Pivotal role of the C-terminal DW-motif in mediating inhibition of pyruvate dehydrogenase kinase 2 by dichloroacetate

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.

Original languageEnglish (US)
Pages (from-to)34458-34467
Number of pages10
JournalJournal of Biological Chemistry
Volume284
Issue number49
DOIs
StatePublished - Dec 4 2009

Fingerprint

Pyruvate Dehydrogenase Complex
Adenosine Diphosphate
Medical problems
Conformations
Phosphorylation
Inhibitory Concentration 50
Neoplasms
Protein Isoforms
Substitution reactions
Nucleotides
Adenosine Triphosphate
Binding Sites
Communication
Mutation
pyruvate dehydrogenase (acetyl-transferring) kinase
Inhibition (Psychology)
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

@article{eca0174f159745da897a9e10d7f193c4,
title = "Pivotal role of the C-terminal DW-motif in mediating inhibition of pyruvate dehydrogenase kinase 2 by dichloroacetate",
abstract = "The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60{\%} residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.",
author = "Jun Li and Masato Kato and Chuang, {David T.}",
year = "2009",
month = "12",
day = "4",
doi = "10.1074/jbc.M109.065557",
language = "English (US)",
volume = "284",
pages = "34458--34467",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "49",

}

TY - JOUR

T1 - Pivotal role of the C-terminal DW-motif in mediating inhibition of pyruvate dehydrogenase kinase 2 by dichloroacetate

AU - Li, Jun

AU - Kato, Masato

AU - Chuang, David T.

PY - 2009/12/4

Y1 - 2009/12/4

N2 - The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.

AB - The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20-60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DW-motif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DW-motif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.

UR - http://www.scopus.com/inward/record.url?scp=71749096219&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=71749096219&partnerID=8YFLogxK

U2 - 10.1074/jbc.M109.065557

DO - 10.1074/jbc.M109.065557

M3 - Article

VL - 284

SP - 34458

EP - 34467

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 49

ER -