From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
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ABSTRACT |
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Pyruvate dehydrogenase phosphatase
(PDP) is one of the few mammalian phosphatases residing within the
mitochondrial matrix space. It is responsible for dephosphorylation and
reactivation of the pyruvate dehydrogenase complex (PDC) and, by this
means, is intimately involved in the regulation of utilization of
carbohydrate fuels in mammals. PDP is a dimeric enzyme consisting of
catalytic and regulatory subunits. The catalytic subunit of PDP is a
Mg2+-dependent enzyme homologous to the
cytosolic phosphatases of the 2C family. In the present study, we
isolated two cDNAs encoding for mitochondrial phosphatases. The
first cDNA is highly homologous to the previously identified
cDNA encoding for the catalytic subunit of PDP (PDP1). The second
cDNA encodes a previously unknown catalytic subunit of PDP (PDP2).
The new phosphatase, expressed as the recombinant protein in
Escherichia coli, shows strict substrate specificity toward
PDC and does not use phosphorylated branched chain -ketoacid dehydrogenase as substrate. Like PDP1, PDP2 is a
Mg2+-dependent enzyme, but its sensitivity to
Mg2+ ions is almost 10-fold lower than that of PDP1. In
contrast to PDP1, PDP2 is not regulated by Ca2+ ions.
Instead, it is sensitive to the biological polyamine spermine, which,
in turn, has no effect on the enzymatic activity of PDP1. Western blot
analysis of PDP extracted from mitochondria isolated from liver and
skeletal muscle revealed that PDP1 is predominantly expressed in
mitochondria from skeletal muscle, whereas PDP2 is much more abundant
in the liver rather than muscle mitochondria. Both isoenzymes are
expressed in mitochondria from 3T3-L1 adipocytes, but the level of
expression of PDP2 is considerably higher. These observations are
consistent with previous findings on the enzymatic parameters of PDP in
adipose tissue. Thus, our results provide the first evidence that there
are at least two isoenzymes of PDP in mammals that are different with
respect to tissue distribution and kinetic parameters and, therefore,
are likely to be different functionally.
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INTRODUCTION |
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The reaction catalyzed by the mammalian pyruvate dehydrogenase complex (PDC)1 links glycolysis with several biochemical pathways. In tissues with large energy demands like brain, muscle, and heart, it supplies the carbon units derived from carbohydrate fuels mainly for complete oxidation by the Krebs cycle. In lipogenic tissues such as adipose, mammary gland, and liver, the metabolic fate of acetyl-CoA derived from carbohydrates is quite different, because it can be used for the biosynthesis of fatty acids and cholesterol (for reviews, see Refs. 1 and 2). Therefore, the reaction catalyzed by PDC should be highly regulated, and this regulation must be sophisticated enough to accommodate the different metabolic requirements of a variety of tissues.
Indeed, several molecular mechanisms have been implicated in the regulation of mammalian PDC. In the short term, the enzyme activity may be regulated by negative feedback (3, 4). The products of the PDC reaction, NADH and acetyl-CoA, inhibit the overall reaction by reversing the partial reactions catalyzed by the dihydrolipoamide dehydrogenase and transacetylase components of the complex (4). This mechanism may be important for the rapid adjustment of flux through PDC, particularly in the liver (3). A perhaps even more important mechanism of PDC regulation is based on reversible phosphorylation (5). Mammalian mitochondria contain two enzymes dedicated to the regulation of PDC: pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) (6). The kinase is an integral part of the multienzyme complex (7). It phosphorylates three serine residues of the decarboxylase component (pyruvate decarboxylase) (8). Phosphorylation completely halts the enzymatic activity of PDC (9, 10). The phosphoenzyme can be reactivated only via dephosphorylation catalyzed by phosphatase (11). In contrast to PDK, PDP is loosely associated with PDC but becomes complex-bound upon stimulation (12). Both PDK and PDP can integrate a considerable number of different regulatory stimuli (reviewed in Ref. 13). The kinase activity is likely to reflect the relative intramitochondrial concentrations of the substrates (pyruvate, NAD+, and CoA) and products (NADH and acetyl-CoA) of the main dehydrogenase reaction. An excess of substrates results in the inhibition of kinase activity with concomitant activation of PDC by the phosphatase. An excess of products activates the kinase and results in the net phosphorylation and inactivation of PDC. The enzymatic activity of PDP depends on intramitochondrial concentrations of Mg2+ and Ca2+ ions (14, 15). The latter promotes the association of PDP with the complex (12) and, by this means, stimulates the rate of dephosphorylation. Another co-factor that may be important for the regulation of PDP is intramitochondrial NADH (16). At least in vitro, NADH inhibits the phosphatase activity and, therefore, may simultaneously modulate the activities of both the kinase and the phosphatase (17). Thus, the phosphorylation state of PDC in mammalian mitochondria reflects the relative activities of the kinase and phosphatase, which, in turn, depend upon the intramitochondrial concentrations of different metabolites and co-factors.
