT2 gene. A complex containing NF1, Smad and Sp proteins binds to the GoR and Sp1 promoter elements in serum-starved cells. Upon activation of cell growth, NF1-Smad2/3 and Sp3 are released from the Go element, leaving a complex of Sp1 and Smad4 bound to the proximal Sp1 transcription-activation elements (AB box). The repressor elements of the GoR and the C box (see the main text) are depicted by grey boxes. The filled ovals are putative Smad binding elements. An arrow indicates the transcription initiation site, a cross indicates transcription inhibition.

Fig. 2 ,Fig. 3 , 24 Biochemical

 2324 Fig. 2, Luciakova et al.

Fig. 4 , 25 BiochemicalFig. 6 ,

 4256 Fig. 4, Luciakova et al.

Fig. 7 ,Fig. 8 , 29 BiochemicalFig. 9 , 30 Biochemical

 7829930 Fig. 7, Luciakova et al.

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( 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

This study was supported by the Slovak Science and Technology Assistance Agency (APVT) No. 26-002102 (KL), the Slovak Grant Agency VEGA No. 2/6060/26 (PB) and the Swedish Research Council (BDN). We thank Dr. M. Imagawa (Nagoya), Dr. A. Moustakas (Uppsala) and N. Tanese (New York) for generous gifts of NF1 isoform cDNA vectors, GST-Smad expression vectors and NF1-C antibodies, respectively.

The authors declare no conflict of interest.

The abbreviations used are: ANT, adenine nucleotide translocator; GoR, Go repressor