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Thomas E. Roche - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of the catalytic subunit of bovine Pyruvate Dehydrogenase Phosphatase.
Acta Crystallographica Section F Structural Biology Communications, 2020Co-Authors: Youzhong Guo, Thomas E. Roche, Weihua Qiu, Marvin L. HackertAbstract:Mammalian Pyruvate Dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH Phosphatase isomers, respectively. PDH Phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 A resolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 A. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (Rfree = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the Pyruvate Dehydrogenase complex.
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Formation of a complex of the catalytic subunit of Pyruvate Dehydrogenase Phosphatase isoform 1 (PDP1c) and the L2 domain forms a Ca2+ binding site and captures PDP1c as a monomer.
Biochemistry, 2004Co-Authors: Ali Turkan, Yasuaki Hiromasa, Thomas E. RocheAbstract:Pyruvate Dehydrogenase Phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca(2+)-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca(2+) was evaluated in activity and L2 binding studies. An increase in the Mg(2+) concentration decreased the Ca(2+) concentration required for half-maximal activation of PDP1 from 3 to 1 microM, but this parameter was unchanged at 3 microM with PDP1c. Near 1 microM Ca(2+), tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg(2+). With just Ca(2+) included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c.Ca(2+).L2 complex is supported by micromolar Ca(2+) concentrations and becomes sensitive to the Mg(2+) level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 +/- 2.5 microM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K(d) of approximately 0.3 microM. Isothermal titration calorimetry and (45)Ca(2+) binding studies failed to detect binding of Ca(2+) (
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formation of a complex of the catalytic subunit of Pyruvate Dehydrogenase Phosphatase isoform 1 pdp1c and the l2 domain forms a ca2 binding site and captures pdp1c as a monomer
Biochemistry, 2004Co-Authors: Ali Turkan, Yasuaki Hiromasa, Thomas E. RocheAbstract:Pyruvate Dehydrogenase Phosphatase isoform 1 (PDP1) is a heterodimer with a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). The activities of PDP1 or just PDP1c are greatly increased by Ca(2+)-dependent binding to the L2 (inner lipoyl) domain of the dihydrolipoyl acetyltransferase (E2) core. Using EGTA-Ca buffers, the dependence of PDP1 or PDP1c on the level of free Ca(2+) was evaluated in activity and L2 binding studies. An increase in the Mg(2+) concentration decreased the Ca(2+) concentration required for half-maximal activation of PDP1 from 3 to 1 microM, but this parameter was unchanged at 3 microM with PDP1c. Near 1 microM Ca(2+), tight binding of PDP1 but not PDP1c to gel-anchored L2 required Mg(2+). With just Ca(2+) included, some PDP1c separated from PDP1r and remained more tightly bound to L2 than intact PDP1. Thus, formation of the PDP1c.Ca(2+).L2 complex is supported by micromolar Ca(2+) concentrations and becomes sensitive to the Mg(2+) level when PDP1c is bound to PDP1r. Sedimentation velocity and equilibrium studies revealed that PDP1c exists as a reversible monomer/dimer mixture with an equilibrium dissociation constant of 8.0 +/- 2.5 microM. L2 binds tightly and preferentially to the PDP1c monomer. Approximately 45 PDP1c monomers bind to the E2 60mer with a K(d) of approximately 0.3 microM. Isothermal titration calorimetry and (45)Ca(2+) binding studies failed to detect binding of Ca(2+) (<100 microM) to L2 or PDP1c, alone, but readily detected binding to L2 and PDP1c. Therefore, both proteins are required for formation of a complex with tightly held Ca(2+), and complex formation hinders the tendency of PDP1c to form a dimer.