Tissue-specific aspects of the regulation of mammalian PDC became a subject of interest fairly recently when it was found that there are multiple isoenzymes of PDK in humans and rodents (18, 19). Thus far, four genes showing characteristic patterns of tissue-specific expression have been identified (18, 20). The respective protein products appear to be somewhat different with respect to their enzymatic activities and regulation (19). These observations bring about the very intriguing possibility that regulation of PDC in mammals may be tissue-specific, reflecting the isoenzymic composition of PDK. Whether the diversity of PDKs in mammals is matched by the diversity of PDPs, however, is largely unknown. So far, just one gene encoding PDP has been identified by Lester Reed's laboratory (21). It encodes for a Mg2+-dependent, Ca2+-stimulated form of PDP (22). Some studies, however, have suggested that the complexity of the PDP gene family may be greater. For example, PDP purified from the parasitic nematode Ascaris suum is Ca2+-insensitive but responsive to physiologically relevant concentrations of malate (23). Accordingly, it appears that malate at physiologically relevant concentrations produces marked activation of spermatozoal PDC and does not modify significantly the activity of liver PDC (24). Even more importantly, Rutter et al. (25) reported that the kinetic properties of PDP in mitochondria from adipose tissue are markedly different from those that were described for purified enzyme. Calcium ions at physiologically relevant concentrations activate adipose PDP at low concentrations of Mg2+. However, when Mg2+ was present at saturating concentrations, there was no longer any effect of Ca2+ on PDP activity (25). In contrast, Ca2+-stimulated PDP purified from kidney mitochondria showed a large activation by Ca2+ at all concentrations of Mg2+ (14, 15). These findings prompted us to analyze the diversity of the PDP gene family in mammals. Here we report the first evidence indicating that indeed there is another isoenzyme of PDP in rodents and, probably, in humans. It is abundantly present in mitochondria from liver and 3T3-L1 adipocytes and virtually absent in mitochondria from skeletal muscle. We also show that this isoenzyme, obtained as the recombinant protein, is not sensitive to stimulation by Ca2+ ions but instead can be directly regulated by spermine.
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EXPERIMENTAL PROCEDURES |
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Materials--
5'-STRETCH PLUS gt10 rat heart and rat liver
cDNA libraries, bovine total RNA, QUICK-CloneTM rat
liver cDNA, rat liver 5'-RACE-ReadyTM cDNA, and
TALONTM metal affinity resin were purchased from
CLONTECH Laboratories, Inc. QIAGEN plasmid
purification kits were obtained from QIAGEN Inc. Native Pfu
DNA polymerase was from Stratagene. The reverse transcriptase-PCR kit
was obtained from Perkin-Elmer. Restriction and DNA modifying enzymes
were purchased from Life Technologies, Inc. The bacterial expression
vector pET-28a and Escherichia coli strain BL21(DE3) were
from Novagen. pGroESL plasmid was obtained as a generous gift from Dr.
Antony Guttenbery (DuPont). Recombinant human PDC and PDK3 were
prepared as described elsewhere. 125I-Protein A was
obtained from ICN Biochemicals. Radioactive nucleotides were from NEN
Life Science Products. Other reagents used were of the highest purity
available commercially.
