Facilitated Interaction between the Pyruvate Dehydrogenase Kinase Isoform 2 and the Dihydrolipoyl Acetyltransferase
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m212733200
ISSN1083-351X
AutoresYasuaki Hiromasa, Thomas E. Roche,
Tópico(s)Metabolism and Genetic Disorders
ResumoThe dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd ≃ 175 μm for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd ≃ 3 μm), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in ∼8-fold tighter binding of PDK2 to GST-L2red, which was ∼300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound ∼18 PDK2 dimers with a Kd similar to GST-L2. E2·E1 bound more PDK2 (∼27.6) than E2 with ∼2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2·E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3–5-fold and adenosine 5′-(β,γ-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2·E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed “hand-over-hand” movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate. The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd ≃ 175 μm for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd ≃ 3 μm), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in ∼8-fold tighter binding of PDK2 to GST-L2red, which was ∼300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound ∼18 PDK2 dimers with a Kd similar to GST-L2. E2·E1 bound more PDK2 (∼27.6) than E2 with ∼2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2·E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3–5-fold and adenosine 5′-(β,γ-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2·E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed “hand-over-hand” movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate. The mitochondrial pyruvate dehydrogenase complex (PDC) 1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1 domain, NH2-lipoyl domain of dihydrolipoyl acetyltransferase component; L2 domain, interior lipoyl domain of dihydrolipoyl acetyltransferase component; E3BP, dihydrolipoyl dehydrogenase-binding protein; L3, N-terminal lipoyl domain of dihydrolipoyl dehydrogenase-binding protein; GST, glutathione S-transferase; GST-L1, GST-L2, and GST-L3, glutathione S-transferase fused to L1, L2, and L3 domains, respectively; E2red, L2red, and GST-L2red, etc., lipoyl groups reduced to dihydrolipoyl groups; E3, dihydrolipoyl dehydrogenase; PDK, pyruvate dehydrogenase kinase; PDK1, PDK2, PDK3, and PDK4, pyruvate dehydrogenase kinase isoforms 1, 2, 3, and 4; PDP, pyruvate dehydrogenase phosphatase; PDP1 and PDP2, pyruvate dehydrogenase phosphatase isoforms 1 and 2; RMSE, root mean square error; AUC, analytical ultracentrifugation; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate).1The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase component; E2, dihydrolipoyl acetyltransferase component; L1 domain, NH2-lipoyl domain of dihydrolipoyl acetyltransferase component; L2 domain, interior lipoyl domain of dihydrolipoyl acetyltransferase component; E3BP, dihydrolipoyl dehydrogenase-binding protein; L3, N-terminal lipoyl domain of dihydrolipoyl dehydrogenase-binding protein; GST, glutathione S-transferase; GST-L1, GST-L2, and GST-L3, glutathione S-transferase fused to L1, L2, and L3 domains, respectively; E2red, L2red, and GST-L2red, etc., lipoyl groups reduced to dihydrolipoyl groups; E3, dihydrolipoyl dehydrogenase; PDK, pyruvate dehydrogenase kinase; PDK1, PDK2, PDK3, and PDK4, pyruvate dehydrogenase kinase isoforms 1, 2, 3, and 4; PDP, pyruvate dehydrogenase phosphatase; PDP1 and PDP2, pyruvate dehydrogenase phosphatase isoforms 1 and 2; RMSE, root mean square error; AUC, analytical ultracentrifugation; AMP-PNP, adenosine 5′-(β,γ-iminotriphosphate). catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD+. Mammalian PDC has a highly organized structure in which the dihydrolipoyl acetyltransferase (E2) component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar). The other components of the complex, required for the overall reaction, include the pyruvate dehydrogenase (E1) component, the dihydrolipoyl dehydrogenase (E3) component, and the E3-binding protein (E3BP). The PDC reaction is regulated by a phosphorylation-dephosphorylation cycle, which is carried out by dedicated kinase and phosphatase components. The desired control in different organs is achieved by the selective expression and the novel regulation of at least four pyruvate dehydrogenase kinase (PDK) isozymes and at least two pyruvate dehydrogenase phosphatase (PDP) isoforms (Refs. 