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First published online May 15, 2003; 10.1104/pp.103.023937 Plant Physiology 132:1097-1106 (2003) © 2003 American Society of Plant Biologists A Conserved 19-Amino Acid Synthetic Peptide from the Carboxy Terminus of Phosphoenolpyruvate Carboxylase Inhibits the in Vitro Phosphorylation of the Enzyme by the Calcium-Independent Phosphoenolpyruvate Carboxylase Kinase1Departamento de Biología Vegetal, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes Number 6, 41012 Seville, Spain (R.A., S.G.M.-M., A.-B.F., C.E.); and Institut de Biotechnologie des Plantes, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 8618, Bâhtiment 630, Université de Paris-Sud, Centre d'Orsay cedex, France (J.V.)
Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 µM). This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize [Zea mays]) enzymes.
In C4 plants, the photosynthetic phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a diel posttranslational regulation that alters its functional and regulatory properties (Echevarría et al., 1990  ; Chollet et al., 1996; Vidal et al., 1996). This is performed by a small molecular mass (32 kD) phosphoenolpyruvate carboxylase kinase (PEPCk) which phosphorylates the Ser of the enzyme's N-terminal domain (E/DR/KxxSIDAQL/MR; Jiao et al., 1991b). This Ser/Thr protein kinase is unique in that its regulation is via a fast protein synthesis event (Jiao et al., 1991a; Hartwell et al., 1999) after activation of a complex light transduction cascade (Vidal and Chollet, 1997; Coursol et al., 2000). It has been shown that this kinase belongs to the Ca2+/calmoduline kinase group of protein kinases, albeit that it does not possess any calcium-binding domain, lacks the autoinhibitory region, and does not require calcium for activity (Hartwell et al., 1999). On the other hand, its activity has been shown to be controlled both in vitro and in vivo by metabolites that also affect C4 PEPC activity, e.g. it is inhibited by L-malate, and this effect is counteracted by sugar-P-like glucose-6-phosphate (Echevarría et al., 1994; Chollet et al., 1996). Recently, a protein inhibitor of PEPCk has been characterized in plant extracts, yet its physiological role is still unclear (Nimmo et al., 2001). Furthermore, it has been proposed that the activity of maize (Zea mays) PEPCk could be subject to redox control via a thioredoxin system (Saze et al., 2001).
The molecular mechanism by which phosphorylation modifies C4 PEPC properties has been approached by site-directed mutagenesis. This showed that the introduction of a negative charge at the N-terminal Ser position was required for the changes to be observed (Wang et al., 1992
The PEPC protein kinase is believed to be dedicated to PEPC because it does not phosphorylate a variety of heterologous kinase substrates, whereas all plant PEPC isoforms examined to date serve as substrates in vitro (Chollet et al., 1996 The main objective of the present work was to address the question of the molecular basis for the interaction between the sorghum C4 PEPC and its dedicated PEPCk, focusing on the target's C-terminal domain. It is shown that C-terminal synthetic peptides, C19 and C15 (a truncated peptide lacking the terminal QNTG), specifically inhibit the in vitro phosphorylation of the enzyme in reconstituted assays, and the active domain is located downstream from the QNTG motif.
Partial Purification of Ca2+-Independent PEPCk
We first purified the Ca2+-independent PEPCk from protein extracts of illuminated sorghum leaves by using the blue dextran agarose (BDA) procedure described by Jiao and Chollet (1989
The well-conserved C terminus domain has been implicated in the maintenance of PEPC stability and catalytic activity of the enzyme (Dong et al., 1999
Our results demonstrate that two populations of C4 PEPC coexist in vitro, the less abundant one having an accessible C terminus. As a next step to this study, the hypothesis that the PEPCk may interact with this PEPC domain by using the C-terminal synthetic peptide C19 was assessed.