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Essential roles of lipoyl domains in the activated function and control of Pyruvate Dehydrogenase kinases and Phosphatase isoform 1
FEBS Journal, 2003Co-Authors: Thomas E. Roche, Yasuaki Hiromasa, Ali Turkan, Xiaoming Gong, Tao Peng, Shane A. Kasten, Jianchun DongAbstract:Four Pyruvate Dehydrogenase kinase and two Pyruvate Dehydrogenase Phosphatase isoforms function in adjusting the activation state of the Pyruvate Dehydrogenase complex (PDC) through determining the fraction of active (nonphosphorylated) Pyruvate Dehydrogenase component. Necessary adaptations of PDC activity with varying metabolic requirements in different tissues and cell types are met by the selective expression and pronounced variation in the inherent functional properties and effector sensitivities of these regulatory enzymes. This review emphasizes how the foremost changes in the kinase and Phosphatase activities issue from the dynamic, effector–modified interactions of these regulatory enzymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component.
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Structural Requirements within the Lipoyl Domain for the Ca2+-dependent Binding and Activation of Pyruvate Dehydrogenase Phosphatase Isoform 1 or Its Catalytic Subunit
The Journal of biological chemistry, 2002Co-Authors: Ali Turkan, Xiaoming Gong, Tao Peng, Thomas E. RocheAbstract:Abstract The inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2) 60-mer forms a Ca2+-dependent complex with the Pyruvate Dehydrogenase Phosphatase 1 (PDP1) or its catalytic subunit, PDP1c, in facilitating large enhancements of the activities of PDP1 (10-fold) or PDP1c (6-fold). L2 binding to PDP1 or PDP1c requires the lipoyl-lysine prosthetic group and specificity residues that distinguish L2 from the other lipoyl domains (L1 in E2 and L3 in the E3-binding component). The L2-surface structure contributing to binding was mapped by comparing the capacities of well folded mutant or lipoyl analog-substituted L2 domains to interfere with E2 activation by competitively binding to PDP1 or PDP1c. Our results reveal the critical importance of a regional set of residues near the lipoyl group and of the octanoyl but not the dithiolane ring structure of the lipoyl group. At the other end of the lipoyl domain, substitution of Glu182 by alanine or glutamine removed L2 binding to PDP1 or PDP1c, and these substitutions for the neighboring Glu179 also greatly hindered complex formation (E179A > E179Q). Among 11 substitutions in L2 at sites of major surface residue differences between the L1 and L2 domains, only the conversion of Val-Gln181 located between the critical Glu179 and Glu182 to the aligned Ser-Leu sequence of the L1 domain greatly reduced L2 binding. Certain modified L2 altered E2 activation of PDP1 differently than PDP1c, supporting significant impact of the regulatory PDP1r subunit on PDP1 binding to L2. Our results indicate hydrophobic binding via the extended aliphatic structure of the lipoyl group and required adjacent L2 structure anchor PDP1 by acting in concert with an acidic cluster at the other end of the domain.
Lester J. Reed - One of the best experts on this subject based on the ideXlab platform.
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One-step purification of the recombinant catalytic subunit of Pyruvate Dehydrogenase Phosphatase.
Protein expression and purification, 2000Co-Authors: Wahn Soo Choi, Jiangong Yan, Diane Mccarthy, Seung Hee Park, Lester J. ReedAbstract:A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine Pyruvate Dehydrogenase Phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian Pyruvate Dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.
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Cloning, Expression, and Properties of the Regulatory Subunit of Bovine Pyruvate Dehydrogenase Phosphatase
The Journal of biological chemistry, 1997Co-Authors: Janet E. Lawson, Jiangong Yan, Seung Hee Park, Angela R. Mattison, Lester J. ReedAbstract:cDNA encoding the regulatory subunit of bovine mitochondrial Pyruvate Dehydrogenase Phosphatase (PDPr) has been cloned. Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA. The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculatedM r of 95,656. This value is in agreement with the molecular mass of native PDPr (95,800 ± 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry. The mature form of PDPr was expressed inEscherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity. It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine. A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine Dehydrogenase, which functions in choline degradation.