Polymerase Chain Reaction-- Common PCR primers for amplification of PDP-related genes were designed according to the sequences of polypeptides -A345TDGLWE351- and -M442YRDDIT448- of bovine PDP (GCN ACN GA(C/T) GGN CTN TGG GA and GT(A/G/T) AT(A/G) TC(A/G) TCN CG(A/G) TAC AT, respectively). These primers were used to amplify rat liver QUICK-CloneTM cDNA. The PCR reaction mixture contained 50 pmol of each gene-specific primer and 1 ng of double-stranded cDNA as well as deoxyribonucleic triphosphates, buffer, and 5.0 units of native Pfu DNA polymerase, which were added according to the manufacturer's instructions. 35 cycles of PCR were set up using 1 min at 94 °C for denaturation, 1 min at 50 °C for annealing, and 1 min at 72 °C for extension. The resulting PCR product of approximately 300 bp was subcloned in pUC18 and sequenced. The cDNA for bovine PDP was amplified by reverse transcriptase-PCR using reverse-transcribed total RNA from bovine heart as the template. The upstream (GCT TCC ACA CCG CAG AAG TTT) and downstream (CTG TTC CTG GTT TTG ATA TGC) PCR primers corresponded to the bases 378-398 and 1757-1778 of published cDNA. Reverse transcriptase-PCR reactions were set up according to the manufacturer's instructions. The first strand was primed with random hexamers. The cycling parameters were as follows: 1 min at 94 °C for denaturation, 1 min at 60 °C for annealing, and 3 min at 72 °C for extension with a 10-min final extension at 72 °C. The resulting DNA (1.4 kb) was subcloned in pUC18 and sequenced.
5'-Stretch gt10 cDNA Library Screening--
The rat heart
cDNA library was screened with random-primed
32P-labeled cDNA of bovine PDP (26). Approximately
2-4 × 106 plaque-forming units of cDNA library
were screened as described elsewhere. In order to clone the cDNA
encoding for the PDP-related protein, approximately 0.5-1 × 106 plaque-forming units of rat liver
gt10 cDNA
library were screened with 32P-labeled 0.3-kb PCR product,
which was obtained as described above. Positive plaques from both
libraries were purified by four additional rounds of plating and
screening.
DNA was purified as described by Chisholm (27) and
digested with EcoRI. Resulting cDNAs were separated on
TEA-agarose gel, purified, and religated in EcoRI-digested
pUC18 for sequencing.
Rapid Amplification of the 5'-End of PDP2 cDNA (5'-RACE)-- Commercially available 5'-RACE-ReadyTM templates prepared from rat liver cDNA were used in order to obtain the 5'-end of PDP2 cDNA. The first round of amplification was set up according to the manufacturer's instructions with gene-specific primer (TAG GTC AAG GAG TTC CTG CCA ATA) corresponding to bases 663-686 of PDP2 cDNA. For the second round of amplification, gene-specific primer (CCA CTG CAG GAT GGG CAG CAA) corresponding to positions 558-596 was used. Several PCR products ranging in size from 300 to approximately 700 bp were separated on TEA-agarose gel, purified, and subcloned in pUC18 for sequencing. The PCR products obtained from three independent amplifications were analyzed.