When added to a reconstituted assay containing partially purified BDA-PEPCk, immunopurified nonphosphorylated C4 PEPC, and the components of the phosphorylation reaction, the peptide C19 was able to markedly block PEPC phosphorylation by BDA-PEPCk (Fig. 3A, lane 3). The inhibitory effect of this peptide was also observed when in vitro phosphorylation was performed with a desalted protein extract from illuminated sorghum leaves (Fig. 3A, lanes 5 and 6). In contrast, neither the activity of the partially purified PEPC-CDPK (Fig. 3A, lanes 8 and 9) nor that of the catalytic subunit of the cAMP-dependent protein kinase (PKA; shown to be an efficient PEPC kinase, although so far, not found in plants; Terada et al., 1990
Because peptide C19 is rather hydrophobic, the specificity of its inhibitory action was investigated by the use of a set of synthetic peptides, designed from the reported three-dimensional structure of E. coli PEPC. Computerized modeling of the sorghum C4 PEPC using the bacterial enzyme structure indicated the presence of several exposed additional loops (these were used to define peptides L1, L2, and L3). Peptides L2 and L3 were as hydrophobic as peptide C19. When assayed in the reconstituted assay, they showed no effect on the phosphorylation reaction (Fig. 4a, lanes 2–4).
In addition, the BDA-PEPCk fraction was affinity purified using peptide C19 immobilized on agarose (HiTrap, Amersham-Pharmacia Biotech). This column partially, but significantly, retained the enzyme that was subsequently eluted by water (Fig. 4b, lanes 2 and 3). Elution by water (low ionic strength) suggested that the interaction mainly involved hydrophobic bonding. No unspecific binding to the gel matrix was observed because chromatography on the HiTrap resin lacking peptide C19 did not retain BDA-PEPCk (Fig. 4b, lanes 4 and 5). These experiments show that BDA-PEPCk was specifically retained by the presence of peptide C19. Finally, the 50% inhibition of initial activity (IC50) for peptide C19 was estimated in a phosphorylation assay containing a desalted protein extract from illuminated leaves, supplemented or not with 0.2 units of immunopurified, non-phosphorylated C4 PEPC and the components of the phosphorylation reaction. It was found to be close to 230 µM (Fig. 5).
The highly conserved C-terminal domain (highly hydrophobic alpha-helix) of E. coli PEPC and maize PEPC is buried within the core hydrophobic region of the subunit. At its extremity is the non-helical tetrapeptide RNTG (QNTG in the plant enzyme) where the N residue is involved in the binding of the allosteric inhibitor Asp (Dong et al., 1999
Collectively, our results show for the first time, to our knowledge, that a synthetic peptide corresponding to the C-terminal end of C4 PEPC interacts specifically with the genuine Ca2+-independent PEPCk and inhibits in vitro C4 PEPC phosphorylation. Among the 19 amino acids, the presence of the QNTG tetrapeptide is not mandatory to promote this inhibitory effect.
Studies on mutated C4 PEPCs in the phosphorylation domain have led to the proposition that the interaction of PEPCk with its target is not restricted to this domain. However, no information is available as to where this could occur on PEPC (Wang et al., 1992 ; Li et al., 1997). The C-terminal domain is highly conserved in all PEPCs sequenced so far. It has been implicated in the maintenance of the stability of the protein (Grisvard et al., 1998; Dong et al., 1999), the maintenance of the catalytic activity, and the oligomerization state of the enzyme (Dong et al., 1999). However, to date, the possible implication of the C-terminal tail in the phosphorylation of PEPC by plant PEPCk has not been studied. In the present work, this issue was investigated by using synthetic peptides (based on the C-terminal sequence of C4 PEPC from sorghum) and corresponding antibodies. In a first set of experiments, specific antibodies raised against peptide C19 were used. It was assumed that this antibody might react with the putative interaction domain and affect PEPC phosphorylation. However, when added to the reconstituted assay system, these antibodies barely affected the phosphorylation reaction (Fig. 2a). This result raised the question of whether the antibodies could bind to the corresponding C4 PEPC epitopes. In fact, blotting experiments showed that C19 IgGs cross-reacted with a heat-denatured C4 PEPC but much less with the native enzyme. This indicated that the C-terminal tail domain is embedded in the native enzyme, in good agreement with the recently reported three-dimensional structures of E. coli (Kai et al., 1999) and maize PEPCs (Matsumura et al., 2002). However, the antibody still could bind, and this may also be valid for the PEPCk. On the other hand, if PEPCk binding to the C4 PEPC C-terminal is required for catalytic efficiency, the peptide should compete with the target, thus decreasing the catalytic rate in a phosphorylation assay. Peptide C19 (containing the last 19-amino acid sequence of the C-terminal tail of sorghum C4 PEPC) inhibited in a dose-dependent manner (IC50 = 230 µM) the in vitro phosphorylation of C4 PEPC by the BDA-PEPCk. Interestingly, the peptide did not have any effect on the other kinases capable of phosphorylating the C4 PEPC e.g. PEPC-CDPK and PKA. Because peptide C19 is highly hydrophobic, much like the BDA-PEPCk, a nonspecific interaction could occur. This was checked by using synthetic peptides (L1, L2, and L3) designed from computerized modeling of the C4 PEPC (by reference to the three-dimensional structure of E. coli PEPC and from loops that are not present in the bacterial PEPC). In addition, peptides L2 and L3 were as hydrophobic as peptide C19. It was observed that neither of them decreased the phosphorylation rate of PEPC in the reconstituted assay. Furthermore, when the BDA-PEPCk was subjected to affinity chromatography on the peptide C19-agarose column, the enzyme was partially but significantly retained and the bound fraction subsequently eluted by water. Altogether, these results show that peptide C19 interacts with the BDA-PEPCk in a very specific manner.
