JXB Advance Access originally published online on October 30, 2006
Journal of Experimental Botany 2006 57(15):4079-4088; doi:10.1093/jxb/erl175
RESEARCH PAPER |
Autophosphorylation of Solanum chacoense cytosolic nucleoside diphosphate kinase on Ser117
1IRBV, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, QC, H1X 2B2 Canada
2Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, QC, H4P 2R2 Canada
* To whom correspondence should be addressed. E-mail: jean.rivoal{at}umontreal.ca
Received 24 July 2006; Accepted 30 August 2006
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Abstract |
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NDPK catalyses the interconversion of NTPs and NDPs using a phosphohistidine intermediate as part of its catalytic site. Recombinant Solanum chacoense cytosolic NDPK incubated with [
-32P]ATP was allowed to autophosphorylate and 32P-labelled P-Ser was identified in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of 32P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive peptide allowed the identification of a single and conserved P-Ser residue at position 117. Analysis of site-directed mutants where Ser117 was substituted to Asp indicated that the presence of a negative charge at position 117 dramatically lowered the enzyme's catalytic efficiency. Ser autophosphorylation was markedly reduced with increasing ADP concentrations in the autophosphorylation assay. These findings provide evidence that autophosphorylation of cytosolic NDPK on Ser117 could constitute a regulatory mechanism for this important enzyme and that autophosphorylation of Ser117 is modulated by NDP availability. Key words: Enzymology, nucleoside diphosphate kinase, plant, phosphoamino acid, phosphorylation, protein sequencing, site-directed mutagenesis
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Introduction |
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Nucleoside diphosphate kinase (NDPK, EC 2.7.4.6 [EC] ) is found in animals, fungi, plants, and prokaryotes. This enzyme catalyses the transfer of the
-phosphate of a donor nucleoside triphosphate (NTP) to an acceptor nucleoside diphosphate (NDP). NDPK is believed to have a housekeeping function in the general homeostasis of cellular nucleoside triphosphate pools (Dancer et al., 1990; Lambeth et al., 1997; Roberts et al., 1997; Bernard et al., 2000). In plants, NDPK1 is found in the cytosol (Sweetlove et al., 2001; Dorion et al., 2006), whereas NDPK2 and NDPK3 have N-terminal extensions that suggest organellar targeting (Sweetlove et al., 2001). Cytosolic NDPK1 has been shown to be the main NDPK isoform in plants, accounting for more than 70% total NDPK activity in various tissues (Dorion et al., 2006). In addition to its housekeeping function, high levels of NDPK1 protein and NDPK activity in meristematic tissues suggest that this isoform fulfils a role in early growth (Dorion et al., 2006). The NDPK2 isoform was shown to be involved in various signalling events (Tanaka et al., 1998; Choi et al., 1999; Zimmermann et al., 1999; Matsushita et al., 2002; Moon et al., 2003; Novikova et al., 2003). NDPK3 is found in the mitochondrial intermembrane space where it interacts with the adenine nucleotide translocator (Sweetlove et al., 2001; Knorpp et al., 2003). Despite their importance in a number of cellular and metabolic processes, our understanding of regulatory mechanisms for plant NDPKs remains rudimentary. NDPK has long been recognized as a phosphoprotein (Gilles et al., 1991; Moisyadi et al., 1994; Matsushita et al., 2002). Autophosphorylation of the enzyme occurs as part of the catalytic mechanism because NDPK's enzymatic reaction involves the formation of a P-His intermediate which is found at position 118 in the human enzyme (Gilles et al., 1991). In the mammalian NDPK homologue Nm23, a regulator of breast carcinoma metastatic activity, several Ser residues appeared autophosphorylated (MacDonald et al., 1993). Ser phosphorylation of Nm23 was shown to correlate with the suppression of the tumour metastatic potential (MacDonald et al., 1993). Ser phosphorylation has also been detected in bacterial and in plant mitochondrial NDPKs (Munoz-Dorado et al., 1993; Struglics and Hakansson, 1999). Recently, evidence has been provided that a recombinant S119A