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Role of the regulatory subunit of bovine Pyruvate Dehydrogenase Phosphatase
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Jiangong Yan, Janet E. Lawson, Lester J. ReedAbstract:Abstract Bovine Pyruvate Dehydrogenase Phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein Phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.
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Pyruvate Dehydrogenase Phosphatase
Alpha-Keto Acid Dehydrogenase Complexes, 1996Co-Authors: Lester J. Reed, Janet E. Lawson, Xiaoda Niu, Jiangong YanAbstract:Four classes of protein serine/threonine Phosphatases have been identified in eukaryotic cells on the basis of substrate specificities and sensitivity to activators and inhibitors (Cohen, 1989; Shenolikar and Nairn, 1991). Protein Phosphatase 1 is sensitive to the thermostable proteins inhibitor 1 and inhibitor 2, and protein Phosphatases 1 and 2A are sensitive to okadaic acid. Protein Phosphatase 2B is Ca2+/calmodulin-regulated and protein Phosphatase 2C is Mg2+-dependent. The protein serine/threonine Phosphatases of known sequence comprise two distinct gene families, a major family that includes protein Phosphatases 1, 2A, and 2B and isoforms thereof, and a smaller family that includes protein Phosphatase 2C and Pyruvate Dehydrogenase (PDH) Phosphatase.
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Molecular cloning and expression of the catalytic subunit of bovine Pyruvate Dehydrogenase Phosphatase and sequence similarity with protein Phosphatase 2C
Biochemistry, 1993Co-Authors: Janet E. Lawson, Xiaoda Niu, Jiangong Yan, Karen S. Browning, Hai Le Trong, Lester J. ReedAbstract:After many unsuccessful attempts to detect cDNA encoding the catalytic subunit of bovine Pyruvate Dehydrogenase Phosphatase (PDPc) in bovine cDNA libraries, an approach based on the polymerase chain reaction (PCR) was undertaken. Overlapping DNA fragments were generated by PCR from bovine genomic DNA and from cDNA synthesized from total RNA with synthetic oligonucleotide primers on the basis of experimentally determined amino acid sequences. The DNA fragments were subcloned and sequenced. The complete cDNA is 1900 base pairs in length and contains an open reading frame of 1614 nucleotides encoding a putative presequence of 71 amino acid residues and a mature protein of 467 residues with a calculated M(r) of 52,625. Hybridization analysis showed a single mRNA transcript of about 2.0 kilobases. Comparison of the deduced amino acid sequences of the mitochondrial PDPc and the rat cytosolic protein Phosphatase 2C indicates that these protein serine/threonine Phosphatases evolved from a common ancestor. The mature form of PDPc was coexpressed in Escherichia coli with the chaperonin proteins groEL and groES. The recombinant protein (rPDPc) was purified to near homogeneity. Its activity toward the bovine 32P-labeled Pyruvate Dehydrogenase complex was Mg(2+)-dependent and Ca(2+)-stimulated and comparable to that of native bovine PDP. An active, truncated form of rPDPc, with M(r) approximately 45,000, was produced in variable amounts during growth of cells and/or during the purification procedure.
Lianyu Chen - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of Pyruvate Dehydrogenase Phosphatase 1 promotes the progression of pancreatic adenocarcinoma by regulating energy-related AMPK/mTOR signaling.