Construction of the Bacterial Expression Vectors for Rat PDP1 and PDP2 cDNAs-- NcoI and XhoI restriction sites flanking the coding region of PDP1 cDNA corresponding to the mature polypeptide were constructed by PCR using the cDNA of rat PDP1 (cloned in the present study) as the template and native Pfu DNA polymerase. The sense primer (AAA CCA TGG CTT CTA CGC CTC AGA AAT TTT AC) containing the NcoI restriction site (both restriction sites are underlined) corresponded to bases 340-363 of rat PDP1 cDNA. The antisense primer (TTT CTC GAG CTG TTC CTG GTT TTG ATA TGC) carrying the XhoI restriction site corresponded to bases 1720-1740. The resulting cDNA was digested with NcoI and XhoI restrictases and subcloned between the NcoI and XhoI sites of the pET-28a expression vector in order to produce the carboxyl-terminal fusion with His6 tag encoded by the vector (plasmid PDP1). The cDNA for rat PDP2 was amplified using primers AAA TCA TGA CAT CAA CCG AGG AAG AGG ATT (bases 225-247) and TTT CTC GAG ACC CTC CTT AAA ATA GGT ATC (bases 1597-1617). The sense primer contained the BspHI restriction site, and the antisense primer contained the XhoI restriction site (both restriction sites are underlined). Amplification reactions were performed using rat liver QUICK-CloneTM cDNA as the template with native Pfu DNA polymerase. The resulting cDNA of approximately 1.4 kb was digested with BspHI (New England BioLabs) and XhoI restrictases and subcloned between NcoI and XhoI sites of pET-28a (plasmid PDP2). The resulting plasmid directs the synthesis of PDP2 with a carboxyl-terminal His6 tag. The fidelity of the pPDP1 and pPDP2 vectors was established by nucleotide sequencing.
Nucleotide Sequencing-- Sequencing of double-stranded plasmid DNA was carried out by the Biochemistry Biotechnology Facility (Indiana University) using a Taq DyeDeoxy terminator cycle sequencing kit with AmpliTaq DNA polymerase, FS (Perkin-Elmer) following the manufacturer's instructions. Both strands were sequenced.
Expression and Purification of Recombinant Proteins--
To
establish expressing cell lines, competent BL21(DE3) cells were
co-transformed with one of the pPDP vectors and pGroESL containing the
inducible genes for the molecular chaperonins GroEL and GroES.
Resulting transformants were selected on TY agar plates containing
kanamycin (45 µg/ml) and chloramphenicol (35 µg/ml). Several
colonies displaying resistance to both antibiotics were tested for
their ability to express phosphatases and were used to prepare glycerol
stocks. In order to express the recombinant phosphatases, 10 µl of
the respective glycerol stock was inoculated into 1 liter of M9ZB
medium containing kanamycin (45 µg/ml) and chloramphenicol (35 µg/ml). The cells were allowed to grow at 37 °C in an incubator
with constant shaking at 200 rpm. After the OD of the culture at 600 nm
reached 0.5-0.6, the flasks were transferred to a shaker at room
temperature and induced with 0.4 mM
-D-thiogalactopyranoside. Incubation was continued for
another 20-24 h at room temperature.
Preparation of Phosphorylated
Substrate--
32P-Labeled substrate was prepared by
incubating human recombinant PDC (2-5 mg) in MEMG buffer supplemented
with 2.5 mM MgCl2, 0.1 mM
[-32P]ATP (specific activity ~1,000 cpm/pmol) and
recombinant PDK3 (10-25 µg) in a final volume of 1.0 ml at 37 °C
for 30 min. By the end of the incubation, PDC was precipitated with
polyethylene glycol-8000 (12% final concentration) for 1 h on
ice. The precipitate was collected by centrifugation at 12,000 rpm for
10 min at 4 °C, dissolved in 0.5 ml of MEMG, and passed through a
PD-10 column equilibrated with MEMG to remove the radioactive ATP. The
resulting complex contained 4-9 nmol of phosphoryl group/mg of
protein.
PDP Assay-- The PDP activity assay was performed essentially as described by Yan et al. (22). Briefly, the extract containing PDP was diluted in MEMG buffer containing 1 mg/ml bovine serum albumin, and aliquots containing approximately 1.0 µg of total protein were preincubated at 30 °C for 2 min with the indicated concentrations of MgCl2 and effectors. The reaction was initiated by adding 32P-labeled PDC (final concentration, 1.0 mg/ml) in a final volume of 40 µl. The reaction was allowed to proceed for 90 s and then terminated with 200 µl of 20% trichloroacetic acid. Precipitated protein was removed by centrifugation at 12,000 rpm for 2 min. Aliquots of the supernatant were counted in order to determine the PDP activity. All experiments were conducted in duplicate. Under the conditions described, the time course of the reaction was linear for several minutes of incubation, and the rate of the reaction was proportional to the amount of PDP added. The results of kinetic experiments were fitted and analyzed using GraFit software (Eritacus Software).