mutant of pea mitochondrial NDPK incorporated less radioactivity in autophosphorylation assays than the corresponding wild-type protein (Johansson et al., 2004). These data could indicate that Ser119 is a target for autophosphorylation, but could also mean that this residue is structurally or catalytically involved in the autophosphorylation of another residue without becoming phosphorylated itself. Indeed, because Ser119 lies within a few Ångströms of the catalytic His117 residue (Johansson et al., 2004), it could possibly influence enzyme activity. The issue of NDPK Ser phosphorylation has been made even more puzzling by a recent finding suggesting that Arabidopsis NDPK2, which is involved in phytochrome signal transduction, is not Ser phosphorylated (Shen et al., 2006). All these data highlight the fact that NDPK Ser phosphorylation has not been completely elucidated. Indeed, to date, the biochemical identification of plant NDPK phosphorylated Ser residue(s) has not been achieved and the possible effect of such phosphorylation on NDPK activity is still unknown. The present study was initiated to investigate these issues. At the outset, the aim was to determine if the plant cytosolic NDPK isoform that had recently been characterized (ScNDPK1) (Dorion et al., 2006) was able to autophosphorylate on a Ser residue. The next objective was to identify this phosphorylation site and to investigate the possible role of Ser phosphorylation on ScNDPK1 activity using a site-directed mutagenesis approach.
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Chemicals and biological materials
Except when mentioned otherwise, buffers, chemicals, and reagents were of analytical grade from Sigma Chemical Co. (St Louis, MO) or Fisher Scientific (Nepean, ON, Canada). The Platinum Pfx DNA polymerase, the vector pProEx HTb and Ni2+-nitrilotriacetic acid agarose were from Invitrogen Canada Inc. (Burlington, ON, Canada). Restriction enzymes were from MBI Fermentas (Burlington, ON, Canada) and Invitrogen Canada Inc. Primers for PCR and sequencing were from Sigma Genosys (The Woodlands, TX).
Site-directed mutagenesis of ScNDPK1
The construct carrying Solanum chacoense (6xHis)-tagged ScNDPK1 in the bacterial expression vector pProEX HTb was obtained as described before (Dorion et al., 2006) and is also referred to as WT below. Site-directed mutagenesis of the WT sequence was done using the QuickChange mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's directions and the mutated nucleotide primers described in Table 1. All the mutations were verified by sequencing.
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Production and purification of recombinant proteins
Recombinant WT and mutant NDPKs were produced in E. coli (DH5
strain) and purified as described by Dorion et al. (2006) except for the S117D mutant, which was induced with isopropyl ß-D-thiogalactoside at 22 °C to improve recovery of the protein.
In vitro phosphorylation of recombinant proteins and SDS/PAGE analysis
Unless otherwise mentioned, autophosphorylation reactions were conducted using 2 µg of WT or mutant ScNDPK1s which were incubated in a 20 µl reaction mixture containing 50 mM TRIS-Cl, pH 7.5, 5 mM EDTA, 0.05 mM MgATP, and 4 µCi [32P]-ATP. After 30 min at 25 °C, the reactions were stopped by adding Laemmli sample buffer (Laemmli, 1970) for subsequent SDS/PAGE analysis. The protein sample was denatured by incubation at 95 °C for 5 min and immediately loaded on the gel. In some cases (see below), the autophosphorylated protein was denatured by incubation at 25 °C for 60 min prior to SDS/PAGE analysis. Samples were run on 15% acrylamide SDS/PAGE according to Laemmli (Laemmli, 1970), dried and scanned using a Typhoon 9200 phosphorImager (GE Healthcare, Baie d'Urfé, QC, Canada).
TLC analysis of protein-bound ATP
To investigate the possibility that [32P]-ATP could remain bound to ScNDPK1 after autophosphorylation, the autophosphorylated protein was subjected to SDS/PAGE and electrotransfer to PVDF membrane. The piece of membrane corresponding to the protein band was visualized using a phosphorImager and cut out. This piece of membrane was washed for 1 h in 200 µl H2O. [32P]-ATP released in H2O was analysed by TLC (Norman et al., 1974) followed by scanning with a phosphorImager.
