Báo cáo Y học: Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase ppt

Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase Alejandro Tovar-Mendez 1 , Jan A. Miernyk 1,2 and Douglas D. Randall 1 1 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA; 2 USDA, Agricultural Research Service, Plant Genetics Research Unit, Columbia, MO 65211, USA Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehy- drogenase complex (PDC). Analysis of the primary amino- acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1a subunit of the PDC. This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phos- pho-His intermediate. The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Fur- thermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1a. Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser- kinase activity. Keywords: autophosphorylation; protein kinase; pyruvate dehydrogenase complex; regulatory phosphorylation; site- directed mutagenesis. The reaction catalyzed by the pyruvate dehydrogenase complex (PDC) occupies a key position in intermediary metabolism, and is subject to multiple layers of regulation [1,2]. Reversible phosphorylation is a particularly important control mechanism for mitochondrial PDC [3,4]. Multisite serine phosphorylation of the E1a subunit of PDC by the intrinsic pyruvate dehydrogenase kinase (PDK) inactivates the complex, which can then be re-activated by an intrinsic phosphopyruvate dehydrogenase phosphatase [5]. Steady- state activity of the PDC is determined by the sum of the PDK and phosphopyruvate dehydrogenase phosphatase reactions. Sequence analysis and biochemical characterization have shown that phosphopyruvate dehydrogenase phosphatase is a unique member of the type 2C class of phosphoprotein phosphatases [6,7]. In contrast, however, PDK, and the closely related branched-chain a-ketoacid dehydrogenase kinase (BCKDK), remain enigmatic. Despite exclusively phosphorylating Ser residues in the E1a subunits of the target complexes, these kinase sequences lack motifs typically conserved in protein Ser/Thr-kinases [8–10]. Instead, they include the cardinal motifs of protein His-kinases (PHKs) [11]. In addition, site-directed mutagenesis of conserved amino-acid residues within the N, D, F and G boxes of PHKs has confirmed that these motifs comprise the catalytic domain [12–14]. These motifs are located in the C-terminal region of PDK, as with PHKs, with the presumptive H box distal to the catalytic domain [15]. In addition to the sequence resemblance to PHKs, treatment with His-directed reagents inactivated PDK [16]. However, mutagenesis of the con- served His121 residue, a potential site of His autophos- phorylation, did not abolish PDK activity [17]. Autophosphorylation has been reported for PDKs from several sources [17–19]. It has been determined that one or more Ser residues near the N-terminus of PDK, and the closely related BCKDK, are autophosphorylated. Surpris- ingly, however, this Ser phosphorylation seems to be stable; the phosphate is not transferred to E1a [17,19]. If, in addition to Ser autophosphorylation, there was also His autophosphorylation, it would not have been detected in previous experiments [17]. The most simple interpretation of the existing data is that PDK is a protein Ser-kinase (PSK) with a phospho-His catalytic intermediate. The experiments described here were designed to test this hypothesis. Four His residues are conserved among PDK sequences from various organisms; His121, His168, His231 , and His233 [numbered according to the Arabidopsis thaliana (AtPDK) sequence] [20]. His121, is  40 residues N-terminal to the H box, His168 is within the H box, and His231 and His233 are immediately N-terminal to the N box. It has been established that His233 co-operates with Glu238 in acting as a general base catalyst in the PDK reaction [12]. We report the results of pH-stability studies, which indicate that there is not a phospho-His reaction intermediate. In addition, the activity of AtPDK was not abolished by the H121Q mutation, the H168Q mutation, or the H121Q/ H168Q double mutation. Thus, despite the sequence resemblance to PHKs, PDK does not have a phospho-His intermediate and is instead a unique type of PSK. Correspondence to D. D. Randall, Department of Biochemistry, Schweitzer Hall, University of Missouri, Columbia, MO 65211, USA. Fax: + 1 573 882 5635, Tel.: + 1 573 882 4847, E-mail: randalld@missouri.edu Abbreviations: AtPDK, Arabidopsis thaliana pyruvate dehydrogenase kinase; BCKDK, branched-chain a-ketoacid dehydrogenase kinase; E1, pyruvate dehydrogenase; kd-PDC, kinase-depleted pyruvate dehydrogenase complex; MBP, maltose-binding protein; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PHK, protein histidine-kinase; PSK, protein serine-kinase. Note: a web page is available at http://www.biochem.missouri.edu/ (Received 6 February 2002, revised 4 April 2002, accepted 15 April 2002) Eur. J. Biochem. 269, 2601–2606 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02933.x MATERIALS AND METHODS Reagents Restriction endonucleases were from New England Biolabs (Beverly, MA, USA). PCR primers were from Integrated DNA Technology, Inc. (Coralville, IA, USA). Unless otherwise noted, reagents were provided by the Sigma Chemical Company (St Louis, MO, USA). In silico analyses Amino-acid sequences were retrieved from the National Center for Biotechnology Information web page (http:// www.ncbi.nlm.nih.gov/). Sequences were initially aligned using PHYLIP [21], and the alignments polished manually. For phylogenetic comparisons, the AtPDK amino-acid sequence wasusedasthequeryfora BLAST [22] search of the NCBI database. The resulting sequences were aligned with PHYLIP , then subjected to 500 rounds of bootstrap analysis using the SEQBOOT function of DNAMAN (version 2.71; Lynnon BioSoft, Vaudreui, Quebec, Canada). The phylogenetic tree was constructed using TREEVIEW [23]. Secondary-structure- based phylogenetic comparisons were conducted using PSI - BLAST [24] coupled to GenTHREADER [25]. Bacterial expression and purification of maltose-binding protein (MBP)–AtPDK MBP–AtPDK was expressed and purified as described previously [17]. After affinity purification, the protein was concentrated using an ultrafiltration membrane with a 10-kDa cut-off (Amicon, Beverly, MA, USA), then stored in aliquots at )20 °C. Site-directed mutagenesis The AtPDK reading frame, previously cloned into pMal- cRI [17], was used as the template for mutagenesis. The BamHI–PstI fragment containing the sequence that encodes the C-terminal half of AtPDK (G160 to P366) was PCR- amplified using primers DDR229 (5¢-ctcgagctgcagctat taTCGGGTAAAGGCTCTTGCGA-3¢) and DDR 309 (5¢-CGAATCGGGATCCGGATGCTTATTGGGCAGC AAGTTGAGTTGCAT-3¢). The DDR309 primer includes the H168Q mutation (the base changed is in bold). Thermal cycling was conducted using the ACCUZYME DNA polymerase (Midwest Scientific, St Louis, MO, USA). The PCR product was inserted into pGEM-3Zf(+) and sequenced to verify the H168Q mutation and that no additional changes had been introduced. The H168Q C-terminus fragment was then transferred to pBluescript II SK+. The