Site Specificity of Four Pyruvate Dehydrogenase Kinase Isoenzymes toward the Three Phosphorylation Sites of Human Pyruvate Dehydrogenase
2001; Elsevier BV; Volume: 276; Issue: 40 Linguagem: Inglês
10.1074/jbc.m103069200
ISSN1083-351X
AutoresLioubov G. Korotchkina, Mulchand S. Patel,
Tópico(s)Electrolyte and hormonal disorders
ResumoActivity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the α subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4≈PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states. Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the α subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4≈PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states. pyruvate dehydrogenase complex pyruvate dehydrogenase dihydrolipoamide acetyltransferase dihydrolipoamide dehydrogenase E3-binding protein pyruvate dehydrogenase kinase 4-morpholinepropanesulfonic acid The mammalian multienzyme pyruvate dehydrogenase complex (PDC)1, which plays an important role in carbohydrate metabolism, catalyzes the oxidative decarboxylation of pyruvic acid with formation of CO2, acetyl-CoA, and NADH. PDC is composed of multiple copies (per complex) of three catalytic components, pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3); a binding protein, referred to as E3-binding protein (E3BP); and two regulatory enzymes, pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase (1Reed L.J. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (648) Google Scholar, 2Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (486) Google Scholar). E1 is a thiamine pyrophosphate-requiring α2β2heterotetramer with two active sites that interact with each other during catalysis (3Butler J.R. Pettit R.H. Davis P.F. Reed L.J. Biochem. Biophys. Res. Commun. 1977; 74: 1667-1674Crossref PubMed Scopus (34) Google Scholar, 4Khailova L.S. Korochkina L.G. Severin S.E. Ann. N. Y. Acad. Sci. 1989; 573: 36-54Crossref PubMed Scopus (34) Google Scholar). There are three specific serine residues in the α subunit of E1 that are subject to ATP-dependent phosphorylation and inactivation by PDK (5Yeaman S.J. Hutcheson E.T. Roche T.E. Pettit F.H. Brown J.R. Reed L.J. Watson D.C. Dixon G.H. Biochemistry. 1978; 17: 2364-2370Crossref PubMed Scopus (203) Google Scholar). Phospho-pyruvate dehydrogenase phosphatase dephosphorylates these three serine residues and reactivates PDC (1Reed L.J. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (648) Google Scholar).PDK exists as the multiple isoenzymes in several organisms, four isoenzymes of PDK in humans (PDK1, PDK2, PDK3, PDK4), three in rodents (PDK1, PDK2, PDK4 (PDK3 was not cloned, but its existence is not excluded)), two in plants (two in Zea mays and one inArabidopsis thaliana), one in nematodes (Ascaris suum and Caenorhabditis elegans), and one in fruit fly (6Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 7Rowles 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, 8Thelen J.J. Muszynski M.G. Miernyk J.A. Randall D.D. J. Biol. Chem. 1998; 273: 26618-26623Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Thelen J.J. Miernyk J.A. Randall D.D. Biochem. J. 2000; 349: 195-201Crossref PubMed Scopus (53) Google Scholar, 10Katsube T. Nomoto S. Togashi S. Ueda R. Kobayashi M. Takahisa M. DNA Cell Biol. 1997; 16: 335-339Crossref PubMed Scopus (10) Google Scholar, 11Chen W. Huang X. Komuniecki P.R. Komuniecki R. Arch. Biochem. Biophys. 1998; 353: 181-189Crossref PubMed Scopus (15) Google Scholar). Although PDKs phosphorylate specific serine residues of the E1α subunit, they show very little amino acid sequence similarity with eukaryotic Ser/Thr protein kinases. PDKs were suggested to belong to the ATPase/kinase superfamily (composed of bacterial histidine protein kinase, DNA gyrases, and molecular chaperone Hsp90) based on similarity of their catalytic domains (12Bowker-Kinley M. Popov K.M. Biochem. J. 1999; 344: 47-53Crossref PubMed Scopus (51) Google Scholar).PDKs are bound to the PDC core through the lipoyl domains of E2, and this interaction is important for their efficient catalytic function (13Yang 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). PDK activity is enhanced through changes in the