Chemical stability of 32P-labelled WT ScNDPK1
Autophosphorylated ScNDPK1 was subjected to SDS/PAGE and transferred to PVDF. To investigate the stability of the radioactive label of the protein to acidic and alkaline conditions, radioactive bands were first localized using a phosphorImager and cut out of the membrane. Alkaline treatments were done by incubating the membrane pieces with 2 M NaOH pH 13.5 at 42 °C whereas acidic treatments were done by incubation with 0.2 M citrate buffer pH 2.4 at 42 °C (Kumble et al., 1996). Following incubation, the solution was removed and the membrane pieces were rapidly washed with 50 mM KH2PO4, pH 7.0 at room temperature and dried. Radioactivity remaining on the membrane was assayed by Cerenkov counting.
Phosphoprotein hydrolysis and TLC analysis of phosphoamino acids
To detect the presence of P-Ser in ScNDPK1 after autophosphorylation, the protein was subjected to SDS/PAGE and transferred to a PVDF membrane. The radioactive band was localized using a phosphorImager, cut out, deposited in a 2 ml Wheaton vacule vial and hydrolysed in vacuo for 60 min with 250 µl 5.7N HCl at 110 °C. The hydrolysate was dried under vacuum and resuspended in 20 µl H2O containing 40 nmol of P-Ser, P-Thr, and P-Tyr standards. Phosphoamino acids were separated at room temperature by two-dimensional TLC using cellulose-coated plates (Duclos et al., 1991). The first dimension was developed for 7.5 h in isobutyric acid:0.5M NH4OH (5:3, v/v). After drying overnight, the plate was subjected to the second dimension for 10 h in 2-propanol:11.6 N HCl:H2O (7:1.5:1.5, by vol.). Radioactive spots were visualized using a phosphorImager and phosphoamino acids standards by spraying the plate with a ninhydrin solution followed by 10 min incubation at 65 °C for colour development (Duclos et al., 1991).
HPLC purification and N-terminal sequencing of phosphopeptides
Phosphopeptides analysis was performed after SDS/PAGE analysis of autophosphorylated ScNDPK1. The gel was stained with Coomassie Blue R-250 and destained for 30 min in 30% methanol. The protein band was excised from the gel and subjected to DTT reduction and iodoacetamide alkylation followed by overnight digestion at 37 °C with 0.2 µg of sequencing grade trypsin (Promega, Nepean, ON, Canada) (Hellman et al., 1995). Tryptic peptides were extracted from the gel and separated by reverse-phase HPLC as described by (Gopalbhai et al., 2003), except that the column flow rate was 0.1 ml min–1. The peptides were detected by absorbance at 220 nm. The peaks were collected manually and subjected to Cerenkov counting to identify fractions containing radioactive phosphopeptides. A single fraction contained radioactivity. An aliquot of this fraction was applied to a TFA-treated glass fibre filter coated with Biobrene Plus (0.5 mg of polybrene (Applied Biosystems, Foster City, CA) and 0.03 mg of NaCl) and subjected to automated Edman degradation on a model 494 CLC Procise sequencer using the general protocol of (Hewick et al., 1981). The PTH amino acid derivatives were analysed on line using a capillary separation system (Applied Biosystems model 140 D) and ultraviolet detector (Applied Biosystems model 785A) at 269 nm. The remainder of the fraction was covalently attached to a Sequelon-AA PVDF matrix derivatized with an aryl amine group (Millipore, Nepean, ON, Canada) and submitted to Edman degradation (Aitken and Learmonth, 1997). In this case, the anilinothiazolinone amino acid derivative was collected after each cycle (Admon and King, 1992) and subjected to Cerenkov counting.
Kinetic analyses
Kinetic constants were determined using a spectrophotometric coupled enzyme assay (Dorion et al., 2006). Values for Vmax and Km were calculated from the Michaelis-Menten equation using a non-linear least-squares regression program (SigmaPlot 8.0, SPSS Inc., Chicago, IL, USA). The kcat values were calculated using a subunit molecular mass of 19.3 kDa.