N-terminus (XbaI–BamHI fragment) of either the wild-type (wt) or the H121Q AtPDK sequence was then ligated to the pBluescript H168Q C-terminus, generating an ORF encoding the H168Q or H121/168Q AtPDK mutants. These constructs were transferred to pMal-cRI for expression. Phosphorylation/inactivation of the PDC Activity of the PDC was measured spectrophotometrically as the increase in A 340 due to reduction of NAD + [26]. As a substrate for recombinant PDK, kinase-depleted (kd)-PDC was prepared from purified pea seedling mitochondria as described previously [27]. For phosphorylation assays, MBP–AtPDK was incubated with kd-PDC for 5 min at 25 °C, then reaction was initiated by adding [c- 32 P]ATP (Perkin Elmer Life Sciences; 2.2 TBqÆmmol )1 ). Reactions were stopped by adding an equal volume of 2 · sample buffer [8 M urea, 4% (w/v) SDS, 4% (v/v) 2-mercaptoeth- anol, 10 m M EDTA] followed by heating at 70 °Cfor 20 min. Proteins were resolved by SDS/PAGE, transferred to nitrocellulose membranes, then examined by autoradio- graphy. Incorporation of 32 P into proteins was quantified by liquid-scintillation spectrometry of excised bands. ATPase activity was assayed essentially as described in [12], using [c- 32 P]ATP as the substrate. The 32 P i released from ATP was quantified by liquid-scintillation spectrometry. Determination of the pH stability of autophosphorylated AtPDK To investigate which amino-acid residues were involved, 1.4 lg of either wild-type MBP–PDK or the H121Q/ H168Q mutant protein was autophosphorylated with [c- 32 P]ATP, resolved by SDS/PAGE, transferred to poly(vinylidene difluoride) membranes (Millipore Corp.). The membranes were treated with 50 m M KCl/HCl, pH 1, 100 m M Tris/HCl, pH 7, or 1 M NaOH, pH 14, in the presence of 10% methanol for 2 h at 45 °C. Radioactivity was then quantified by liquid-scintillation spectrometry of the corresponding excised bands. In parallel with AtPDK, Escherichia coli CheA was autophosphorylated for use as a bona fide PHK control. RESULTS Alignment of the AtPDK sequence with those of orthologs from maize, mouse, and Ascaris suum reveals substantial primary sequence conservation (Fig. 1). Sequence identity is notably high within the H, N, D, and G boxes, which comprise the cardinal motifs of PHKs (Fig. 1). When the AtPDK sequence was used as the search term in BLAST analysis of GenBank, the only sequences retrieved that had significant E-values (<0.026) were those of other PDKs, the closely related BCKDKs, and several signal-transducing PHKs. Significance was determined using the student’s 2-tailed t test. The phylogenetic relationships among these sequences are presented in Fig. 2. Similar results were obtained when predicted secondary structures were used for comparisons (data not presented). From the results of previous analyses, it was concluded that both native and recombinant PDK autophosphory- late Ser residues. The methods used, however, would not have specifically detected the occurrence of phospho-His. To re-evaluate this question, AtPDK was autophosphor- ylated using [c- 32 P]ATP, then subjected to acidic (pH 1), neutral (pH 7), or alkaline (pH 14) treatment before quantitative analysis. The E. coli CheA protein, a PHK involved in chemotaxis, was included as a control. The 32 P labeling of AtPDK was acid-stabile but alkali-labile (Fig. 3). The extent of 32 P incorporation into the AtPDK H121Q/H168Q mutant protein was much lower than with the wild-type protein; however, the pattern of pH stability was identical. Under the same experimental conditions, 2602 