Antisera and Immunoblotting-- Antisera against recombinant PDP1 and PDP2 were produced in New Zealand White rabbits following a standard immunization protocol (28). Antisera obtained were specific for each of these isoenzymes and showed little if any cross-reactivity under the loading and detection conditions used in the present study for immunoblotting. Western blot analysis was carried out essentially as described previously (29). Briefly, the mitochondria purified from rat skeletal muscle, liver, or 3T3-L1 adipocytes were solubilized in 50 mM Tris-HCl, pH 6.8, 1.0 mM EDTA, 2% (w/v) SDS at 100 °C for 5 min. Unsolubilized proteins were removed by centrifugation at 12,000 × g for 10 min at room temperature. Mitochondrial proteins (~10 µg of total mitochondrial protein/lane) were separated on SDS-polyacrylamide gel electrophoresis according to Laemmli (30). Separated proteins were transferred to a nitrocellulose membrane. Blots were blocked in 50 mM Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.05% (w/v) Tween 20 (TBST) plus 5.0% (w/v) bovine serum albumin for 2-4 h at room temperature and then incubated with anti-PDP1 or anti-PDP2 antiserum diluted 1:1000 in TBST/bovine serum albumin at 4 °C for 10-12 h. Antibody-antigen complexes were visualized after incubation with 125I-protein A (0.5 µCi/ml) in TBST/bovine serum albumin followed by autoradiography. Under the described conditions, there was a linear relationship between the relative densities of the bands corresponding to PDP1 or PDP2 and the amount of total mitochondrial protein loaded per lane.
Other Techniques-- 3T3-L1 cells purchased from ATCC were maintained and differentiated as described by Kedishvili et al. (31). Mitochondria from skeletal muscle, liver, and 3T3-L1 adipocytes were isolated according to Refs. 32-34, respectively. Protein concentration was determined according to Lowry et al. (35).
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RESULTS AND DISCUSSION |
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Molecular Cloning of Rat PDP1 and PDP2 cDNAs-- Results from several laboratories have indicated that in mammals, and possibly in other higher eukaryotes, there may be more then one gene encoding for the phosphatase regulating the activity of PDC (23-25). These studies prompted us to search for genes that are homologous to the bovine PDP characterized recently (21). Thus, we employed a PCR-based approach using common primers designed according to the sequences of the two polypeptides (-A345TDGLWE351- and -M442YRDDIT448-). These sequences were chosen based on the recently solved three-dimensional structure of phosphoprotein phosphatase 2C (PP2C) (36). The bovine PDP is a metal-dependent enzyme showing some sequence similarity to PP2C, as pointed out by Lawson and colleagues (21). This suggested that PDP may have a folding pattern reminiscent of other metal-dependent phosphatases (36). The two polypeptides chosen for the primer design in this study are likely to be a part of the binuclear metal-binding center, residues Asp347 and Asp445 of bovine PDP, and, therefore, should be conserved among PDP-related proteins (see the model below). The use of these primers has an additional advantage, because, in contrast to the cytosolic phosphatases, PDP has a 56-amino acid long insertion in this region. This, therefore, allows the identification of PDP-related cDNAs according to their size (approximately 300 bp). As discussed under "Experimental Procedures," amplification of rat liver cDNA indeed yielded a product 300 bp long. Sequencing of this cDNA revealed that it encodes for the protein, showing about 60% similarity to the previously reported sequence of bovine PDP. This finding indicated that there may be another isoenzyme of PDP in mammals or, alternatively, that the rat enzyme is quite distantly related to the bovine one.