In the phosphorylation assay, peptide C15 (peptide C19 lacking the QNTG motif) had a similar inhibitory effect to that of peptide C19, thereby showing that the interaction domain is located downstream from the QNTG end. The addition of specific QNTG IgGs to a reconstituted assay did not affect the phosphorylation of C4 PEPC by the PEPCk. Along these lines, Dong et al. (1999 To our knowledge, the results in this work show for the first time that a peptide containing the 19-amino acid sequence from the C-terminal tail of C4 PEPC interacts with the genuine Ca2+-independent PEPCk. In addition, we show by using the C19-IgGs that the C-terminal domain is partially offset in the native enzyme but that it could still bind the antibodies. This result suggests that C4 PEPC could be, in vitro, in two different conformational states. In one of them, the C-terminal end is accessible to IgGs and, therefore, to the PEPCk. However, this C-terminal-accessible form of PEPC seems to be in low abundance in our reconstituted assay, and no effect of the specific C19-IgGs was detected.
It has been shown that metabolites affect the in vitro phosphorylation of C4 PEPC by PEPCk, e.g. it is inhibited by L-malate, and this effect is counteracted by sugar-P-like glucose-6-phosphate (Echevarría et al., 1994 It could be that the hydrophobic C-terminal domain is only accessible in specific cellular environmental conditions depending on pH, substrate and effector concentrations, and/or phosphorylation, oligomerization, or redox state of the PEPC. The hypothesis is a possible and specific dialogue between the cell and the PEPC via the degree of accessibility of its C-terminal end. Further studies are necessary to determine the exact conditions that could make the C-terminal tail accessible and if these different conformational states operate in vivo. Alternatively, it can be hypothesized that the inhibitory effect exerted by the peptide C19 on C4 PEPC phosphorylation is related to the active site of PEPCk. This can be viewed as an auto-inhibitory-like effect (when the C-terminal domain of PEPC is made available for interaction with the active site of the PEPCk). In this case, the hypothesis that the allosteric interaction of the protein kinase with PEPC governs PEPC phosphorylation is no longer valid. The active site inhibition hypothesis would confer what has been called a " unique protein kinase" (because no classical regulatory mechanism applies to this kinase), a variation of a regulatory mechanism that is commonly found for protein kinases.
