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. 2000 Jul 1;349(Pt 1):195–201. doi: 10.1042/0264-6021:3490195

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues.

J J Thelen 1, J A Miernyk 1, D D Randall 1
PMCID: PMC1221137  PMID: 10861228

Abstract

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

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Selected References

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  1. Appleby J. L., Parkinson J. S., Bourret R. B. Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell. 1996 Sep 20;86(6):845–848. doi: 10.1016/s0092-8674(00)80158-0. [DOI] [PubMed] [Google Scholar]
  2. Bourret R. B., Hess J. F., Simon M. I. Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY. Proc Natl Acad Sci U S A. 1990 Jan;87(1):41–45. doi: 10.1073/pnas.87.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Camp P. J., Randall D. D. Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex : A Source of Acetyl-CoA and NADH for Fatty Acid Biosynthesis. Plant Physiol. 1985 Mar;77(3):571–577. doi: 10.1104/pp.77.3.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang C., Kwok S. F., Bleecker A. B., Meyerowitz E. M. Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science. 1993 Oct 22;262(5133):539–544. doi: 10.1126/science.8211181. [DOI] [PubMed] [Google Scholar]
  6. Chen W., Huang X., Komuniecki P. R., Komuniecki R. Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum. Arch Biochem Biophys. 1998 May 1;353(1):181–189. doi: 10.1006/abbi.1998.0627. [DOI] [PubMed] [Google Scholar]
  7. Davie J. R., Wynn R. M., Meng M., Huang Y. S., Aalund G., Chuang D. T., Lau K. S. Expression and characterization of branched-chain alpha-ketoacid dehydrogenase kinase from the rat. Is it a histidine-protein kinase? J Biol Chem. 1995 Aug 25;270(34):19861–19867. doi: 10.1074/jbc.270.34.19861. [DOI] [PubMed] [Google Scholar]
  8. Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
  9. Elich T. D., Chory J. Phytochrome: if it looks and smells like a histidine kinase, is it a histidine kinase? Cell. 1997 Dec 12;91(6):713–716. doi: 10.1016/s0092-8674(00)80458-4. [DOI] [PubMed] [Google Scholar]
  10. Gamble R. L., Coonfield M. L., Schaller G. E. Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7825–7829. doi: 10.1073/pnas.95.13.7825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jackson J. C., Vinluan C. C., Dragland C. J., Sundararajan V., Yan B., Gounarides J. S., Nirmala N. R., Topiol S., Ramage P., Blume J. E. Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues. Biochem J. 1998 Sep 15;334(Pt 3):703–711. doi: 10.1042/bj3340703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Liu S., Baker J. C., Roche T. E. Binding of the pyruvate dehydrogenase kinase to recombinant constructs containing the inner lipoyl domain of the dihydrolipoyl acetyltransferase component. J Biol Chem. 1995 Jan 13;270(2):793–800. doi: 10.1074/jbc.270.2.793. [DOI] [PubMed] [Google Scholar]
  13. Loomis W. F., Shaulsky G., Wang N. Histidine kinases in signal transduction pathways of eukaryotes. J Cell Sci. 1997 May;110(Pt 10):1141–1145. doi: 10.1242/jcs.110.10.1141. [DOI] [PubMed] [Google Scholar]
  14. Miernyk J. A., Randall D. D. Some properties of pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase. Plant Physiol. 1987 Feb;83(2):311–315. doi: 10.1104/pp.83.2.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mizuno T. His-Asp phosphotransfer signal transduction. J Biochem. 1998 Apr;123(4):555–563. doi: 10.1093/oxfordjournals.jbchem.a021972. [DOI] [PubMed] [Google Scholar]
  16. Mooney B. P., David N. R., Thelen J. J., Miernyk J. A., Randall D. D. Histidine modifying agents abolish pyruvate dehydrogenase kinase activity. Biochem Biophys Res Commun. 2000 Jan 19;267(2):500–503. doi: 10.1006/bbrc.1999.1994. [DOI] [PubMed] [Google Scholar]