status of the lipoyl domains from oxidized to reduced and acetylated forms, which in turn depend on the NADH/NAD+ and acetyl-CoA/CoA ratios (14Ravindran 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). Mammalian PDK isoenzymes differ in their catalytic activity, responsiveness to the modulators such as NADH and acetyl-CoA, and tissue-specific expression (15Bowker-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). PDK1 is present mostly in heart, whereas PDK2 is found in most tissues. PDK3 is predominantly expressed in testis, whereas heart and skeletal muscle have the highest amount of PDK4 (6Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 15Bowker-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).The E1 component has six potential phosphorylation sites (3 sites per α subunit) namely, site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203) (5Yeaman S.J. Hutcheson E.T. Roche T.E. Pettit F.H. Brown J.R. Reed L.J. Watson D.C. Dixon G.H. Biochemistry. 1978; 17: 2364-2370Crossref PubMed Scopus (203) Google Scholar). The rates of phosphorylation of the three sites in mammalian E1 were shown to be different (4.6-fold higher for site 1 than for site 2 and 16-fold higher for site 1 than for site 3) with PDK preparations (containing more than one isoenzyme) purified from mammalian tissues (16Korotchkina L.G. Patel M.S. J. Biol. Chem. 1995; 270: 14297-14304Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). We showed recently that the mechanism of inactivation appears to be different for phosphorylation of each of the three sites, i.e. phosphorylation of site 1 affects E1 interaction with its substrates, especially lipoyl moieties of E2, whereas phosphorylation of site 3 interferes with thiamine pyrophosphate binding (17Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 276: 5731-5738Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Because phosphorylation of any one site nearly completely renders E1 (and hence PDC) inactive, the presence of the three phosphorylation sites in E1 remains an enigma. It has been suggested that hyperphosphorylation of E1 may play a role in regulation of PDC in certain pathological conditions (18Randle P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar, 19Wu P. Inskeep K. Bowker-Kinley M.M. Popov K.M. Harris R.A. Diabetes. 1999; 48: 1593-1599Crossref PubMed Scopus (240) Google Scholar). The existence of the four isoenzymes in mammalian tissues raises an intriguing question about their specificity toward the three phosphorylation sites of mammalian E1. The availability of recombinant human mutant E1s with only one functional phosphorylation site, as well as E1-S264A and E1-S264E, has provided us the unique opportunity to examine the specificity of PDK isoenzymes toward each phosphorylation site. Our findings show that four PDK isoenzymes have different specificity toward the three phosphorylation sites. Site 3 was phosphorylated by PDK1 only. In contrast, the stimulation of PDKs by reduction and acetylation of the lipoyl moieties of E2 is not site-specific. Presence of glutamate at site 1 as a phosphorylation mimic did not interfere with phosphorylation of sites 2 and 3 by PDKs.DISCUSSIONThe importance of regulation of PDC activity in metabolism of carbohydrates and some amino acids is evident from the complexity of its regulation by phosphorylation-dephosphorylation. The heterotetrameric (α2β2) E1 has three phosphorylation sites per α subunit, and of the six potential sites available, phosphorylation of any one site results in near complete inactivation of E1. Recent studies have shown that the rates of phosphorylation are site-specific and that phosphorylation of each of the three sites affects the active site in a site-specific manner for its ability to bind thiamine pyrophosphate and substrates (16Korotchkina L.G. Patel M.S. J. Biol. Chem. 1995; 270: 14297-14304Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 17Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 276: 5731-5738Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Tissue-specific expression of multiple isoenzymes of PDK add yet another complexity to regulation of E1 (and hence PDC). PDK1 is expressed