To explore these possibilities, we undertook a series of cloning experiments designed to obtain the cDNA encoding the entire polypeptide of a new PDP-like protein (PDP2) as well as to check whether rodents have a gene analogous to the bovine PDP (PDP1). In order to clone the full-length cDNA encoding rat PDP1, a heart cDNA library was screened with bovine cDNA obtained by PCR. As reported previously by Lawson et al. (21), for unknown reasons the cDNA for PDP1 is underrepresented in commercially available libraries. This appeared to be the case for the rat cDNA as well. Screening of approximately 2-4 × 106 plaque-forming units of the cDNA library resulted in just one positive clone. Fortunately, the resulting cDNA was 2295 bp long and contained the entire coding region of rat PDP1 (Fig. 1A). The putative translation initiation signal is located at base 127 of the rat cDNA, corresponding to the one suggested for the bovine sequence. Its position, however, can now be identified precisely, because, in contrast to the bovine cDNA, the rat cDNA does not have an open reading frame and an alternative translation initiation signal upstream from the ATG at base 127. The putative translation termination signal is located at base 1741 of the rat cDNA. It corresponds to the respective signal in the bovine sequence (21). The 3'-noncoding region of the cloned cDNA is 619 bp long and lacks an apparent polyadenylation signal.
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Primary Structures of PDP1 and PDP2-- The cDNAs of PDP1 and PDP2 predict polypeptides of 538 and 530 amino acids long with calculated molecular masses of 61,207 and 59,654 Da, respectively. Like most mitochondrial proteins, PDP1 contains the mitochondrial targeting sequence at its amino terminus, which is cleaved after translocation into the mitochondrial matrix space (37). PDP1 has a relatively long leader peptide of 71 amino acids. Its cleavage should generate the mature protein with a predicted molecular mass of 52,618 Da (the amino acid sequence of the mature polypeptide of PDP1 as determined by Lawson et al. (21) for bovine enzyme is underlined in Fig. 1A). Unfortunately, the isoenzyme PDP2, in contrast to PDP1, has never been purified from native sources, and thus, its amino-terminal sequence is currently unknown. Some predictions, however, can be made based on analysis of its primary structure. As shown in Fig. 1B, the sequence of the first 65-70 amino acids of PDP2 is almost completely devoid of negatively charged amino acids and enriched in positively charged residues, which are spread along the entire 70-amino acid-long peptide. These features are consistent with the idea that this region comprises the mitochondrial targeting sequence of PDP2 and should direct the polypeptide into the mitochondrial matrix space (37). Since Western blot analysis suggests that the molecular weight of PDP2 is close to the molecular weight of PDP1 (see below), it is likely that the length of the PDP2 leader peptide is approximately 65-69 amino acids (the putative amino-terminal sequence of the mature polypeptide of PDP2 is underlined in Fig. 1B).
Alignment of the deduced protein sequences of PDP1 and PDP2 revealed approximately 55% identity within the sequences of the mature polypeptides. In several regions that are presumably functionally important, the identity reaches up to 90% (residues 72-94, 163-180, 196-228, 318-351, 404-417, and 432-448 of PDP1). On the other hand, the similarity to the homologous phosphatase 2C is only about 20% (Fig. 2). Like PDP1, PDP2 has a sequence reminiscent, although imperfect, of the EF-hand motif (residues 173-184 for both isoenzymes) (21), which has been implicated in the regulation of PDP activity by Ca2+ ions. It also has several insertions that are characteristic of PDP versus the PP2C family (Fig. 2). Thus, comparison of the deduced polypeptide sequences of PDP1 and PDP2 clearly indicates that these enzymes are structurally related.
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Hypothetical Three-dimensional Structure of PDP--
Considerable
progress was made recently in the elucidation of the structure and
function of metal-dependent phosphatases when Das et
al. (36) reported the first three-dimensional structure of
phosphatase 2C. It revealed a new fold in the catalytic core in which
two -sheets form a structure called a
-sandwich surrounded by
-helices. The active site of the enzyme appeared to be on top of the
-sandwich and is composed of two metal ions chelated by several acid
residues spread along the sequence of the entire polypeptide (Fig.