Cell & bioscience, 2020Co-Authors: Jia Shen, Chien Shan Cheng, Huifeng Gao, Jiangang Zhao, Lianyu ChenAbstract:Background Human Pyruvate Dehydrogenase Phosphatase 1 (PDP1) plays an important physiological role in energy metabolism; however, its expression and function in human pancreatic adenocarcinoma (PDAC) remain unknown. This study aimed to investigate the expression pattern and mechanisms of action of PDP1 in human PDAC. Methods The expression pattern of PDP1 in PDAC was determined, and its correlation with patient survival was analyzed. Ectopic expression or knockdown of PDP1 was performed, and in vitro proliferation and migration, as well as in vivo tumor growth of PDAC, were measured. The mechanism was studied by biochemical approaches. Results PDP1 was overexpressed in human PDAC samples, and high expression of PDP1 correlated with poor overall and disease-free survival of PDAC patients. PDP1 promoted the proliferation, colony formation, and invasion of PDAC cells in vitro and facilitated orthotopic tumor growth in vivo. PDP1 accelerated intracellular ATP production, leading to sufficient energy to support rapid cancer progression. mTOR activation was responsible for the PDP1-induced tumor cell proliferation and invasion in PDAC. AMPK was downregulated by PDP1 overexpression, resulting in mTOR activation and cancer progression. Conclusion Our findings suggested that PDP1 could be a promising diagnostic and therapeutic target for anti-PDAC treatment.
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overexpression of Pyruvate Dehydrogenase Phosphatase 1 promotes the progression of pancreatic adenocarcinoma by regulating energy related ampk mtor signaling
Cell & Bioscience, 2020Co-Authors: Jia Shen, Chien Shan Cheng, Huifeng Gao, Jiangang Zhao, Lianyu ChenAbstract:Background Human Pyruvate Dehydrogenase Phosphatase 1 (PDP1) plays an important physiological role in energy metabolism; however, its expression and function in human pancreatic adenocarcinoma (PDAC) remain unknown. This study aimed to investigate the expression pattern and mechanisms of action of PDP1 in human PDAC. Methods The expression pattern of PDP1 in PDAC was determined, and its correlation with patient survival was analyzed. Ectopic expression or knockdown of PDP1 was performed, and in vitro proliferation and migration, as well as in vivo tumor growth of PDAC, were measured. The mechanism was studied by biochemical approaches. Results PDP1 was overexpressed in human PDAC samples, and high expression of PDP1 correlated with poor overall and disease-free survival of PDAC patients. PDP1 promoted the proliferation, colony formation, and invasion of PDAC cells in vitro and facilitated orthotopic tumor growth in vivo. PDP1 accelerated intracellular ATP production, leading to sufficient energy to support rapid cancer progression. mTOR activation was responsible for the PDP1-induced tumor cell proliferation and invasion in PDAC. AMPK was downregulated by PDP1 overexpression, resulting in mTOR activation and cancer progression. Conclusion Our findings suggested that PDP1 could be a promising diagnostic and therapeutic target for anti-PDAC treatment.
Caroline S. Hill - One of the best experts on this subject based on the ideXlab platform.
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Turning off Smads: identification of a Smad Phosphatase.
Developmental cell, 2006Co-Authors: Caroline S. HillAbstract:Although the activation of Smad signaling pathways downstream of TGF-beta superfamily ligands by receptor-mediated Smad phosphorylation is well understood, nothing is known about Smad Phosphatases that turn off Smad activity. Recently in Genes & Development, describe Pyruvate Dehydrogenase Phosphatase as a Smad1 Phosphatase that functions in Drosophila in the Decapentaplegic pathway and in mammalian cells in the BMP pathway.
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Turning off Smads: identification of a Smad Phosphatase.
Developmental cell, 2006Co-Authors: Caroline S. HillAbstract:Although the activation of Smad signaling pathways downstream of TGF-β superfamily ligands by receptor-mediated Smad phosphorylation is well understood, nothing is known about Smad Phosphatases that turn off Smad activity. Recently in Genes & Development , Chen et al. (2006) describe Pyruvate Dehydrogenase Phosphatase as a Smad1 Phosphatase that functions in Drosophila in the Decapentaplegic pathway and in mammalian cells in the BMP pathway.
Kirill M. Popov - One of the best experts on this subject based on the ideXlab platform.