Finally, it also could be that in vivo PEPC proteolysis generates a C-terminal peptide, thereby resulting in a negative feedback on PEPC phosphorylation. This could also be performed by a polypeptide carrying a similar sequence. For instance, the occurrence in plant extracts of a protein inhibiting the PEPCk activity in vitro has been described (Nimmo et al., 2001
Materials
Peptide C19 contains the entire C-terminal sequence {amino acids 942–960 [(Y)942EDTLILTMKGIAAGMQNTG960]} of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC. Peptide C15 is peptide C19 lacking the terminal QNTG [(Y)942EDTLILTMKGIAAGM956]. Both polypeptides and the anti-peptide C19 antiserum were from NEOSYSTEM S.A. (Strasbourg, France). The APS-IgGs were raised against the N-terminal [4ERHHSIDAQLRALAPGKVSEE24] of sorghum C4 PEPC containing the phosphorylation motif (Pacquit et al., 1995
Sorghum plants were grown under controlled environmental conditions in a greenhouse under a 12-h photoperiod (350 µmoL m-2 s-1, photosynthetically active radiation) and a temperature of 28°C/20°C (light/dark). The plants were irrigated with a nutrient solution (Hewitt, 1966
Fully expanded youngest leaves were excised and dark adapted or illuminated (750 µmoL m-2 s-1) for 2 h. Leaf tissue (8 cm2) was immediately ground in a chilled mortar with 1 mL of buffer A (100 mM Tris-HCl [pH 7.5], 20% [v/v] glycerol, 1 mM EDTA, 10 mM MgCl2, 14 mM
The PEPC from darkened sorghum leaves was purified by affinity chromatography on an immuno-adsorbent column (Vidal et al., 1981
This was essentially done according to the procedure of Jiao and Chollet (1989
All steps were performed at 4°C. Illuminated (350 µmol m-2 s-1) sorghum leaves were chopped (2 g) and ground thoroughly in a chilled mortar with washed sand in 10 mL of buffer F (100 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 1 mM EDTA, 5% [v/v] glycerol, 14 mM L-malate (5 mM) and 0.01% (w/v) bromphenol blue were added to desalted extract (450 µg) and loaded onto a native-PAGE (5% [w/v] acrylamide). After electrophoresis (100 V for 3 h), the PEPC band was located by incubation of the gel for 5 min in a medium containing the assay components of PEPC activity and 0.16 mM CaCl2. Pi released by the PEPC reaction precipitated as a white calcium phosphate band. The PEPC band was excised from the gel and proteins were electro-eluted for 12 h at 5 V (model 422 Electro-eluter, Bio-Rad). Proteins (1.3 mg) were recovered in Tris-Gly buffer (25 mM Tris-HCl and 192 mM Gly [pH 8.3]) and stored at -20°C. This highly purified fraction contained PEPC and a Ca2+-dependent C4 PEPCk that comigrate during PAGE with C4 PEPC. It was used in the in vitro phosphorylation assay as PEPC-CDPK fraction.
One milligram of peptide C19 was dissolved in 900 µL of 100 mM HEPES-NaOH (pH 8.0) and covalently linked to a HiTrap/NHS affinity column (1 mL) according to the manufacturer's instructions. The calculated molecular mass for peptide C19 was 2,127.4 D. One mg of the lyophylized powder contained 0.85 mg/0.39 µmol of peptide. The coupling reaction was performed at room temperature for 5 h, blocked with 1 M Tris-HCl (pH 8), and the column (peptide C19 affinity column) was equilibrated in the appropriate buffer. In parallel, the same treatment was performed to another column (a control column) where the peptide C19 was omitted. The partially purified BDA-PEPCk (600 µg of protein/100 µL) was loaded (0.05 mL min-1) onto the peptide C19 affinity column equilibrated with buffer H (50 mM Tris-HCl [pH 7.8], 1 mM DTT, and 20% [w/v] glycerol). The column was washed with 5 mL of buffer H supplemented with 0.5 M NaCl using a flow rate of 0.2 mL min-1. The bound proteins were eluted with 3 mL of distilled water. The eluted fractions were concentrated to a volume of 100 µL by centrifugation at 15,000g for 1 h at 4°C using a Millipore filter (10,000 D).
One milligram of peptide C19 was dissolved in 400 µL of 0.2 M NaHCO3 and 0.5 M NaCl (pH 8.3) and covalently linked to a HiTrap/NHS affinity column (1 mL) according to the procedure described above. After coupling, the column was equilibrated with 4 mL of phosphate-buffered saline (PBS; 0.25 M NaH2PO4 and 0.15 M NaCl [pH 7.5]), and 1 mL of C19-IgGs (5 mg) was loaded onto the column. Fixation was performed at room temperature for 2 h. Unfixed material was washed with 3 mL of PBS, and the bound IgGs were eluted with 0.2 M citric acid (pH 2.6). Peptide C19-specific IgGs were precipitated by ammonium sulfate at 60% (w/v) saturation, collected by centrifugation, and dissolved in 200 µL of PBS. These highly purified C19-IgGs were loaded onto a peptide C15 column (peptide C15 covalently linked to a HiTrap/NHS affinity column as described above). The calculated molecular mass for peptide C15 was 1,727 D. One milligram of lyophylized powder contained 0.57 µmol peptide. The unbound IgGs containing specific antibody against the last four amino acids (QNTG) were precipitated with ammonium sulfate to 60% (w/v) saturation, dissolved in PBS, and stored at -20°C until use.