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Results |
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WT ScNDPK1 autophosphorylates in a His115-dependent manner and contains phosphoamino acids sensitive to acidic and alkaline pHs
Recombinant WT and H115A mutant were purified to homogeneity. The WT protein displayed NDPK activity (see below), whereas the H115A mutant had no detectable activity (not shown). Both proteins were incubated with [
-32P]ATP in an autophosphorylation reaction and analysed by SDS/PAGE (Fig. 1A). As expected, the WT protein incorporated 32P label whereas the H115A mutant did not. 32P label incorporation was time-dependent and proportional to the amount of protein in the assay (not shown). These results indicate that ScNDPK1 is able to autophosphorylate like all other NDPKs tested for this property. This capacity depends on the catalytic His residue found at position 115 in the S. chacoense enzyme. The nature of the phosphoamino acid(s) in ScNDPK1 was further examined by investigating the pH stability of the phosphoryl-protein linkage. P-His (but not P-Ser, P-Thr, or P-Tyr) is hydrolysed under acidic conditions (Hultquist et al., 1966). P-Ser, and to a lesser extent P-Thr, are sensitive to alkaline pHs whereas P-Tyr is stable under acidic and alkaline conditions (Duclos et al., 1991). Autophosphorylated WT protein was transferred to PVDF membrane and incubated in acidic or alkaline conditions. The stability of 32P on ScNDPK1 as a function of time and treatment is represented in Fig. 1B. The data demonstrate that autophosphorylated WT ScNDPK1 was sensitive to both acid and alkaline treatments and that a greater proportion of the 32P label incorporated in the protein was labile under alkaline conditions, suggesting the presence of P-Ser or P-Thr in ScNDPK1 in addition to P-His.
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Conditions for sample preparation are important for ScNDPK1 phosphorylation studies
It was recently suggested that Ser phosphorylation of A. thaliana NDPK2 could be due to a non-enzymatic process in which the phosphorylated catalytic His residue would donate its phosphate to Ser residues upon denaturation of the enzyme at 95 °C in SDS sample buffer (Shen et al., 2006). In order to examine this possibility, ScNDPK1 was autophosphorylated, then subjected to a denaturation at 25 °C for 60 min or 95 °C for 5 min as outlined in Fig. 2A. The samples were subjected to SDS/PAGE and transferred to PVDF membranes. Radioactive membranes were treated under acidic and neutral conditions to evaluate the effect of denaturation temperature on acid-stable phospho-label. Our results contrast with those obtained by Shen et al. (2006) under similar conditions. Indeed, ScNDPK1 denatured at 25 °C or 95 °C contained comparable acid-resistant phospho-label, arguing against a non-enzymatic process. Furthermore, samples treated at neutral pH contained more radioactivity when denatured at 25 °C compared with 95 °C. These data are consistent with the characteristic instability of P-His to high temperatures (Duclos et al., 1991; Wieland et al., 1993). Nevertheless, the nature of the radioactivity bound to the enzyme denatured at 95 °C or 25 °C and subjected to SDS/PAGE and electrotransfer was investigated further. The particular aim of this experiment was to determine whether [32P]-labelled ATP could still be associated to the enzyme denatured under mild conditions (60 min at 25 °C). Membrane pieces carrying the electrotransferred enzyme were incubated in water in order to separate any soluble material from the protein on the membrane. Analysis of the eluate by TLC revealed the presence of radioactive ATP in the protein sample denatured at 25 °C whereas the enzyme denatured at 95 °C did not contain any detectable radioactive ATP. Thus, denaturation of ScNDPK1 at 25 °C was not sufficient to denature the enzyme fully and to dissociate it from strongly bound ATP. This was not the case with denaturation carried out at 95 °C. The subsequent experiments were therefore performed with samples denatured at 95 °C in order to avoid the possible interference of [32P]-ATP bound to ScNDPK1 in our phosphorylation studies.