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 5Harris R.A. Huang B. Wu P. Adv. Enzyme Regul. 2001; 41: 269-288Crossref PubMed Scopus (84) Google Scholar, 6Rowles J. Scherer S.W. Xi T. Majer M. Nickle D.C. Rommens J.M. Popov K.M. Harris R.A. Riebow N.L. Xia J. Tsui L.-C. Bogardus C. Prochazka M. J. Biol. Chem. 1996; 271: 22376-22382Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 7Wu P. Blair P.V. Sato J. Jaskiewicz J. Popov K.M. Harris R.A. Arch. Biochem. Biophys. 2000; 381: 1-7Crossref PubMed Scopus (151) Google Scholar, 8Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (438) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 279: 37223-37229Abstract Full Text Full Text PDF Scopus (160) Google Scholar, 11Huang B. Gudi R. Wu P. Harris R.A. Hamilton J. Popov K.M. J. Biol. Chem. 1998; 273: 17680-17688Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar and references therein). The kinases act to inactivate PDC and the phosphatases to reactivate PDC by phosphorylation and dephosphorylation of the E1 component.The PDKs and the related kinase regulating the branched-chain α-keto acid dehydrogenase complex are unrelated to cytoplasmic Ser/Thr/Tyr kinases; however, the C-terminal half of these kinases share structural motifs and fold in a similar manner (12Bower-Kinley M. Popov K.M. Biochem. J. 1999; 344: 47-53Crossref PubMed Google Scholar, 13Wynn R.M. Chaung J.L. Cote C.D. Chuang D.T. J. Biol. Chem. 2000; 275: 30512-30519Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 14Steussy C.N Popov K.M. Bowker-Kinley M.M. Sloan R.B. Harris R.A. Hamilton J.A. J. Biol. Chem. 2001; 276: 37443-37450Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 15Machius M. Chaung J.L. Wynn R.M. Tomchick D.R. Chuang D.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11218-11223Crossref PubMed Scopus (68) Google Scholar) to the ATP-binding regions of DNA gyrase B, heat shock protein 90, and histidine kinases such as CheA (16Wigley Davies G.J. Dadson E.J. Maxwell A. Dodson G. Nature. 1991; 351: 624-629Crossref PubMed Scopus (485) Google Scholar, 17Prodromou C. Roe M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1106) Google Scholar, 18Bilwes A.M. Alex L.A. Crane B.R. Simon M.I. Cell. 1999; 96: 131-141Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Indeed, within the three-dimensional structure of PDK2 (14Steussy C.N Popov K.M. Bowker-Kinley M.M. Sloan R.B. Harris R.A. Hamilton J.A. J. Biol. Chem. 2001; 276: 37443-37450Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), the C-terminal domain, excluding the last 34 residues for which structure has not been resolved, are folded in a very similar manner to these ATP-binding regions as is a C-terminal domain in the branched-chain kinase (15Machius M. Chaung J.L. Wynn R.M. Tomchick D.R. Chuang D.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11218-11223Crossref PubMed Scopus (68) Google Scholar). PDK2 was interpreted as a dimer (14Steussy C.N Popov K.M. Bowker-Kinley M.M. Sloan R.B. Harris R.A. Hamilton J.A. J. Biol. Chem. 2001; 276: 37443-37450Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), although definitive evidence was not presented for this oligomeric state in solution. Here we establish that functional PDK2 is a stable dimer in solution; the other three PDK isoforms have a greater tendency to form larger aggregates.Markedly different effector sensitivities have been uncovered for the different PDK isoforms (3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 7Wu P. Blair P.V. Sato J. Jaskiewicz J. Popov K.M. Harris R.A. Arch. Biochem. Biophys. 