A. Tovar-Mendez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 labeling of CheA was, as expected, alkali-stable and acid-labile. Recombinant AtPDK was capable of both time-depend- ent inactivation of kd-PDC (Fig. 4) and incorporation of radioactivity from [c- 32 P]ATP into E1a (Fig. 5). The kd-PDC used as the substrate for PDK had virtually no associated kinase activity. Residual PDC activity after a 60-min incubation in vitro was essentially identical with or without MgATP (Fig. 4). The H121Q/H168Q mutant of MBP–AtPDK inactivated kd-PDC less rapidly than did wild-type MBP–AtPDK (Fig. 4), although, if assays were extended, the same end-point was ultimately reached (data not presented). Whereas the MBP–AtPDK chimera had full catalytic capability, removal of the fusion partner increased the rate of PDC inactivation approximately fourfold. The lower rate of the mutant kinase was more pronounced when tested as the MBP fusion. Mutagenesis of conserved residues His121 and His168 differentially affected autophosphorylation and trans- phosphorylation of E1a (Fig. 5). The AtPDK H121Q mutant displayed an  80% reduction in the extent of autophosphorylation after 5 min, but had only a small effect on transphosophorylation of E1a. Similar results were obtained in experiments of longer duration. The H168Q mutant had less inhibition of autophosphorylation but more of transphosphorylation (Fig. 5). Inhibition of both autophosphorylation and transphosphorylation by the H121/168Q double mutant was approximately additive. In contrast with a previous report of low-level ATPase activity of rat PDK2 [12], we were unable to detect any significant hydrolysis of ATP to ADP plus P i by AtPDK (data not shown). The recombinant enzyme preparations used for ATPase assays were, however, fully capable of inactivating kd-PDC. It is not clear if the previously reported ATPase activity was the result of a minor contaminant, or if this result indicates an actual difference between the rat and A. thaliana enzymes. This possibility seems unlikely, considering the extent of protein sequence conservation. DISCUSSION A prominent regulatory mechanism for the mitochondrial PDC is multisite serine phosphorylation [2–5]; thus, PDK is a PSK. The domains that define PSK enzymes include 12 conserved subdomains which fold into a common catalytic core structure [8]. After cloning and sequence analysis, however, it was clear that mammalian PDK lacks the AtPDK MAVKKACEMFPKSLIEDVHKWGCMKQTGVSLRYM MEFGSKPTERNLLISAQFLHKELP ZmPDK2 MASEPVARAVAEEVARWGAMRQTGVSLRYM MEFGARPTERTLLLAAQFLHKELP MmPDK2 MRWVRALLKNASLAGAPKYIEHFSKFSPSPLSMKQFLDFGSSNACEKTSF TFLRQELP AsPDK MFLTRRLLGPFTSAIARKLEHYSQFQPSSLTIQQYLDFGQTGTMKSSFL FLKNELL consensus L + ++FG+ T + +L FL++ELP IRVARRAIELQTLPYGLSDKPAVLKVRDWYLESFRDMRAF PEIKDSGDEKDFTQ IRIARRALDLDSLPFGLSTKPAILKVKDWYVESFREIRSF PEVRNQKDELAFTQ VRLANIMKEINLLPDRVLGTPSVQLVQSWYVQSLLDIMEF LDKDPEDHRTLSQFTDAL VRLANIMQEISLLPPTLLKMPSRRLVSNWYCESFEDLLQFEHAQVEPDIMSKFNDQLQTI +R+A+ E+ LP +L P++ V WY+ESF D+ F PE+ + T MIKAVKVRHNNVVPMMALGVNQLKK GMN SGNLDEIHQ-FLDRFYLSRIGIRMLIG MIKMIRVRHTNVVPAIALGVQQLKKDLGGPKAFPPGIHEIHQ-FLDRFYMSRIGIRMLIG VT IRNRHNDVVPTMAQGVLEYKDTYGDD PVSNQNI-QYFLDRFYLSRISIRMLIN L— KRHSRVVETMAEGLIELRESEGVD IASERGI-QYFLDRFYINRISIRMLQN + ++ RH +VVP+MA GV LK G + + I Q FLDRFY+SRI+IRMLI ______ QHVELHNP NP PLHTVGYIHTKMSPMEVARNASEDARSICFREYGSAPEINIYGDPS QHVALHDPD P-EPGVI-GLINTKMSPMTVARIASEDARAICMREYGSSPDVDIYGDPG QHTLIFDGSTNPAHPKHI-GSIDPNCSVSDVVKDAYDMAKLLCDKYYMASPDLEIQ-EVN QHLVVF-GVVLPESPRHI-GCIDPGCDVESVVHDAYENARFLCERYYLTAPGMKL EMH QH+ + P P HI G I + S V + A E AR +C R Y ++P++ I + H-box _ FTFP–-YV PTHL-HLMMYELVKNSLRAVQERFVDSDRVAPPIRIIVADGIEDVT FTFPYVTP-HL-HLMIFELVKNSLRAVQERYMDSDKLAPPVRIIVADGAEDVT -ATNANQPIHMVYVPSHLYHML-FELFKNAMRATVESHESSLTL-PPIKIMVALGEEDLS NSVNPGMPISIVAVPSHLYHIM-FELFKNSMRATVENHGADEDL-PPIKVMVVRGAEDLS P +V P+HL H+M FEL+KNS+RA E S+ L PPI+I+VA GAED++ N-box D-box- IKVSDEGGGIARSGLPRIFTYLYSTARNPLEEDVDLGIADVPVTMAGYGYGLPISRLYAR IKISDEGGGIPRSGLSRIFTYLYSTAENPPD LDGHNEG-VTMAGYGYGIPISRLYAR IKMSDRGGGVPLRRIERLFSYMYSTAPTPQPGTGG TPLAGFGYGLPISRLYAK IKISDRGGGVSRTILDRLFTYMYSTAPPPPRDGTQPP LAGYGYGLPLSRLYAR IK+SD GGG+ R+ L RIFTY+YSTA P +AGYGYGLPISRLYAR G1-box G2-box YFGGDLQIISMEGYGTDAYLHL-SRLGDSQEPLP YFGGDLQIISMEGYGTDAYLHL-SRLGDSEEPLP YFQGDLQLFSMEGFGTDAVIYLKALSTDSVERLPVYNKSAWRHHYQTIQEAGDWCVPSTE YFHGDMYLVSMEGYGTDAMIFLKAIPVEASEVLPIYSTSSRRQLTMSPQAADWSHQLPNH YF GDLQ++SMEGYGTDA++ L + DS E LP PKNTSTYRVS GNRNL Fig. 1. Comparison of selected PDK sequences. At, A. thaliana PDK (GI:4049631); Zm, Zea mays PDK2 (GI:3695005); Mm, Mus musculus PDK2; As, Ascaris suum PKD (GI:1945392). The sequences were aligned using PHYLIP [21], then the alignments were optimized manually. The conserved His residues are shown in bold. The locations of sequences corresponding to the five cardinal motifs of PHKs [11,15] are indicated by underlines. When at least three residues belong to the same family, the consensus is indicated as (+). Ó FEBS 2002 A. thaliana pyruvate dehydrogenase kinase (Eur. J. Biochem. 269) 2603 defining domain organization of PSKs [9]. Instead PDK sequences include the five canonical domains (H, N, D, G1, and G1 boxes) of PHKs [11,15]. It was subsequently found that this same organization is shared by mammalian, nematode [28], fly [29], and plant (Figs 1 and 2) [10] PDK sequences, as well as those of the related BCKDKs [19]. Thus, PDK is a conundrum. The results from primary sequence analysis and at least some biochemical experi- ments [16] are consistent with PDK as a PHK. At the same time, it has been unequivocally established that PDK phosphorylates multiple Ser residues in the E1a regulatory target. The simplest resolution of these apparent contradic- tions would be the occurrence of phospho-His as a reaction intermediate. This, however, does not seem to be the case. Previous analyses employed pH conditions that would not have allowed detection of the transient occurrence of phospho-His. We directly addressed this by incubating AtPDK with [c- 32 P]ATP and then determining the stability of the resultant phosphoenzyme. Recombinant CheA was included in these assays as a PHK control. From previous results [17] we expected that most of the radiolabel in AtPDK would be acid-stable phospho-Ser. This was the case, and we observed no significant alkali-stable labeling of pH1 Radioactivity (cpm x 10 3 ) 0 3 6 9 12 15 CheA wild-type H121Q/ H168Q MBP-AtPDK pH7 pH14 Fig. 3. Effect of pH on the stability of AtPDK autophosphorylated using [c- 32 P]ATP. Approximately 1.4 lg wild-type or the H121Q/H168Q mutant of MBP–AtPDK, or E. coli CheA, was incubated with 2 pmol Mg[c- 32 P]ATP (0.22 TBqÆpmol )1 ) for 1 h at 25 °C. After SDS/PAGE, the proteins were transferred to poly(vinylidene difluoride) mem- branes, which were then treated with 50 m M KCl/HCl(pH 1),100m M Tris/HCl (pH 7), or 1 M NaOH (pH 14) in the presence of 10% (v/v) methanol for 2 h at 45 °C. The radiolabeled proteins were detected by autoradiography, and the bands excised and quantified by liquid- scintillation spectrometry. Relative PDC activity 0 0.2 0.4 0.6 0.8 1.0 0 Time (min) 10 20 30 40 50 60 Fig. 4. Inactivation of kd-PDC by AtPDK. All assay mixtures con- tained 45 lg kd-PDC and 20 l M MgATP. Assays contained 53 lg MBP–AtPDK (wt and mutant) or 13 lg AtPDK (wt or mutant). At the indicated time points, samples were taken for spectrophotometric assay of PDC activity. (n)+ATP;(m) – ATP + MBP–AtPDK; (j) + ATP + MBP–AtPDK; (d) + ATP + MBP–AtPDK H121Q/ H168Q; (h) + ATP + AtPDK; (s) + ATP + AtPDK H121Q/ H168Q. Data are representative results. 