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Detection of P-Ser in autophosphorylated WT ScNDPK1
To seek further confirmation about the identity of the acid-stable phosphoamino acid(s) detected in Figs 1B and 2A, autophosphorylated WT ScNDPK1 was subjected to acid hydrolysis followed by 2-dimensional TLC separation of phosphoamino acids (Fig. 3). Two radioactive signals were observed. The weakest corresponds to free Pi, most probably arising from P-His hydrolysed during the acid treatment. The strongest signal co-migrated with the P-Ser standard. These results confirm that after acid hydrolysis, the major phosphoamino acid recovered from labelled ScNDPK1 was P-Ser.
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Ser117 is the acid-stable phosphorylation site in WT ScNDPK1 and is conserved in all NDPKs
To identify the P-Ser residues in autophosphorylated WT ScNDPK1, the protein was subjected to trypsin digestion. Tryptic peptides were separated by reverse phase HPLC. Fractions corresponding to each peak detected at 220 nm were recovered and counted for radioactivity (Fig. 4A). A single radioactive fraction, eluting at approximately 25.5 min, was identified during this separation. The fraction was subjected to automated Edman degradation using procedures that allowed the identification of the amino acid and the measurement of the radioactivity released at each degradation cycle (Fig. 4B). The fraction contained a single peptide with the sequence NH2-NVIHGSDAVESAR, which corresponds to Asn112-Arg124 region in the ScNDPK1 sequence (Dorion et al., 2006). This sequence contained 2 Ser residues at positions 117 and 122. Only Ser117 appeared phosphorylated because the peak of released 32P coincided with the Ser117 cycle and the amounts of 32P associated with Ser122 were close to background levels (Fig. 4B). These results unambiguously demonstrate that ScNDPK1 only autophosphorylates on Ser117 in vitro (besides H115). The conservation level of this Ser residue in various NDPKs was examined by aligning the sequence of the tryptic peptide from Fig. 4B with the homologous region present in NDPKs from a wide range of organisms (Fig. 5). The data demonstrate that Ser117, which lies two amino acids from the catalytic His115 residue, is conserved in all plant cytosolic and organellar NDPK isoforms. It is also strictly conserved in NDPKs from prokaryotic, animal and higher plant origins. In the region containing the catalytic site, only Asn112 and His115 (marked by arrows) share the same degree of conservation.
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The presence of a negative charge at position 117 affects autophosphorylation and kinetic properties of ScNDPK1
In order to examine the possible effects of Ser117 phosphorylation on ScNDPK1, a pseudophosphorylated mutant (S117D) was generated using site-directed mutagenesis (Table 1). Another mutant (S117A) was also generated to obtain a protein that could not carry a negative charge at position 117. WT ScNDPK1 and the two mutant proteins were purified from E. coli and used in an autophosphorylation assay (Fig. 6). When analysed by SDS/PAGE, the three preparations contained a single protein band with an identical molecular mass of 19.3 kDa (Fig. 6, top panel). As expected, the WT protein displayed a higher level of autophosphorylation than the two mutants (Fig. 6, bottom panel). These data are consistent with the fact that the two mutants have lost their capacity to autophosphorylate on position 117. Consequently, the remaining radioactivity would probably be due to incorporation at His115.
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In order to determine whether ScNDPK1 phosphorylation occurs via an intra- or intermolecular reaction, 1 µg of the S117A mutant was incubated with 1 µg of the H115A mutant in the presence of [
32P]-ATP. Control reactions contained [32P]-ATP with only 1 µg of a single mutant protein. Analysis of phosphorylation levels did not show any increase in acid-stable phosphorylation when the two mutants were incubated together compared with controls (data not shown). These data suggest that in vitro ScNDPK1 phosphorylation occurs via an intramolecular process. The kinetic properties of the WT, S117A, and S117D recombinant proteins were examined using a coupled enzyme assay (Table 2). The apparent Km values for ATP and TDP determined for the WT protein are similar to the values that were recently reported for this enzyme (Dorion et al., 2006). The Vmax and kcat values obtained were the highest for the WT protein. The S117A and S117D mutants had kcat values, respectively, 3-fold and 2–3 orders of magnitude lower than the WT protein, depending on the substrate considered. Therefore, the presence of a negative charge at position 117 had a large negative impact on the enzyme turnover number. This effect on apparent Km values was different for TDP and ATP. There was a clear effect on TDP affinity: compared with the WT enzyme, the S117D mutant had much lower affinity for TDP whereas the S117A mutant had 2-fold higher affinity for this substrate. Both mutant proteins, however, had higher apparent affinities for ATP than the WT enzyme. The inhibitory effect of having a permanent negative charge at position 117 was also evident from the catalytic efficiency values of the different enzymes. While the Vmax/Km ratios for WT and S117A were in the same range, that of the S117D mutant was 450- to 1600-fold lower than WT values, depending on the substrate considered.