3, top panel). It was
suggested that metal-bound water coordinates the phosphate group and
also serves as a nucleophile for the hydrolytic reaction. The authors
also suggested that several other phosphatases, including PDP, should
share a similar folding pattern (36). The alignment shown in Fig. 2,
made according to Das et al. (36), shows that indeed the
secondary structure of the catalytic domain of PDP may be similar to
that of PP2C. Both 51- and 56-residue-long insertions within the PDP
sequence can be accommodated as loops between helices A1a and A2 and
between helix A6 and strand B11, respectively. The alignment also shows that phosphatase 2C contains a carboxyl-terminal domain built of
helices A7, A8, and A9, which is absent in the structure of PDP. The
information concerning the sequence alignment and the three-dimensional
atomic coordinates from the study of protein phosphatase 2C by Das
et al. (36) were used to produce a putative three-dimensional structure of PDP (Fig. 3, middle panel).
The coordinates for PP2C were kindly given to us by Dr. David Barford (University of Oxford). The three-dimensional structure of
sequence-aligned PDP was obtained by optimization of a molecular
probability density function using the program MODELLER, version 4.0 (38). Despite the speculative character of this model, some of the
details appear to be quite revealing. First of all, the residues
Asn49, Glu53, Asp54,
Asp73, Gly74, Asp347, and
Asp445 of PDP are likely to form the binuclear metal center
(Asp73, Gly74, Asp347, and
Asp445) and to bind the phosphate ion (Asn49,
Glu53, and Asp54). This explains why some of
the corresponding regions have been previously identified as subdomains
of PDP through sequence alignment (21). Second, it appears that the
previously mentioned EF-hand motif-like sequence is located within
helix A2 and, therefore, that its involvement in the regulation of PDP
activity by Ca2+ ions is highly unlikely. Third, the
56-residue-long loop 2 (L2), which is absent in the structure of PP2C,
appears to be in proximity to the active site (Fig. 3, bottom
panel). Therefore, it may shield the active site and require
movement in order to make the active site accessible to the protein
substrate. This, in turn, should affect the position of
Asp445, which is part of the binuclear metal-binding center
and, therefore, change the affinity of PDP for the metal ion. It is
interesting to note in this respect that the regulation of PDP activity
by Ca2+ ions, spermine, or the regulatory subunit is
associated with changes in the affinity for the metal co-factor that
are often accompanied by changes in Vmax (16,
22). These observations are consistent with the idea that binding of
Ca2+ ions and/or the regulatory subunit affects the
position of L2, making the active site more or less accessible to
substrate. Taking into account that part of L2 is involved in chelating
Mg2+ ions, these movements should simultaneously affect the
affinity for the metal co-factor. It is even possible that L2 itself
may be a part of the structure responsible for the binding of
Ca2+ ions because it contains multiple negatively charged
residues spread along its entire length.
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Enzymatic Activities of PDP1 and PDP2-- The physiological role of the isoenzymes of PDP can not be understood without analysis of their kinetics and regulation. Thus far, these data are available only for isoenzyme PDP1, which has been quite extensively characterized primarily through the efforts of Lester Reed's laboratory (12, 14, 21, 22). The comparable analysis for isoenzyme PDP2 is complicated by fact that this isoenzyme has never been purified from native sources. Thus, to initiate these studies, we developed an expression system allowing production of the recombinant isoenzymes in E. coli. Both cDNAs were expressed in the BL21(DE3) strain under control of the strong bacteriophage T7 promoter. This system allowed us to purify approximately 10-20 mg of each phosphatase from 1 liter of culture using immobilized metal affinity chromatography. For kinetic experiments, we analyzed the enzymes in E. coli extracts without further purification to avoid interference with phosphatase activity that could be caused by Co2+ or Ni2+ ions commonly used in immobilized metal affinity chromatography.