  17. Popov K. M., Kedishvili N. Y., Zhao Y., Shimomura Y., Crabb D. W., Harris R. A. Primary structure of pyruvate dehydrogenase kinase establishes a new family of eukaryotic protein kinases. J Biol Chem. 1993 Dec 15;268(35):26602–26606. [PubMed] [Google Scholar]
  18. Popov K. M. Regulation of mammalian pyruvate dehydrogenase kinase. FEBS Lett. 1997 Dec 15;419(2-3):197–200. doi: 10.1016/s0014-5793(97)01453-1. [DOI] [PubMed] [Google Scholar]
  19. Popov K. M., Zhao Y., Shimomura Y., Kuntz M. J., Harris R. A. Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases. J Biol Chem. 1992 Jul 5;267(19):13127–13130. [PubMed] [Google Scholar]
  20. Radke G. A., Ono K., Ravindran S., Roche T. E. Critical role of a lipoyl cofactor of the dihydrolipoyl acetyltransferase in the binding and enhanced function of the pyruvate dehydrogenase kinase. Biochem Biophys Res Commun. 1993 Feb 15;190(3):982–991. doi: 10.1006/bbrc.1993.1146. [DOI] [PubMed] [Google Scholar]
  21. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stone J. M., Walker J. C. Plant protein kinase families and signal transduction. Plant Physiol. 1995 Jun;108(2):451–457. doi: 10.1104/pp.108.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sugden P. H., Kerbey A. L., Randle P. J., Waller C. A., Reid K. B. Amino acid sequences around the sites of phosphorylation in the pig heart pyruvate dehydrogenase complex. Biochem J. 1979 Aug 1;181(2):419–426. doi: 10.1042/bj1810419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. The electronic plant gene register. Plant Physiol. 1998 Dec;118(4):1533–1536. doi: 10.1104/pp.118.4.1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thelen J. J., Miernyk J. A., Randall D. D. Molecular cloning and expression analysis of the mitochondrial pyruvate dehydrogenase from maize. Plant Physiol. 1999 Feb;119(2):635–644. doi: 10.1104/pp.119.2.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thelen J. J., Muszynski M. G., David N. R., Luethy M. H., Elthon T. E., Miernyk J. A., Randall D. D. The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain. J Biol Chem. 1999 Jul 30;274(31):21769–21775. doi: 10.1074/jbc.274.31.21769. [DOI] [PubMed] [Google Scholar]
  27. Thelen J. J., Muszynski M. G., Miernyk J. A., Randall D. D. Molecular analysis of two pyruvate dehydrogenase kinases from maize. J Biol Chem. 1998 Oct 9;273(41):26618–26623. doi: 10.1074/jbc.273.41.26618. [DOI] [PubMed] [Google Scholar]
  28. Thelen JJ, Miernyk JA, Randall DD. Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex . Plant Physiol. 1998 Apr;116(4):1443–1450. doi: 10.1104/pp.116.4.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Uhl M. A., Miller J. F. Integration of multiple domains in a two-component sensor protein: the Bordetella pertussis BvgAS phosphorelay. EMBO J. 1996 Mar 1;15(5):1028–1036. [PMC free article] [PubMed] [Google Scholar]
  30. Williams M., Randall D. D. Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L. Plant Physiol. 1979 Dec;64(6):1099–1103. doi: 10.1104/pp.64.6.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yeaman S. J., Hutcheson E. T., Roche T. E., Pettit F. H., Brown J. R., Reed L. J., Watson D. C., Dixon G. H. Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry. 1978 Jun 13;17(12):2364–2370. doi: 10.1021/bi00605a017. [DOI] [PubMed] [Google Scholar]
  32. Yeh K. C., Lagarias J. C. Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13976–13981. doi: 10.1073/pnas.95.23.13976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yeh K. C., Wu S. H., Murphy J. T., Lagarias J. C. A cyanobacterial phytochrome two-component light sensory system. Science. 1997 Sep 5;277(5331):1505–1508. doi: 10.1126/science.277.5331.1505. [DOI] [PubMed] [Google Scholar]

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