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Ser phosphorylation of ScNDPK1 is reduced in the presence of UDP and ADP
ADP is the most abundant NDP in plant tissues (Farré et al., 2001). Since NDPK is involved in the metabolism of NDPs and because autophosphorylation on Ser117 could potentially compete with phosphorylation of NDPs, the effect of including various concentrations of ADP on the capacity of ScNDPK1 to autophosphorylate on Ser was investigated. ScNDPK1 was allowed to autophosphorylate in reactions containing 0–1 mM ADP. Subsequently, the labelled protein was separated by SDS/PAGE and transferred to PVDF membrane. The membrane was then subjected to an acid treatment to hydrolyse P-His residues. The radioactivity remaining in ScNDPK1 bands and corresponding to P-Ser was visualized using a phosphorImager (Fig. 7, top panel). There was a marked decrease in the autophosphorylation of ScNDPK1 with increasing concentrations of ADP in the assay. Subsequent treatment of the membrane under alkaline conditions (Fig. 7, bottom panel) eliminates the radioactive signal present in the acid-washed membrane. This confirms that the radioactivity detected in the top panel of the figure corresponds to P-Ser. Similar results were obtained in the presence of UDP (not shown).
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ScNDPK1 autophosphorylates on Ser117
NDPK is an important enzyme of energy metabolism since it is involved in the generation of NTPs from NDPs using ATP as phosphate donor (Lascu and Gonin, 2000). This housekeeping reaction is critical in the generation of precursors for nucleic acid synthesis (Bernard et al., 2000). In plants, the cytosolic isoform is found at high levels in root and shoot meristematic regions and provascular tissues (Dorion et al., 2006). In these tissues, it may contribute to the synthesis of the cell wall precursor UDP-Glc by supplying UTP to the enzyme UDP-Glc pyrophosphorylase (EC 2.7.7.9 [EC] ) (Dorion et al., 2006). Apart from this biochemical activity towards NTPs and NDPs, some plant NDPKs also display a protein binding capacity (Choi et al., 1999; Escobar Galvis et al., 2001; Moon et al., 2003) that possibly mediates the involvement of NDPK in various signal transduction pathways (Choi et al., 1999; Moon et al., 2003). However, despite its importance in several critical aspects of metabolism and cell biology, the regulation of plant NDPKs is still poorly understood. In the present study, it has been demonstrated that recombinant ScNDPK1 incubated with [
32P]-ATP incorporates label on the Ser residue at position 117 (Fig. 4). To our knowledge, this constitutes the first direct and unambiguous evidence that NDPK Ser117 is phosphorylated. Characterization of the structure of the plant mitochondrial NDPK isoform has provided evidence that both Ser69 and Ser119 (equivalent to Ser67 and Ser117 in ScNDPK1) participate in autophosphorylation (Johansson et al., 2004). Using a direct sequencing approach of ScNDPK1, autophosphorylation was only detected on Ser117. Ser67 is a strictly conserved residue that plays an important role in NDPK's oligomeric state and is also necessary for the full activity of the enzyme (Johansson et al., 2004). If the cytosolic and mitochondrial isoforms autophosphorylate in a similar way, these results imply that Ser67/Ser69 participation in autophosphorylation is only due to the involvement of this residue in the structure and/or activity of the enzyme. The formation of P-Ser117 was dependent on the catalytic His115 residue (Fig. 1A). His115 becomes phosphorylated as part of the enzyme's catalytic mechanism and the phosphate is normally transferred to an NDP acceptor molecule (Parks and Agarwal, 1973). Because there was no evidence of phosphotransfer between the catalytically active S117A mutant and the catalytically inactive H115A mutant, it is concluded that an intramolecular phosphotransfer from H115 to Ser117 is the mechanism by which Ser phosphorylation of ScNDPK1 occurs. Autophosphorylation of ScNDPK1 on Ser appears quite specific because a single labelled tryptic peptide was recovered from the autophosphorylated WT protein (Fig. 4A). This peptide contains two Ser residues (Ser117 and Ser122). Only the strictly conserved Ser117 was found to be labelled (Fig. 4B). The specificity of the Ser autophosphorylation process argues against the recently suggested non-enzymatic mechanism (Shen et al., 2006). Discrepancies between the two studies could be due in part to differences in overall structure and/or function in NDPK1 and NDPK2. In addition, the fact that ATP could remain strongly bound to the enzyme after NDPK autophosphorylation assay and denaturation at 25 °C (Fig. 2) shows that bound ATP could interfere with the estimation of autophosphorylation under these conditions. This observation also underlines that protein phosphorylation experiments in which proteins have been incompletely denatured should generally be interpreted with caution.