0.1 Sp-PDK Mm-BCKDK Nc-BCKDK Ec-SHK Ss-SHK Am-SHK Sc-SHK Bh-SHK St-SHK At-PDK Zm-PDK2 As-PDK Dm-PDK Mm-PDK Fig. 2. Phylogenetic relationships among PDKs, BCKDKs, and sensor histidine kinases (SHK). Sp, Schizosaccharomyces pombe (GI:7708590); Mm, M. musculus BCKDK (GI:6753164); Nc, Neurospora crassa (GI:12718471); Ec, E. coli BaeS (GI:2507376); Ss, Synechocystis sp. (strain PCC 6803) SHK (GI:7469378); Am, Amycolatopsis mediterra- nei kA SHK (GI:7339510); Sc, Streptomyces coelicolor SHK (GI:7799270); Bh, Bacillus halodurans ResE (GI:15614144); St, Strep- tococcus thermophilus Hpk2 (GI:13324643); At, A. thaliana PDK (GI:4049631); Zm, Z. mays PDK2 (GI:3695005); As, A. suum PDK (GI:1945392); Dm, Drosophila melanogaster PDK (GI:7303893); Mm, M. musculus PDK2 (GI:8096763). The sequences were aligned using PHYLIP ,thenanalyzedwith SEQBOOT (500 rounds of bootstrapping). The scale bar corresponds to the number of substitutions per site. The tree was constructed using TreeView. 2604 A. Tovar-Mendez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 AtPDK, although this was seen with the CheA control. The method used for these analyses does not yield an all or nothing result, so it is not possible to unequivocally state that there is not a phospho-His intermediate, although the results do not support this possibility. The phospho-His contingency was further addressed using site-directed mutagenesis. Four His residues are conserved among PDK sequences; His121, His168, His231, and His233 (numbered according to the A. thaliana PDK sequence [20]). It has been established that His233 co-operates with Glu238 in acting as a general base catalyst in the PDK reaction [12], so we discounted His231 or His233 as sites for His autophosphorylation. The remaining possi- bilities are His121 and His168, which are upstream of and within the H box, respectively. If one of these His residues were a site of autophosphorylation, it would be expected that mutagenesis would inactivate AtPDK. Although the H121Q, H168Q, and H121Q/H168Q mutant proteins had reduced PDK activity, they were each capable of both autophosphorylation and transphosphorylation of E1a. At this point, the function of Ser autophosphorylation in PDK activity is unclear, although it has been reported with PDKs from several sources [17–19]. Ser autophosphoryla- tion is apparently stable, and the phosphate group is not subsequently transferred to E1a [17,19]. The His mutagen- esis of AtPDK had distinct effects on autophosphorylation and transphosphorylation of E1a. It seemed possible that the apparently different effects were an artefact arising from differential phosphorylation of the recombinant proteins during bacterial expression. This possibility was tested by adding 32 P i to bacterial cultures before induction of AtPDK synthesis. However, none of the resulting AtPDK proteins, wild-type or any of the His mutants, showed any 32 P labeling. Thus the role of Ser autophosphorylation remains enigmatic, although the results with the His mutant AtPDK proteins further support dissociation of autophosphoryla- tion from transphosphorylation of PDC. While this manuscript was in preparation, the structure of ADP-ligated