predominantly in heart, PDK2 is the most abundant isoenzyme present in several tissues such as liver, heart, skeletal muscle, etc. with lower amounts in spleen and lung, PDK3 is expressed mostly in the testis and lung, and PDK4 is expressed in heart and skeletal muscle (15Bowker-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). The results presented here demonstrate for the first time (i) the specificity of the four PDK isoenzymes toward the three phosphorylation sites of E1, (ii) the presence of significant level of PDK activity toward free E1 (PDK1, PDK2, and PDK4 toward site 1, PDK4 toward site 2, and PDK1 only toward site 3), and (iii) a lack of influence of site 1 on phosphorylation of the other two sites by PDK isoenzymes. Hence, site specificity for phosphorylation of four PDK isoenzymes with unique tissue distribution and differential regulation provides an added feature for tissue-specific regulation of PDC in normal and pathological conditions.Activities of four PDK isoenzymes were measured previously using wild-type E1 (with all three sites functional) in Tris buffer (15Bowker-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). Recently, activities of PDK2 and PDK3 were compared using wild-type E1 in three different buffer systems (phosphate buffer, MOPS-K+ buffer, and Tris buffer) (22Baker J.C. Yan X. Peng T. Kasten S. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the present study we have determined activities of PDK isoenzymes for phosphorylation sites 1, 2, and 3 individually in human E1. PDK activities were determined in two buffer systems for E1 sites 1, 2, and 3 individually. PDKs displayed different maximal specific activities for E1s with sites 1, 2, and 3 reconstituted in PDC depending on the buffer system used. In phosphate buffer, activities were as follows: PDK2 > PDK4≈PDK1 > PDK3 for site 1 and PDK3 > PDK4 > PDK2 > PDK1 for site 2 (Table I). In MOPS-K+ buffer activities were as follows: PDK1≈PDK3≈PDK2 > PDK4 for site 1 and PDK3 ≫ PDK1 > PDK4 > PDK2 for site 2 (Table I). These data indicate the sensitivity of the PDKs to the slight changes in their microenvironment. Site 3 was phosphorylated only by PDK1 in both buffer systems, and the activities of the other three PDKs toward site 3 were undetectable under our experimental conditions (see Fig. 1 and TableI). PDK1 differs from PDK2, PDK3, and PDK4 by the length of its amino acid sequence (and hence possibly in structure). Human precursor PDK amino acid sequences are composed of 436 residues for PDK1, 407 for PDK2, 406 for PDK3, and 411 for PDK4 (7Rowles 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). Matured PDK1 has the largest molecular mass among the four PDKs and corresponds to the 48-kDa subunit identified in the PDK preparations purified from bovine and porcine tissues (25Stepp L.R. Pettit F.H. Yeaman S.J. Reed L.J. J. Biol. Chem. 1983; 258: 9454-9458Abstract Full Text PDF PubMed Google Scholar), whereas matured PDK2, PDK3, and PDK4 have similar molecular masses and correspond to the 45-kDa subunit.The stimulation of PDK activities with the reduction and acetylation of the lipoyl moieties of E2 was isoenzyme-specific but not site-specific and was dependent on the buffer system employed. The maximal stimulation was observed for PDK2 in both phosphate buffer and MOPS-K+ buffer (Tables I and III). However, the extent of stimulation was not much different for different phosphorylation sites as substrates. Reduction or acetylation of the lipoyl moieties was suggested to induce conformational changes that generated a more active PDK state (14Ravindran 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). Earlier it was shown that stimulation of PDK purified from mammalian tissues by reduction and acetylation of lipoyl moieties of E2 required physiological concentrations of potassium salts (26Pettit F.H. Pelley J.W. Reed L.J. Biochem. Biophys. Res. Commun. 1975; 65: 575-582Crossref PubMed Scopus (192) Google Scholar). However, increasing K+ concentration inhibited bovine PDK activity (27Roche T.E. Reed L.J. Biochem. Biophys. Res. Commun. 