A possible regulation of ScNDPK1 by autophosphorylation on Ser117
It is highly significant that the P-Ser residue identified in this study is strictly conserved in all NDPKs from bacteria to plants and mammals. This suggests that the mechanism that has been described here may also exist in other NDPKs, a possibility that deserves to be investigated. Protein phosphorylation is a widespread regulatory mechanism and is implicated in a growing number of metabolic enzymes in plants (Huber and Hardin, 2004). These results provide evidence that phosphorylation of ScNDPK1 on Ser117 could serve a regulatory purpose. Indeed, the use of a pseudophosphorylated mutant has allowed us to show that a permanent negative charge at position 117 has a strong inhibitory effect on NDPK activity. The S117D mutant was still able to bind its NTP and NDP substrates, but was catalytically inefficient (Table 2). The other mutant used in this study (S117A) appeared to be less active than the WT protein. This may be due to a structural effect of the amino acid substitution. It is interesting that the WT ScNDPK1 appears more active than the S117D mutant carrying a permanent negative charge. This suggests that ScNDPK1 would be fully active when non-phosphorylated on Ser117. This argues in favour of the fact that the proposed intramolecular phosphotransfer from His115 to Ser117 may only occur under certain circumstances. The results suggest that fluctuations in NDP pools could influence ScNDPK1 Ser phosphorylation and, consequently, its activity. Indeed, ScNDPK1 Ser autophosphorylation was decreased when increasing concentrations of ADP or UDP were included in the assay (Fig. 7). Two possible mechanisms can account for these results. The presence of a NDP molecule competes with Ser117 to accept the -phosphate of ATP. In that case, the observed decrease in Ser phosphorylation would be due to the availability of an acceptor NDP molecule in the vicinity of the active site. Alternatively, the presence of a NDP molecule could induce a change of conformation in ScNDPK1 that lowers the capacity of the enzyme to autophosphorylate on Ser117. Whatever the precise mechanism, autophosphorylation of ScNDPK1 on Ser117 is decreased by physiological NDP concentrations. Such a process raises the question about NDPK Ser phosphorylation occurrence in biological systems. Since NDPs are normally always presents in vivo, it is unlikely that NDPK is ever fully phosphorylated on Ser117. Nevertheless, autophosphorylation appears to be inextricably linked to the catalytic mechanism of the enzyme because no autophosphorylation is detected in the His115A mutant (Fig. 1A). This provides a strong indication that NDPK Ser phosphorylation could occur in vivo. It is possible that variations in cellular NDP concentrations could modulate NDPK autophosphorylation on Ser117. In that context, tissues or cellular compartments with high NTP are predicted to contain higher amounts of NDPK phosphorylated on Ser117 whereas high NDP concentrations would inhibit phosphorylation on Ser117. In plants, high ADP concentrations can be observed in tissues that are subjected to O2 deprivation (hypoxia and anoxia) (Drew, 1997).
