Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-Phosphorylation of Pyruvate Dehydrogenase

2009 ◽  
Vol 2 ◽  
pp. PRI.S2799 ◽  
Author(s):  
Jan A. Miernyk ◽  
Mark L. Johnston ◽  
Steve C. Huber ◽  
Alejandro Tovar-Méndez ◽  
Elizabeth Hoyos ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Redox State ◽  
Cultured Cells ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Methionine Sulfoxide ◽  
Tryptic Peptides ◽  
Methionine Residue ◽  
Chemical Agents

A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H2O2, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.

scholarly journals The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex

1988 ◽  
Vol 253 (3) ◽  
pp. 819-825 ◽  
Author(s):  
T Pawelczyk ◽  
R A Easom ◽  
M S Olson
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Maximal Activity ◽  
Hill Coefficient ◽  
Kidney Cortex ◽  
Complex Activity ◽  
Pig Kidney ◽  
Constant Strength ◽  
Dehydrogenase Complex

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.

Phosphorylation of the phosphatase PTPROt at Tyr399is a molecular switch that controls osteoclast activity and bone mass in vivo

2019 ◽  
Vol 12 (563) ◽  
pp. eaau0240 ◽  
Author(s):  
Lee Roth ◽  
Jean Wakim ◽  
Elad Wasserman ◽  
Moran Shalev ◽  
Esther Arman ◽  
...  
Keyword(s):  
Bone Mass ◽  
Cultured Cells ◽  
Phosphorylation Site ◽  
Molecular Switch ◽  
Receptor Type ◽  
Mouse Strains ◽  
Bone Homeostasis ◽  
Osteoclast Activity

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527and its activating residue Tyr416. Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399caused PTPROt to dephosphorylate Src at the activating site pTyr416. In contrast, phosphorylation of PTPROt at Tyr399enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

2001 ◽  
Vol 358 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Elena KOLOBOVA ◽  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Enzymic Activity ◽  
Mammalian Tissues ◽  
Activity State ◽  
The Individual ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Thiamin Pyrophosphate ◽  
Dehydrogenase Complex

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4PDK3> PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

scholarly journals Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation

1980 ◽  
Vol 190 (3) ◽  
pp. 705-710 ◽  
Author(s):  
P A Lazo ◽  
A Sols
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metabolic Regulation ◽  
Pyruvate Dehydrogenase Complex ◽  
Acetyl Coa ◽  
Substrate Complex ◽  
Ascites Tumour ◽  
Rate Limiting Step ◽  
Lipoamide Dehydrogenase ◽  
Dehydrogenase Complex

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich—Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17—36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme—substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.

scholarly journals Huzhangoside A Suppresses Tumor Growth through Inhibition of Pyruvate Dehydrogenase Kinase Activity

Cancers ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 712 ◽  
Author(s):  
Choong-Hwan Kwak ◽  
Jung-Hee Lee ◽  
Eun-Yeong Kim ◽  
Chang Woo Han ◽  
Keuk-Jun Kim ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Pyruvate Dehydrogenase ◽  
Malignant Tumors ◽  
Aerobic Glycolysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
In Vitro Assays ◽  
In Vivo Models

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.

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