1974; 59: 1341-1348Crossref PubMed Scopus (48) Google Scholar). The reduction in activities of all four PDKs observed in our study with MOPS-K+ buffer compared with phosphate buffer is consistent with an earlier study (22Baker J.C. Yan X. Peng T. Kasten S. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Although PDK is found to be present in limiting amounts in PDC (1–2 PDK molecules per complex), it was proposed to phosphorylate several molecules of E1 in PDC by moving on the surface of E2 core through interactions with the lipoyl domains (13Yang 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). In our experiments PDK isoenzymes displayed cooperative interaction with its substrate E1 (apparent S0.5 values were determined instead of apparentKm values). If we calculate the E1 concentration under our experimental conditions in phosphate buffer at which the amount of PDK is only one molecule per PDC, the calculated value for E1 concentration of 0.33 μm would be close to the observed apparent S0.5 values for E1 for PDK1, PDK2, and PDK4 toward site 1 and PDK4 for site 2. The presence of the substrates in the range of Km values under physiological conditions provides a higher degree of sensitivity for regulation. In the mitochondria the ratio of PDK isoenzyme activities will depend upon the following: (i) the relative amount of each PDK isoenzyme expressed, (ii) the activity of each PDK isoenzyme at the concentrations of E1 and ATP present, (iii) the level of reduced and acetylated lipoyl moieties of E2, and (iv) the concentration of pyruvate that has been shown to be inhibitory to PDKs (28Pratt M.L. Roche T.E. J. Biol. Chem. 1979; 254: 7191-7196Abstract Full Text PDF PubMed Google Scholar).PDKs are known to be activated by binding to E2 (13Yang 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). The activation of PDK by E2 was explained by enhancement of the catalytic efficiency of PDK and colocalization of PDK and E1 bound to the subunit-binding domain of E2 (22Baker J.C. Yan X. Peng T. Kasten S. Roche T.E. J. Biol. Chem. 2000; 275: 15773-15781Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). When saturating concentrations of E1 and ATP were used, activities of PDK1 and PDK4 toward site 1 of E1 by itself and E1 reconstituted in PDC were almost identical (Tables I and III). In contrast PDK3 was maximally activated by E2. Previously highly purified PDK preparations (mixtures of isoenzymes) purified from bovine tissues and recombinant PDK2 were found to phosphorylate only site 1 of wild-type E1 in the absence of E2 (5Yeaman S.J. Hutcheson E.T. Roche T.E. Pettit F.H. Brown J.R. Reed L.J. Watson D.C. Dixon G.H. Biochemistry. 1978; 17: 2364-2370Crossref PubMed Scopus (203) Google Scholar, 16Korotchkina L.G. Patel M.S. J. Biol. Chem. 1995; 270: 14297-14304Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The results presented here show clearly that site 2 is also phosphorylated by all four PDK isoenzymes, and site 3 is phosphorylated by PDK1 only in the absence or presence of E2-E3BP, and the level of E2-mediated activation is higher for phosphorylation of site 2 by all PDK isoenzymes and for phosphorylation of site 3 by PDK1 only. Interestingly, activity of PDK4 toward site 2 of E1 in the absence of E2-E3BP was much higher (from 4.3 to 9.2-fold) than activities of other three PDK isoenzymes (Table I). Recent evidence showed the presence of a large amount of free branched-chain α-keto acid-dehydrogenase kinase in rat liver mitochondria (29Obayashi M. Sato Y. Harris R.A. Shimomura Y. FEBS Lett. 2001; 491: 50-54Crossref PubMed Scopus (29) Google Scholar). Earlier it was found that the protein found in rat heart and liver mitochondrial extracts was able to increase phosphorylation of PDC. This protein was identified later as free PDK (30Jones B.S. Yeaman S.J. Biochem. J. 1991; 275: 781-784Crossref PubMed Scopus (15) Google Scholar). PDK4 has higher expression in heart and skeletal muscle compared with other tissues, whereas PDK2 is present in large amounts in different tissues including heart, liver, and kidney (15Bowker-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). The amounts of PDK4 in rat heart are increased during starvation and diabetes (4-fold), hyperthyroidism (3.5-fold), and high fat feeding (3.2-fold) (31Wu