In addition to its involvement in the maintenance of cellular NTP pools, NDPK is known to be involved in a remarkable diversity of other cellular processes. For instance, various NDPKs have been shown to have DNA binding capacity and to act as transcriptional regulators (Agou et al., 2000). Others interact with protein partners (Lombardi and Mileo, 2003) and have a protein phosphotransferase activity (Freije et al., 1997). The mammalian NDPK NM23-H1 has been shown to have a Ser protein phosphotransferase activity that depends on the catalytic His residue (Hartsough et al., 2002). In plants, recombinant Brassica campestris NDPK3 was shown to phosphorylate the self-incompatibility factor SRK in vitro (Matsushita et al., 2002). It is currently unknown if ScNDPK1 bears such alternative function(s), but it is tempting to speculate that Ser phosphorylation could participate in the manifestation of these diverse activities. For example, the S117D mutant was shown to have a lower affinity for TDP and higher affinity for ATP than the WT enzyme (Table 2). A possible consequence of these changes in substrate affinities would be to decrease the capacity of ScNDPK1 to phosphorylate NDPs while at the same time increasing its capacity to transfer a phosphate from ATP to an alternative substrate such as a protein. The importance of NDPK Ser phosphorylation has already been demonstrated in animal systems (MacDonald et al., 1993). An increased level of a mutant form of nm23-H1 can be found in advanced stage neuroblastoma (Chang et al., 1994). This mutation is a Ser120 (homologue to ScNDPK1 Ser117) to Gly substitution and it affects enzyme stability (Chang et al., 1996; Lascu et al., 1997) as well as its capacity to interact with a 28 kDa protein partner (Chang et al., 1996). Breast carcinoma cells carrying mutant Nm23H1S120G or Nm23H1S120A have also been shown to have increased tumour cell motility (Freije et al., 1997).
In the plant literature, a few His-to-Ser phosphotransferase activities have been characterized to various degrees. The pyruvate dehydrogenase kinase (PDK, EC 2.7.1.99 [EC] ) from Arabidopsis was originally suspected to catalyse autophosphorylation on Ser using P-His intermediates (Thelen et al., 2000), but further analysis demonstrated that it was not the case (Tovar-Mendez et al., 2002). Ser/Thr and His autophosphorylation activities have been described for the ethylene receptor protein NTHK2 from tobacco (Zhang et al., 2004). However, it is not known in this case if Ser/Thr autophosphorylation actually depends on His autophosphorylation. Another example concerns higher plant phytochromes which bear a histidine-kinase-related module in their C-terminus (Lamparter, 2004). Studies on purified Avena sativa recombinant phytochrome have shown that the protein exhibits Ser/Thr kinase activity and that autophosphorylation is light- and chromophore-regulated (Yeh and Lagarias, 1998). It thus appears that His-to-Ser phosphotransferase activity is often involved in signal transduction events in plants. Future studies on ScNDPK1 autophosphorylation will address this issue.
Taken as a whole, this study's data lead to the conclusion that the S117D mutant carrying a permanent negative charge at position 117 is less catalytically efficient compared with the other two proteins. Indeed, turnover numbers obtained with S117D was several orders of magnitude lower than the WT and S117A enzymes (Table 2). It is tempting to speculate that Ser autophosphorylation of ScNDPK1 occurs in vivo as part of a metabolic control mechanism over this enzyme. In this case, it will be important to investigate the effect of a negative charge at position 117 in plant tissues. It will also be of interest to examine if ScNDPK1 has other functions than NTP synthesis. The generation of transgenic plants overexpressing WT ScNDPK1 or the various site-directed mutants generated in this study should be helpful to elucidate this problem.
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Acknowledgements |
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This work was supported by a Discovery Grant from NSERC to JR. SD was the recipient of a Postdoctoral Fellowship from NSERC. This is NRC paper number 47516.
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Abbreviations |
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HPLC, high pressure liquid chromatography; NDP, nucleoside diphosphate; NDPK, nucleoside diphosphate kinase; NTP, nucleoside triphosphate; P-His, phosphohistidine; PHT, phenylthiohydantoin; P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine; PVDF, polyvinylidene fluoride; TLC, thin layer chromatography; WT, wild type.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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