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Probing a putative active site of the catalytic subunit of Pyruvate Dehydrogenase Phosphatase 1 (PDP1c) by site-directed mutagenesis
Biochimica et biophysica acta, 2004Co-Authors: Tatiana S. Karpova, Boli Huang, Svitlana Danchuk, Kirill M. PopovAbstract:Abstract The catalytic subunit of Pyruvate Dehydrogenase Phosphatase 1 (PDP1c) is a magnesium-dependent protein Phosphatase that regulates the activity of mammalian Pyruvate Dehydrogenase complex. Based on the sequence analysis, it was hypothesized that PDP1c is related to the mammalian magnesium-dependent protein Phosphatase type 1, with Asp54, Asp347, and Asp445 contributing to the binuclear metal-binding center, and Asn49 contributing to the phosphate-binding sites. In this study, we analyzed the functional significance of these amino acid residues using a site-directed mutagenesis. It was found that substitution of each of these residues had a significant impact on PDP1c activity toward the protein substrate. The activities of Asp54, Asp347, and Asp445 mutants were decreased more than 1000-fold. The activity of Asn49 mutant was 2.5-fold lower than the activity of wild-type PDP1c. The decrease in activity of Asp54 and Asp347 came about, most likely, as a result of impaired magnesium binding. Unexpectedly, it was found that the Asp445 mutant bound Mg2+ ions similarly to the wild-type enzyme. Accordingly, the Asp445 mutant was found to be active with the artificial substrate p-nitrophenyl phosphate (pNPP). Asp54 and Asp347 mutants did not demonstrate any appreciable activity with pNPP. Together, these observations strongly suggest that Asn49, Asp54, and Asp347 are important for the catalysis of the Phosphatase reaction, contributing to the phosphate- and metal-binding centers of PDP1c. In contrast, Asp445 is not required for catalysis. The exact role of Asp445 remains to be established, but indirect evidence suggests that it might be involved in the control of interactions between PDP1c and the protein substrate Pyruvate Dehydrogenase.
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Characterization of the isozymes of Pyruvate Dehydrogenase Phosphatase: implications for the regulation of Pyruvate Dehydrogenase activity.
Biochimica et biophysica acta, 2003Co-Authors: Tatiana S. Karpova, Svitlana Danchuk, Elena Kolobova, Kirill M. PopovAbstract:Abstract The activity of mammalian Pyruvate Dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of Pyruvate Dehydrogenase Phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the α chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of Pyruvate Dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of Pyruvate Dehydrogenase.
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Starvation and Diabetes Reduce the Amount of Pyruvate Dehydrogenase Phosphatase in Rat Heart and Kidney
Diabetes, 2003Co-Authors: Boli Huang, Kirill M. Popov, Robert A. HarrisAbstract:The Pyruvate Dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes to conserve three-carbon compounds for gluconeogenesis. This is achieved by an increase in the extent of PDC phosphorylation caused in part by increased Pyruvate Dehydrogenase kinase (PDK) activity due to increased PDK expression. This study examined whether altered Pyruvate Dehydrogenase Phosphatase (PDP) expression also contributes to changes in the phosphorylation state of PDC during starvation and diabetes. Of the two PDP isoforms expressed in mammalian tissues, the Ca 2+ -sensitive isoform (PDP1) is highly expressed in rat heart, brain, and testis and is detectable but less abundant in rat muscle, lung, kidney, liver, and spleen. The Ca 2+ -insensitive isoform (PDP2) is abundant in rat kidney, liver, heart, and brain and is detectable in spleen and lung. Starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. These findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes.
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Isoenzymes of Pyruvate Dehydrogenase Phosphatase DNA-DERIVED AMINO ACID SEQUENCES, EXPRESSION, AND REGULATION
The Journal of biological chemistry, 1998Co-Authors: Boli Huang, Robert A. Harris, Ramadevi Gudi, Jean A. Hamilton, Kirill M. PopovAbstract: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 inEscherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain α-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.