2000; 381: 1-7Crossref PubMed Scopus (151) Google Scholar, 8Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (438) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 279: 37223-37229Abstract Full Text Full Text PDF Scopus (160) Google Scholar). The functioning of each PDK isoform in salient roles is corroborated by the strong conservation of the primary structure of each isoform in mammals (3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 6Rowles J. Scherer S.W. Xi T. Majer M. Nickle D.C. Rommens J.M. Popov K.M. Harris R.A. Riebow N.L. Xia J. Tsui L.-C. Bogardus C. Prochazka M. J. Biol. Chem. 1996; 271: 22376-22382Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). PDK2 is the most widely distributed among the four characterized PDK isoforms (5Harris R.A. Huang B. Wu P. Adv. Enzyme Regul. 2001; 41: 269-288Crossref PubMed Scopus (84) Google Scholar, 7Wu P. Blair P.V. Sato J. Jaskiewicz J. Popov K.M. Harris R.A. Arch. Biochem. Biophys. 2000; 381: 1-7Crossref PubMed Scopus (151) Google Scholar, 8Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (438) Google Scholar). PDK2 is particularly sensitive to effector regulation, with NADH and acetyl-CoA enhancing PDC inactivation by PDK2 (8Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (438) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 279: 37223-37229Abstract Full Text Full Text PDF Scopus (160) Google Scholar, 19Popov K.M. FEBS Lett. 1997; 419: 197-200Crossref PubMed Scopus (23) Google Scholar) and pyruvate and ADP acting synergistically to prevent inactivation (8Bowker-Kinley M.M. Davis W.I. Wu P. Harris R.A. Popov K.M. Biochem. J. 1998; 329: 191-196Crossref PubMed Scopus (438) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Specialized interactions within the PDC assemblage operate to enhance PDK activities and are required for the selective processing of specific regulatory effects (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 279: 37223-37229Abstract Full Text Full Text PDF Scopus (160) Google Scholar, 19Popov K.M. FEBS Lett. 1997; 419: 197-200Crossref PubMed Scopus (23) Google Scholar, 20Hucho F. Randall D.D. Roche T.E. Burgett M.W. Pelley J.W. Reed L.J. Arch. Biochem. Biophys. 1972; 151: 328-340Crossref PubMed Scopus (157) Google Scholar, 21Cate R.L. Roche T.E. J. Biol. Chem. 1978; 253: 496-503Abstract Full Text PDF PubMed Google Scholar, 22Rahmatullah M. Roche T.E. J. Biol. Chem. 1985; 260: 10146-10152Abstract Full Text PDF PubMed Google Scholar, 23Rahmatullah M. Radke G.A. Andrews P.C. Roche T.E. J. Biol. Chem. 1990; 265: 14512-14517Abstract Full Text PDF PubMed Google Scholar, 24Radke G.A. Ono K. Ravindran S. Roche T.E. Biochem. Biophys. Res. Commun. 1993; 190: 982-991Crossref PubMed Scopus (38) Google Scholar, 25Ono K. Radke G.A. Roche T.E. Rahmatullah M. J. Biol. Chem. 1993; 268: 26135-26143Abstract Full Text PDF PubMed Google Scholar, 26Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Yang D. Gong X. Yakhnin A. Roche T.E. J. Biol. Chem. 1998; 273: 14130-14137Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Of particular importance is the functional interplay of PDKs with E2. E2 transforms kinase function and regulation through serving as an adaptable binding framework that directly abets efficient phosphorylation, acting as a processing unit in translating and transmitting effector signals, and modifying the sensitivity to allosteric effectors that directly bind to the kinases (reviewed in Refs. 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar and 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar). Thus, it is important for understanding these roles to elucidate the nature of the dynamic interactions of the kinases with E2.E2 and the E3BP component have remarkable structures that allow them to carry out their related general roles. These components form an adjustable framework that binds the other components and integrates the sequential reactions in the assembled complex. When expressed by itself, 60 E2 subunits associate by their C-terminal domains as 20 trimers assembled in the form of a dodecahedron; each C-terminal domain of this inner core connects by a series of mobile linker regions to an E1-binding domain and then to two lipoyl domains (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 28Thekkumkara T.J. Ho L. Wexler I.D. Pons G. Lui T.