rat PDK2 was solved at 2.5 A ˚ resolution [30]. The crystallographic data support earlier sequence-based structural predictions that PDK is a member of the PHK/ ATPase superfamily [14]. The authors conclude that His115 (His121 in AtPDK) is not solvent accessible, and is involved in important structural hydrogen bonds within the N-terminal domain [30]. Thus, despite the sequence similarity to PHKs, results from both structural analyses and use of site-directed mutagenesis argue against any role for His phosphorylation in the PDK reaction. ACKNOWLEDGEMENTS G. L. Hazelbauer generously provided recombinant E. coli CheA. This research was supported by National Science Foundation grant IBN- 9876680, the Missouri Agricultural Experiment Station, and the Food for 21st Century Program. REFERENCES 1. Reed, L.J. (2001) A trail of research from lipoic acid to a-keto acid dehydrogenase complexes. J. Biol. Chem. 276, 38329–38336. 2. Mooney, B., Miernyk, J.A. & Randall, D.D. (2002) The complex fate of a-ketoacids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 53, 357–375. 3. Korotchkina, L.G. & Patel, M.S. (2001) Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase. J. Biol. Chem. 276, 37223–37229. 4. Kolobova, E., Tuganova, A., Boulatnikov, I. & Popov, K.M. (2001) Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites. Biochem. J. 358, 69–77. 5. Roche,T.E.,Baker,J.C.,Yan,X.,Hiromasa,Y.,Gong,X.,Peng, T., Dong, J., Turkan, A. & Kasten, S.A. (2001) Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. Prog. Nucleic Acid Res. Mol. Biol. 70, 33–75. 6. Lawson,J.E.,Niu,X.D.,Browning,K.S.,Trong,H.L.,Yan,J.& Reed, L.J. (1993) Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C. Biochemistry 32, 8987–8993. 7. Das,A.K.,Helps,N.R.,Cohen,P.T.&Barford,D.(1996)Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A ˚ resolution. EMBO J. 15, 6798–6809. 8. Hanks, K. & Hunter, T. (1995) Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 9, 576–596. 9. Harris, R.A., Popov, K.M., Zhao, Y., Kedishvili, N.Y., Shimomura, Y. & Crabb, D.W. (1995) A new family of protein kinases-the mitochondrial protein kinases. Adv. Enzyme Regul. 35, 147–162. 10. Thelen,J.J.,Muszynski,M.G.,Miernyk,J.A.&Randall,D.D. (1998) Molecular analysis of two pyruvate dehydrogenase kinases from maize. J. Biol. Chem. 273, 26618–26623. 11. Stock, A.M., Robinson, V.L. & Goudreau, P.N. (2000) Two- component signal transduction. Annu. Rev. Biochem. 69, 183–215. kDa 97.6 66 45 29 MBP-AtPD K E1α H121Q H168Q H121Q/ H168Q wt A B Relative incorporation 0 0.2 0.4 0.6 0.8 1.0 wt H121Q H168Q H121Q/ H168Q E1α MBP-AtPDK Fig. 5. Autophosphorylation of MBP–AtPDK and transphosphoryla- tion of PDC E1a. (A) 3 lg kd-PDC was incubated with 1 lgMBP– AtPDK plus 200 l M Mg[c- 32 P]ATP (18 GBqÆmmol )1 )for5minat 25 °C. A set of representative autoradiographs are presented. (B) Relative incorporation of 32 PintoMBP–AtPDKorE1a. Data are means ± SD from three independent enzyme preparations. The same patterns of results were obtained using either 1.5 or 6 lgofMBP– AtPDK. Ó FEBS 2002 A. thaliana pyruvate dehydrogenase kinase (Eur. J. Biochem. 269) 2605 12. Tuganova, A., Yoder, M.D. & Popov, K.M. (2001) An essential role of Glu-243 and His-239 in the phosphotransfer reaction cat- alyzed by pyruvate dehydrogenase kinase. J. Biol. Chem. 276, 17994–17999. 13. Wynn, R.M., Chuang, J.L., Cote, C.D. & Chuang, D.T. (2000) Tetrameric assembly and conservation in the ATP-binding domain of rat branched-chain alpha-ketoacid dehydrogenase kinase. J. Biol. Chem. 275, 30512–30519. 