Aliquots of crude extracts from darkened leaves (0.045 units of C4 PEPC) were heated at 50°C for 2 min (denatured PEPC) or kept on ice (native PEPC). C19-IgGs (28 µg/20 µL) or APS-IgGs (30 µg/20 µL) were added and incubated for 30 min at 4°C. After the incubation, 4% (w/v) protein A-Sepharose was added to the incubated sample and vortexed briefly (5-min interval) for 15 min. The beaded immunocomplexes were sedimented by centrifugation at 12,000g for 5 min, washed once with buffer (0.5 M Tris-HCl [pH 8.0], 1.5 M NaCl, and 1% [v/v] Triton X-100), and twice with 0.1 M HEPES-NaOH (pH 7.1). The pellets were dissolved in SDS sample buffer (50 mM Tris-HCl [pH 8.8], 20% [v/v] glycerol, 1% [w/v] SDS, 14 mM
The samples were subjected to SDS-PAGE (10% [w/v] acrylamide) according to the method of Laemmli (1970
PEPC activity was measured spectrophotometrically at optimal pH (8.0) using the NAD-malate dehydrogenase-coupled assay at 2.5 mM phosphoenolpyruvate (PEP; Echevarría et al., 1994
An aliquot of the BDA-PEPCk (30 µg of protein), PEPC-CDPK (an aliquot that contained 0.1 units of PEPC), or 5 units of the catalytic subunit of PKA from bovine heart was pre-incubated at 4°C for 15 min in the presence of similar amounts of peptide C19 or C15. After pre-incubation was completed, reconstituted assays were performed in 70 µL of 100 mM Tris-HCl (pH 7.8), 20% (v/v) glycerol, and 5 mM MgCl2. In vitro phosphorylation using desalted protein extracts from illuminated leaves (aliquots containing 0.1 units of PEPC) was performed as described above in presence of 0.25 mM P1P5-di(adenosine-5')-pentaphosphate (adenylate kinase inhibitor), 4 mM phosphocreatine, and 10 mM creatine phosphokinase (components of the ADP scavenging system; Echevarría et al., 1990 The effect of C19-IgGs on C4 PEPC phosphorylation was checked after a 10 min-pre-incubation (4°C) of affinity-purified C19-IgGs (40 µg) with the BDA-PEPCk (30 µg) or PEPC-CDPK (aliquot containing 0.1 units of PEPC) and the component of the phosphorylation reaction before the addition of 1 µCi [gamma-32P]ATP. The effects of QNTG-IgGs (40 µg) on the in vitro phosphorylation assay were performed using desalted extracts (aliquots containing 0.1 units of PEPC) in the presence of 0.25 mM P1P5-di(adenosine-5')-pentaphosphate (adenylate kinase inhibitor), 4 mM phosphocreatine, and 10 mM creatine phosphokinase (components of the ADP scavenging system).
The procedure for the "in-gel" assay of protein kinase activity after SDS-PAGE and subsequent renaturation of protein in the gel was essentially the same as described previously by Wang and Chollet (1993
Protein determinations were performed by the method of Bradford (1976
The authors thank Dr Michael Hodges for valuable comments, the selection of peptides L1, L2, and L3, and critical reading and correction of the manuscript. Received March 21, 2003; returned for revision March 25, 2003; accepted March 25, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.023937.
1 This work was supported by the Dirección General de Investigación Cientifica y Técnica (grant nos. PB97–0745–CO2–02 and BCM2001–2366–CO3–02), by the "Acción Integrada HispanoFrancesa HF2000–0009," and by the "Grupo de Investigación de Fisiología Vegetal CVI134" from La Junta de Andalucía. * Corresponding author; e-mail echeva{at}us.es; fax 34–954615780.
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ABSTRACT
RESULTS
-mercaptoethanol, 1 mM phenylmethysulfonylfluoride [PMSF], 10 µg mL-1 chymostatin, 10 µg mL-1 leupeptin, 2 µg mL-1 pepstatin, and 10 mM potassium fluoride). The homogenate was centrifuged at 15,000g for 2 min, and the supernatant was filtered through Sephadex G-25 equilibrated with buffer A without 