-C. Patel M.S. FEBS Lett. 1988; 240: 45-48Crossref PubMed Scopus (73) Google Scholar). The N-terminal lipoyl domain is designated L1, then the second lipoyl domain, L2. L2 is flanked by linker regions connecting to L1 on one side and the E1-binding domain on the other. The E3BP component has a similar segmented structure with globular domains connected by linker regions (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 29Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). E3BP associates with the inner E2 core via its C-terminal domain, binds E3 via a binding domain, and has a single N-terminal lipoyl domain (designated L3) (29Harris R.A. Bowker-Kinley M.M. Wu P. Jeng J. Popov K.M. J. Biol. Chem. 1997; 272: 19746-19751Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Jilka J.M. Rahmatullah M. Kazemi M. Roche T.E. J. Biol. Chem. 1986; 261: 1858-1867Abstract Full Text PDF PubMed Google Scholar, 31Rahmatullah M. Gopalakrishnan S. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 1245-1251Abstract Full Text PDF PubMed Google Scholar, 32Powers-Greenwood S.L. Rahmatullah M. Radke G.A. Roche T.E. J. Biol. Chem. 1989; 264: 3655-3657Abstract Full Text PDF PubMed Google Scholar, 33Gopalakrishnan S. Rahmatullah M. Radke G.A. Powers-Greenwood S.L. Roche T.E. Biochem. Biophys. Res. Commun. 1989; 160: 715-721Crossref PubMed Scopus (39) Google Scholar, 34Lawson J.E. Behal R.H. Reed L.J. Biochemistry. 1991; 30: 2834-2839Crossref PubMed Scopus (64) Google Scholar). Besides serving as substrates and intermediate carriers, the mobile lipoyl domains are involved in crucial interactions with the PDKs and PDP1 (3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar).The binding of PDK2 by lipoyl domains of E2 and binding of E1 by binding domain of E2 results in much faster phosphorylation than when free PDK2 phosphorylates free E1 (9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This difference in kinase activity is increasingly amplified when samples with equivalent E1 but including or not including E2 are evaluated under increasingly more dilute conditions. 2Y. Hiromasa and T. E. Roche, manuscript in preparation. Portions of this work have been described in abstract form (52Hiromasa Y. Roche T.E. FASEB J. 2001; 15: A196Google Scholar).2Y. Hiromasa and T. E. Roche, manuscript in preparation. Portions of this work have been described in abstract form (52Hiromasa Y. Roche T.E. FASEB J. 2001; 15: A196Google Scholar). Additionally, PDK2 responsiveness to regulatory effectors both requires (NADH and acetyl-CoA effects) and is altered (pyruvate and ADP) by the E2-confining interactions within the complex (3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 279: 37223-37229Abstract Full Text Full Text PDF Scopus (160) Google Scholar, 19Popov K.M. FEBS Lett. 1997; 419: 197-200Crossref PubMed Scopus (23) Google Scholar, 26Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Specifically, NADH and acetyl-CoA stimulate PDK2 activity by increasing the state of reduction and acetylation of the lipoyl domains, particularly the L2 domain (9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Popov K.M. FEBS Lett. 1997; 419: 197-200Crossref PubMed Scopus (23) Google Scholar, 26Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). E2 activation of PDK2 activity transforms PDK2 from being poorly inhibited by pyruvate or dichloroacetate to being markedly inhibited (9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Synergistic inhibition by allosteric binding of pyruvate or dichloroacetate results from the binding of these inhibitors by PDK2·ADP (and PDK2·ATP) but not by free PDK2, which slows down ADP dissociation. 3X. Yan, H. Bao, S. A. Kasten, and T. E. Roche, unpublished work. Portions of this work have been described in abstract form (53Yan X. Bao H. Kasten S.A. Roche T.E. FASEB J. 2001; 15: A197Crossref Scopus (28) Google Scholar).3X. Yan, H. Bao, S. A. Kasten, and T. E. Roche, unpublished work. Portions of this work have been described in abstract form (53Yan X. Bao H. Kasten S.A. Roche T.E. FASEB J. 2001; 15: A197Crossref Scopus (28) Google Scholar).To investigate the dynamic interactions of PDK2, we utilize free monomeric lipoyl domains, dimeric fusion constructs (GST-lipoyl domains), as well as E2 60-mer and E2·E3BP subcomplex with their lipoyl groups in an oxidized