14. Bowker-Kinley, M. & Popov, K.M. (1999) Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily. Biochem. J. 344, 47–53. 15. West, A.H. & Stock, A.M. (2001) Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem. Sci. 26, 369–376. 16.Mooney,B.P.,David,N.R.,Thelen,J.J.,Miernyk,J.A.& Randall, D.D. (2000) Histidine modifying agents abolish pyruvate dehydrogenase kinase activity. Biochem. Biophys. Res. Commun. 267, 500–503. 17. Thelen, J.J., Miernyk, J.A. & Randall, D.D. (2000) Pyruvate dehydrogenase kinase from Arabidopsis thaliana:aproteinhisti- dine kinase that phosphorylates serine residues. Biochem. J. 349, 195–201. 18. Jackson, J.C., Vinluan, C.C., Dragland, C.J., Sundararajan, V., Yan,B.,Gounarides,J.S.,Nirmala,N.R.,Topiol,S.,Ramage,P., Blume, J.E., Aicher, T.D., Bell, P.A. & Mann, W.R. (1998) Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues. Biochem. J. 334, 703–711. 19. Davie, J.R., Wynn, R.M., Meng, M., Huang, Y.S., Aalund, G., Chuang, D.T. & Lau, K.S. (1995) Expression and characterization of branched-chain alpha-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase? J. Biol. Chem. 270, 19861– 19867. 20. Thelen, J.J., Miernyk, J.A. & Randall, D.D. (1998) Nucleotide and deduced amino acid sequences of the pyruvate dehydrogenase kinase from Arabidopsis thaliana (accession, AF039406) (PGR 98–192). Plant Physiol. 118, 1533. 21. Felsenstein, J. (1989) PHYLIP-Phylogeny Inference Package Version 3.2. Cladistics 5, 164–166. 22. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215,403– 410. 23. Page, R.D. (1996) TreeView: an application to display phylo- genetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358. 24. Altschul,S.F.,Madden,T.L.,Schaffer,A.A.,Zhang,J.,Zhang,Z., Miller, W. & Lipman, D.J. (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. 25. Jones, D.T. (1999) GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J. Mol. Biol. 287, 797–815. 26. Miernyk, J.A. & Randall, D.D. (1987) Some kinetic and reg- ulatory properties of the pea mitochondrial pyruvate dehy- drogenase complex. Plant Physiol. 83, 306–310. 27. Thelen, J.J., Miernyk, J.A. & Randall, D.D. (1998) Partial pur- ification and characterization of the maize mitochondrial pyruvate dehydrogenase complex. Plant Physiol. 116, 1443–1450. 28. Chen, W., Huang, X., Komuniecki, P.R. & Komuniecki, R. (1998) Molecular cloning, functional expression, and character- ization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum. Arch. Biochem. Biophys. 353, 181–189. 29. Katsube, T., Nomoto, S., Togashi, S., Ueda, R., Kobayashi, M. & Takahisa, M. (1997) Sequence and expression of a gene encoding a pyruvate dehydrogenase kinase homolog of Drosophila melano- gaster. DNA Cell Biol. 16, 335–339. 30. Steussy, C.N., Popov, K.M., Bowker-Kinley, M.M., Sloan, R.B. Jr, Harris, R.A. & Hamilton, J.A. (2001) Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase. J. Biol. Chem. 276, 37443–37450. 2606 A. Tovar-Mendez et al.(Eur. J. Biochem. 269) Ó FEBS 2002 . randalld@missouri.edu Abbreviations: AtPDK, Arabidopsis thaliana pyruvate dehydrogenase kinase; BCKDK, branched-chain a-ketoacid dehydrogenase kinase; E1, pyruvate dehydrogenase; kd-PDC, kinase- depleted pyruvate dehydrogenase. LAGYGYGLPLSRLYAR IK+SD GGG+ R+ L RIFTY+YSTA P +AGYGYGLPISRLYAR G1-box G2-box YFGGDLQIISMEGYGTDAYLHL-SRLGDSQEPLP YFGGDLQIISMEGYGTDAYLHL-SRLGDSEEPLP YFQGDLQLFSMEGFGTDAVIYLKALSTDSVERLPVYNKSAWRHHYQTIQEAGDWCVPSTE . IKVSDEGGGIARSGLPRIFTYLYSTARNPLEEDVDLGIADVPVTMAGYGYGLPISRLYAR IKISDEGGGIPRSGLSRIFTYLYSTAENPPD LDGHNEG-VTMAGYGYGIPISRLYAR IKMSDRGGGVPLRRIERLFSYMYSTAPTPQPGTGG TPLAGFGYGLPISRLYAK IKISDRGGGVSRTILDRLFTYMYSTAPPPPRDGTQPP