Both isoenzymes appeared to be highly specific for PDC and showed little if any activity for the related mitochondrial multienzyme complex, the branched chain
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Western Blot Analysis of PDP1 and PDP2 in Mitochondria from Skeletal Muscle, Liver, and 3T3-L1 Adipocytes-- Considering the unusual enzymatic properties of PDP2, we were interested in determining the tissues expressing this isoenzyme. Therefore, we analyzed skeletal muscle, where PDC is primarily a catabolic enzyme and is regulated by calcium ions; liver, where PDC is mainly an anabolic enzyme involved in the biosynthesis of lipids; and 3T3-L1 adipocytes, which are a well established model for studies on the regulation of glycogen and fatty acid synthesis by insulin. Mitochondria from the above sources were prepared following established protocols and analyzed by Western blotting. Approximately 10 µg of mitochondrial protein was loaded per lane. In studies involving animals, two groups consisting of four animals each were analyzed. In studies with 3T3-L1 cells, we analyzed eight independent preparations of adipocytes. Both anti-PDP1 and anti-PDP2 sera appeared to recognize the immunoreactive protein with a molecular mass of approximately 53 kDa (Fig. 5). However, in muscle mitochondria, it was recognized by anti-PDP1 serum (Fig. 5A, top panel), whereas in liver mitochondria staining was observed mainly with anti-PDP2 serum (Fig. 5B, middle panel). These data, therefore, provide strong evidence that isoenzymes of PDP have a different pattern of tissue distribution. It appears that the Ca2+-sensitive isoenzyme PDP1 is preferentially expressed in muscle mitochondria. In mitochondria from liver, in contrast, it was virtually undetectable, suggesting that if it is expressed there, the level of expression must be very low. On the other hand, isoenzyme PDP2 was present much more abundantly in liver than in muscle. The analysis of mitochondria from 3T3-L1 cells showed abundant expression of isoenzyme PDP2 (Fig. 5B, bottom panel) and only marginal expression, compared with muscle mitochondria, of isoenzyme PDP1 (Fig. 5A, bottom panel). These observations may provide a rationale for previous results, which indicated that PDP in adipose tissue responds to Ca2+ stimulation only at low concentrations of Mg2+ and disappears when the concentration of Mg2+ is raised (25). As shown in the present study, PDP2 has a relatively low activity when the concentration of Mg2+ is low. Thus, it is likely that under these conditions the total PDP activity of adipose tissue primarily reflects the activity of PDP1, which is present in adipose mitochondria at low levels. At high concentrations of Mg2+, when the activity of PDP2 is high, total activity should mainly reflect the contribution of PDP2, which is abundant in adipose mitochondria and is insensitive to Ca2+ ions. It should be noted in this respect that adipose is one of the few tissues where insulin directly regulates the activity of PDP. Taking into account the results of the present study indicating that PDP2 is a major isoenzyme in 3T3-L1 adipocytes, it will be interesting to see the effect of insulin on PDC activity using this model, because such a study may shed new light on the role of PDP2 in the physiological regulation of PDC activity.
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ACKNOWLEDGEMENTS |
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We are thankful to Patricia A. Jenkins for help in the preparation of this manuscript and to Nitin Sud for help with the isolation of rat PDP1 cDNA.
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FOOTNOTES |
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* This work was supported by U.S. Public Health Service Grants GM 51262 (to K. M. P.) and DK 47844 (to R. A. H.), a grant from the Grace M. Showalter Trust (to K. M. P.), and an American Heart Association, Indiana Affiliate, Inc., postdoctoral fellowship (to P. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF062740 and AF06741.
Present address: Microbiological Associates, Inc., 9900 Blackwell
Rd., Bethesda, MD 20850.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5122. Tel.: 317-274-6694; Fax: 317-274-4686; E-mail: kpopov{at}iupui.edu.
1 The abbreviations used are: PDC, pyruvate dehydrogenase complex; PDP, pyruvate dehydrogenase phosphatase; PDP1, previously identified cDNA encoding for the catalytic subunit of PDP; PDP2, previously unidentified cDNA encoding for the catalytic subunit of PDP; PDK, pyruvate dehydrogenase kinase; PP2C, phosphoprotein phosphatase 2C; kb, kilobase(s); bp, base pair(s); Mops, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.
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