Sub‐mitochondrial localization of the catalytic subunit of pyruvate dehydrogenase phosphatase

Simonot, Cédric; Lermé, F.; Louisot, Pierre; Gateau-Roesch, Odile

doi:10.1016/s0014-5793(96)01461-5

Cited by 12 publications

(12 citation statements)

References 22 publications

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“…[2][3][4]49 In mitochondria isolated from reperfused tissue, PDH activity was partially recovered when assayed in the absence of the phosphatase inhibitor NaF. In addition, reperfusion-induced declines in PDH activity were associated with an acidic shift in the isoelectric point of the ␣E1 subunit of pyruvate dehydrogenase that was prevented on inhibition of ␦PKC translocation.…”

Section: Discussionmentioning

confidence: 99%

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Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Churchill

Murriel

Chen

et al. 2005

Circulation Research

122

117

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Abstract-Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive ␦-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Key Words: pyruvate dehydrogenase Ⅲ ␦PKC Ⅲ pyruvate dehydrogenase kinase Ⅲ free radicals Ⅲ mitochondria Ⅲ ischemia/reperfusion P yruvate dehydrogenase (PDH) is responsible for the conversion of pyruvate derived from glycolysis to acetylCoA for Krebs cycle activity. Enzyme activity is regulated, in part, by phosphorylation-and dephosphorylation-dependent inhibition and activation, respectively. 1,2 Phosphorylation is catalyzed by 4 PDH-associated pyruvate dehydrogenase kinases (PDK1-4) that exhibit tissue-specific expression patterns and differences in specific activity toward 3 phosphorylation sites on the E1␣ subunit of PDH. The PDH complex also contains 2 pyruvate dehydrogenase phosphatases (PDP 1 and PDP 2) responsible for reactivation of PDH. 2-4 PDH therefore represents a highly regulated and critical site for the control of glycolytic flux and ATP production.Cardiac ischemia/reperfusion is associated with alterations in metabolism that, depending on the severity of the ischemic insult, can progress to irreparable myocardial damage. 5 Although PDH activity in myocardial tissue has been reported to decline during flow-induced ischemia, 6 this is not universally observed. [7][8][9] The effects of reperfusion also exhibit considerable variability, with the majority of studies demonstrating a decrease in PDH activity. [7][8][9] Despite the disparity in evidence regarding PDH activity, cardiac efficiency and recovery of contractile function in postischemic hearts can be improved by pharmacological stimulation of PDH 8,10 -16 or infusion of pyruvate. [17][18][19][20][21][22][23] Identification of factors that regulate PDH activity during ischemia/reperfusion may therefore enhance the potential for therapeutic intervention.Reperfusion of ischemic myocardium is associated with enhanced free radical generation. 5,24 Pro-oxidants have been shown to regulate protein function either directly or indirectly through the modulation of other regulatory molecules. [25][26][27] One such example is the novel ␦-isoform of PKC. Exposure of purified ␦PKC to the thiol-specific oxidant diamide and glutathione (GSH) at concentrations that induce inactivation of other PKC isozymes results in ␦PKC activation. 28 Additionally, treatment of various cell types with H 2 O 2 , glutathione depleting agents, or the general PKC activator PMA results in tyrosine phosphorylation and/or activation and translocation of ␦PKC to the mitochondri...

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Section: Discussionmentioning

confidence: 99%

“…The PDH complex also contains 2 pyruvate dehydrogenase phosphatases (PDP 1 and PDP 2) responsible for reactivation of PDH. [2][3][4] PDH therefore represents a highly regulated and critical site for the control of glycolytic flux and ATP production.…”

mentioning

confidence: 99%

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Churchill

Murriel

Chen

et al. 2005

Circulation Research

122

117

View full text Add to dashboard Cite

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“…The clear DLDH-containing supernatant was used for mitochondrial DLDH assays as described in this study. It should be noted that as DLDH is a component of mitochondrial a-keto acid dehydrogenase complexes that are known to be associated with mitochondrial membranes [34][35][36], Triton X-100 (1%, v/v, final concentration) should always be included in the extraction buffer so that a complete mitochondria lysis is ensured [12,30].…”

Section: Preparation Of Mitochondrial Extractsmentioning

confidence: 99%

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

et al. 2007

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Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.

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“…In eukaryotes, Ser/Thr phosphatases are grouped into PPP and PPM families (Barford et al 1998). PDPs belong to the PPM family whose catalytic domains are similar to that of PP2C, and pyruvate dehydrogenase was the only known substrate of PDPs (Lawson et al 1993;Simonot et al 1997;Huang et al 1998). We have thus identified a novel substrate and function for PDP.…”

Section: Pdp Directly Dephosphorylates Dpp-activated Madmentioning

confidence: 99%

“…Two orthologs of Drosophila PDP, PDP1 and PDP2, have been identified in the human genome, and both share ∼40% identity with the Drosophila PDP in amino acid sequences (Lawson et al 1993;Simonot et al 1997;Huang et al 1998). Coimmunoprecipitation between PDP2 and Smad1 was detected in 293T cells, suggesting that the functional interaction between PDP and Smad is conserved in mammalian cells (Fig.…”

Section: Mammalian Pdps Are Involved In Dephosphorylation Of Bmp-actimentioning

confidence: 99%

Identification of phosphatases for Smad in the BMP/DPP pathway

Chen¹,

Shen²,

Ip³

et al. 2006

Genes Dev.

120

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Phosphorylation of the SSXS motif of Smads is critical in activating the transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) pathways. However, the phosphatase(s) involved in dephosphorylating and hence inactivating Smads remained elusive. Through RNA interference (RNAi)-based screening of serine/threonine phosphatases in Drosophila S2 cells, we identified pyruvate dehydrogenase phosphatase (PDP) to be required for dephosphorylation of Mothers against Decapentaplegic (MAD), a Drosophila Smad. Biochemical and genetic evidence suggest that PDP directly dephosphorylates MAD and inhibits signal transduction of Decapentaplegic (DPP). We show that the mammalian PDPs are important in dephosphorylation of BMP-activated Smad1 but not TGF-β-activated Smad2 or Smad3. Thus, PDPs specifically inactivate Smads in the BMP/DPP pathway.

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Sub‐mitochondrial localization of the catalytic subunit of pyruvate dehydrogenase phosphatase

Cited by 12 publications

References 22 publications

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Identification of phosphatases for Smad in the BMP/DPP pathway

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