or reduced state. The L2 domain and its lipoyl group make a critical contribution to the E2-activated function of PDK2 (9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Beyond establishing that PDK2 is a stable dimer, we have used biophysical studies to evaluate the contributions of the interactions of the PDK2 dimer with the free L2 domain, GST-L2 dimer, GST bearing the L1 and L3 domains, the E2–60-mer alone and with E2 binding E1 and phosphorylated E1. We also evaluated the effects of catalytic reduction of the lipoyl domains of these structures on PDK2 binding. Our data establish that preferential binding to a lipoyl domain-bearing structure acutely depends on the oligomeric state of the lipoyl domain source and is markedly influenced by the state of oxidation/reduction state of the lipoyl group. Reduction of lipoyl groups alters PDK2 binding in a manner that fits a gain in cofactor-kinase interactions, which is apparently needed for reduction of E2 lipoyl groups enhancing PDK activity (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar, 9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 21Cate R.L. Roche T.E. J. Biol. Chem. 1978; 253: 496-503Abstract Full Text PDF PubMed Google Scholar, 22Rahmatullah M. Roche T.E. J. Biol. Chem. 1985; 260: 10146-10152Abstract Full Text PDF PubMed Google Scholar, 23Rahmatullah M. Radke G.A. Andrews P.C. Roche T.E. J. Biol. Chem. 1990; 265: 14512-14517Abstract Full Text PDF PubMed Google Scholar, 24Radke G.A. Ono K. Ravindran S. Roche T.E. Biochem. Biophys. Res. Commun. 1993; 190: 982-991Crossref PubMed Scopus (38) Google Scholar, 25Ono K. Radke G.A. Roche T.E. Rahmatullah M. J. Biol. Chem. 1993; 268: 26135-26143Abstract Full Text PDF PubMed Google Scholar, 26Ravindran S. Radke G.A. Guest J.R. Roche T.E. J. Biol. Chem. 1996; 271: 653-662Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Yang D. Gong X. Yakhnin A. Roche T.E. J. Biol. Chem. 1998; 273: 14130-14137Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Having E1 bound to E2 and the state of phosphorylation of E1 also influence PDK2 binding to the complex. As considered under “Discussion,” our results are consistent with a mechanism proposed based on functional studies (1Roche T.E. Liu S. Ravinddran S. Baker J.C. Wang L. Patel M.S. Roche T.E. Harris R.A. Alpha-Keto Acid Dehydrogenase Complexes. Birkhauser Verlag, Basel1996: 33-52Crossref Google Scholar, 2Roche T.E. Cox D.J. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1996: 115-132Google Scholar, 3Roche T.E. Baker J. Yan X. Hiromasa Y. Gong X. Peng T. Dong J. Turkan A. Kasten S.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 33-75Crossref PubMed Google Scholar, 4Roche T.E. Hiromasa Y. Turkan A. Gong X. Peng T. Yan X. Kasten S.A. Bao H. Dong J. Eur. J. Biochem. 2003; 270: 1050-1056Crossref PubMed Scopus (61) Google Scholar), whereby the binding of PDK2 to E2 may aid continued access of PDK2 to many bound E2·E1.EXPERIMENTAL PROCEDURESMaterials—Highly purified human PDK2 was prepared generally as previously described (9Baker J.C. Yan X. Peng T. Kasten S.A. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) but with some minor changes. 3X. Yan, H. Bao, S. A. Kasten, and T. E. Roche, unpublished work. Portions of this work have been described in abstract form (53Yan X. Bao H. Kasten S.A. Roche T.E. FASEB J. 2001; 15: A197Crossref Scopus (28) Google Scholar). The maximal specific activities of all human PDK2 preparations were >1100 nmol·min–1·mg–1 when assayed at 30 °Cin20mm potassium phosphate buffer. 3X. Yan, H. Bao, S. A. Kasten, and T. E. Roche, unpublished work. Portions of this work have been described in abstract form (53Yan X. Bao H. Kasten S.A. Roche T.E. FASEB J. 2001; 15: A197Crossref Scopus (28) Google Scholar). Purified human E2, E1, E2·E3BP, and mutated E2·E3BP were prepared as described elsewhere. 4Y. Hiromasa, H. Bao, X. Yan, X. Gong, A. Yakhnin, J. Dong, S. A. Kasten, L. Hu, T. Peng, J. C. Baker, M. Sadler, and T. E. Roche, manuscript in preparation. An E2·E3BP mutant was substituted for all three lysines that undergo lipoylation in L1 (Lys46) and L2 (Lys173) domains of E2 and L3 (Lys44)
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