Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

Kolobova, Elena; Tuganova, Alina; Boulatnikov, Igor G.; Popov, Kirill M.

doi:10.1042/bj3580069

Cited by 186 publications

(104 citation statements)

References 22 publications

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“…Construction of the expression vectors for E1, the E2-E3BP subcomplex, lipoyl-protein ligase A (LPL_A), the GST-LBD2 fusion protein (amino acids Ser 127-Ile 214), His 6 -LBD2 (amino acids Ser 127-Ile 214), and PDK1-PDK4 was described elsewhere (2,9,12,18,21). The fragments of E2 cDNA (11) encoding LBD1, bases 252 (Ser 1) to 573 (Gln 104), and of E3BP cDNA (12) encoding LBD3, bases 168 (Gly 1) to 483 (Arg 107), were made by polymerase chain reaction (PCR) with Pfu polymerase (Stratagene, La Jolla, CA) and appropriate cDNAs as templates (11,12).…”

Section: Experimental Procedures Plasmids and Strainsmentioning

confidence: 99%

“…General conditions for the expression of wild-type PDC, E1, the E2-E3BP subcomplex, PDK2, His 6 -LBD2, and the GST-LBD2 fusion protein were described previously (2,9,12,18,21). PDK2 carrying various deletions and PDK2 chimeras were expressed following the established protocol (2).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Purification of wild-type PDC, E1, the E2-E3BP sub-complex, PDK1-PDK4, His 6 -LBD2, and the GST-LBD2 fusion protein was described elsewhere (2,9,12,18,21). Truncated variants of PDK2 and PDK2 chimeras carrying the amino-terminal His 6 tags were purified using TALON metal affinity resin (Clontech Laboratories, Inc., Palo Alto, CA) essentially as described for His 6 -PDK2 (2).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…All PDK isozymes phosphorylate E1 strictly on serine residues (18,19). However, neither of them displays an appreciable sequence similarity to the Ser/Thr-specific protein kinases residing in other cellular compartments (1,2).…”

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confidence: 99%

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The Carboxy-Terminal Tail of Pyruvate Dehydrogenase Kinase 2 Is Required for the Kinase Activity

2005

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Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoylbearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoylbearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes.Mammalian mitochondria harbor four closely related protein kinases (isozymes PDK1-PDK4) 1 that regulate the activity of the pyruvate dehydrogenase complex (PDC) (1-3) and thereby control the disposal rates of pyruvate and of other metabolically related three-carbon compounds (4).It is generally believed that at least three of the four isozymes (PDK1-PDK3) are the integral components of a multienzyme complex (3,5). On average, PDC contains just two to three kinase molecules per complex (6). A growing body of evidence strongly suggests that the kinase molecule uses the so-called lipoyl-bearing domains (LBDs) as docking sites for the attachment to the complex (7-9). In PDC, there are three types of LBDs (LBD1-LBD3) (10). Two of those domains (LBD1 and LBD2) are provided by the acetyltransferase component of the complex (E2) (11), and one (LBD3) is provided by the so-called E3-binding protein (E3BP) (12), which is a structural component of PDC tightly integrated with E2 (E2-E3BP subcomplex) (10). LBD2 is thought to be the primary binding site for the kinase molecule (13). Kinase activities

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Section: Experimental Procedures Plasmids and Strainsmentioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

Section: Protein Expression and Purificationmentioning

confidence: 99%

mentioning

confidence: 99%

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The Carboxy-Terminal Tail of Pyruvate Dehydrogenase Kinase 2 Is Required for the Kinase Activity

2005

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“…Evidence from this comes from the observation that PGC-1α regulates the expression of pyruvate dehydrogenase kinase 4, which inactivates the pyruvate dehydrogenase complex, an enzyme responsible for catalyzing the conversion of pyruvate to acetyl coenzyme A and hence glucose oxidation (Kolobova et al, 2001;Wende et al, 2005). Taken together, PGC-1α seems to have an important role in enhancing fat oxidation capacity and may mediate classic adaptation of increased fat utilization following an exercise training regime.…”

Section: Fat Oxidationmentioning

confidence: 99%

PGC-1α Mediated Muscle Aerobic Adaptations to Exercise, Heat and Cold Exposure

Ihsan¹,

Watson²,

Abbiss³

2014

Cell Mol Exerc Physiol

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PGC-1α is regarded as a key regulator of mitochondrial biogenesis due to its central role in regulating the activity of key transcription factors associated with encoding mitochondrial components. Additionally, PGC-1α has shown to mediate adaptations that increase fat metabolism and angiogenesis, contributing to the overall oxidative phenotype of the muscle. While it is well established that exercise is a potent stimulator of PGC-1α, recent evidence indicates that heat and cold exposures may also influence mitochondrial biogenesis through the up-regulation of PGC-1α. This highlights the potential use of these modalities in conjunction with exercise to enhance training adaptations. As such, the purpose of this review is to describe the possible mechanisms and pathways by which exercise, as well as hot and cold exposures may influence mitochondrial biogenesis. It is clear that changes in intracellular calcium, oxidative stress and phosphorylation potential are major up-regulators of PGC-1α during exercise. Moreover, there is evidence implicating calcium signalling, in addition to β-adrenergic activation in cold-induced mitochondrial biogenesis, while PGC-1α during heat exposure is likely triggered by changes in phosphorylation potential and nitric oxide signalling. However, these mechanisms appear to change considerably when cold/heat is administered following exercise, and seem to be dependent on the experimental models used (i.e. in vitro vs. in vivo, rodent vs. human). Understanding the effects heat/cold exposure and its interaction with exercise may lead to the optimisation and development of temperature-related interventions to enhance training adaptations, or aid in the treatment of mitochondrial related diseases.

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Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells

et al. 2022

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Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD+ regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin‐like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP‐activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

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Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

Cited by 186 publications

References 22 publications

The Carboxy-Terminal Tail of Pyruvate Dehydrogenase Kinase 2 Is Required for the Kinase Activity

The Carboxy-Terminal Tail of Pyruvate Dehydrogenase Kinase 2 Is Required for the Kinase Activity

PGC-1α Mediated Muscle Aerobic Adaptations to Exercise, Heat and Cold Exposure

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells

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