Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Baker, Jason C.; Yan, Xiaohua; Peng, Tao; Kasten, Shane A.; Roche, Thomas E.

doi:10.1074/jbc.m909488199

Cited by 110 publications

(203 citation statements)

References 63 publications

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“…In mammals, there are four PDK isozymes, all of which have the characteristic carboxyl tails that are somewhat different in the amino acid sequences (2). Several lines of evidence suggest that PDK isozymes are markedly different with respect to their ability to interact with the lipoyl-bearing domain(s) (3,9). Here, we investigated the ability of the carboxyl tails derived from PDK1, PDK3, and PDK4 to support PDK2 activity and LBD2 binding.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

“…At least three of these isozymes (PDK1-PDK3) were directly shown to exist in a complex-bound state (3,5). All PDK isozymes have characteristic carboxyl tails that are markedly different in their amino acid composition (1,2).…”

Section: Activities and Interactions With Lbd2 Of Pdk2 Chimerasmentioning

confidence: 99%

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

2005

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“…Binding of PDKs to the lipoyl domains of E2 was shown to increase their catalytic efficiency (Baker et al, 2000). Examination of activities of four PDK isoenzymes towards free E1 indicated that not only site 1 can be phosphorylated in the absence of E2, but also site 2 by four PDKs and site 3 by PDK1 (at a slightly higher rate than site 2).…”

Section: Site-specificity Of Four Pdk Isoenzymesmentioning

confidence: 99%

“…for interaction with E1 (Baker et al, 2000). The phosphate group in the E1 active site could affect E1 interaction with the negatively charged residues of the lipoyl domain because of its size and anionic nature (Liu et al, 2001).…”

Section: Mechanism Of Inactivation Of Human E1 By Phosphorylation Of mentioning

confidence: 99%

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

Patel

Korotchkina²

2001

Exp Mol Med

150

129

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This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.

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“…The lipoyl-containing domains of the E2 cores function both as an anchor and a modulator for activities of the mitochondrial kinases. Studies with the lipoylated inner L2 domain have been complicated by the fact that the domain alone does not interact as efficiently as holo-E2p with the PDK isoforms, except for PDK3 (13,18). In this communication, we show that lip-LBD alone does not bind to BCK; however, the inclusion of various lengths of the previously neglected C-terminal hinge region results in the interaction between the lip-LBD constructs and BCK.…”

Section: Interaction Of Lipoic Acid-bearing Domain With Bckd Kinase 3mentioning

confidence: 72%

The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

Chuang

Wynn

Chuang

2002

Journal of Biological Chemistry

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The branched-chain ␣-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 ؋ 10 ؊6 M and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK.The mammalian mitochondrial branched-chain ␣-keto acid dehydrogenase (BCKD) 1 complex catalyzes the rate-limiting step in the oxidation of branched-chain amino acids leucine, isoleucine, and valine (1). Genetic defects in this macromolecular multienzyme complex result in the accumulation of branched-chain amino acids leading to inheritable maple syrup urine disease. The clinical phenotype includes early onset and often fatal acidosis, neurological derangement, and mental retardation among survivors (1). The mammalian BCKD complex is organized around a cubic core of 24-meric dihydrolipoyl transacylase (E2b), to which multiple copies of branched-chain ␣-keto acid decarboxylase (E1b), dihydrolipoamide dehydrogenase (E3), BCKD kinase (abbreviated as BCK), and BCKD phosphatase are attached through ionic interactions (2-4). The activity of the BCKD complex is tightly regulated by a phosphorylation/dephosphorylation cycle in response to dietary and hormonal signals (5). Phosphorylation of Ser-292 and Ser-302 in the ␣ subunit of E1b by BCK results in the inactivation of the BCKD complex (6).The structures of rat BCK in the apo-, ADP-bound, and ATP␥S-bound forms were recently determined (7). The BCK structures feature a nucleotide-binding (K) domain and a fourhelix bundle (B) domain, which are similar to modules found in bacterial protein-histidine kinases (8) and the related pyruvate dehydrogenase kinase 2 (PDK2) of the mitochondrial pyruvate dehydrogenase complex (PDC) (9). The K domain is highly conserved between BCK and members of the GHL ATPase family (10), with the presence of characteristic N1, G1, F, and G2 boxes and an "ATP lid." In BCK, the direct back-to-back interaction of two opposing K domains produces a d...

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Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Cited by 110 publications

References 63 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

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

The C-terminal Hinge Region of Lipoic Acid-bearing Domain of E2b Is Essential for Domain Interaction with Branched-chain α-Keto Acid Dehydrogenase Kinase

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