P. Sato J. Zhao Y. Jaskiewicz J. Popov K.M. Harris R.A. Biochem. J. 1998; 329: 197-201Crossref PubMed Scopus (260) Google Scholar, 32Sugden M.C. Langdown M.L. Harris R.A. Holness M.J. Biochem. J. 2000; 352: 731-738Crossref PubMed Google Scholar). Also, starvation resulted in the increased expression of PDK2 and PDK4 in liver, kidney, and lactating mammary gland but not in brain or adipose tissue (33Wu 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). This increase in PDK4 (and PDK2) may not correlate with the increases, if any, in the amounts of PDC molecules present in the mitochondria, resulting in the presence of more PDK4 (and PDK2) in the free form. The increase in hyperphosphorylation (multiple sites phosphorylation) of E1 on sites 1 and 2 (and hence PDC) not only by PDKs bound to PDC but also by free PDK4 (and possibly by PDK2) is supported by an increased expression of PDK4 in pathological studies.E1 was shown to be phosphorylated only on half of its potentially available phosphorylation sites. This seems to be true for all four PDKs. PDK1 phosphorylated about three sites (of six potential sites) and PDK2, PDK3, and PDK4 phosphorylated no more than two sites (of four potential sites) (Fig. 1A). The three phosphorylation sites of E1 are located not far from each other, especially site 1 (Ser-264) and site 2 (Ser-271), and they were suggested to affect E1 activity by different mechanisms (17Korotchkina L.G. Patel M.S. J. Biol. Chem. 2001; 276: 5731-5738Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). It was of interest to find out whether phosphorylation of one site affected phosphorylation of the other sites. The results presented here indicate that all four PDK isoenzymes had similar activities both in basal and stimulated conditions toward mutant E1s with serine at site 1 replaced with alanine or glutamate (Table IV). The lack of inhibition of site 1 modification on phosphorylation of the other two sites would allow hyperphosphorylation of E1 to proceed, which is important under some pathophysiological conditions, such as diabetes and starvation (18Randle P.J. Biochem. Soc. Trans. 1986; 14: 799-806Crossref PubMed Scopus (240) Google Scholar).Concluding RemarksThe present study demonstrates another aspect involved in the regulation of PDC activity in the mitochondria. Not only do PDK isoenzymes have different specific activities toward E1, but also their activities depend upon the individual phosphorylation site of E1 being modified. With the changes of the ratio of different isoenzymes, depending on the tissue and the metabolic state of the cell, PDC activity would be modulated by the extent of site(s) being phosphorylated. The number of sites phosphorylated could be increased by the presence of free PDKs capable of phosphorylation of three sites of E1 as indicated by this study. We have reported previously that each phosphorylation site is dephosphorylated with the similar rate, but dephosphorylation (and hence activation, as phosphorylation of each site causes inactivation) of all the three sites will require a longer time (16Korotchkina L.G. Patel M.S. J. Biol. Chem. 1995; 270: 14297-14304Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), resulting in PDC inhibition for an extended period in the cell. The mammalian multienzyme pyruvate dehydrogenase complex (PDC)1, which plays an important role in carbohydrate metabolism, catalyzes the oxidative decarboxylation of pyruvic acid with formation of CO2, acetyl-CoA, and NADH. PDC is composed of multiple copies (per complex) of three catalytic components, pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3); a binding protein, referred to as E3-binding protein (E3BP); and two regulatory enzymes, pyruvate dehydrogenase kinase (PDK) and phospho-pyruvate dehydrogenase phosphatase (1Reed L.J. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (648) Google Scholar, 2Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (486) Google Scholar). E1 is a thiamine pyrophosphate-requiring α2β2heterotetramer with two active sites that interact with each other during catalysis (3Butler J.R. Pettit R.H. Davis P.F. Reed L.J. Biochem. Biophys. Res. Commun. 1977; 74: 1667-1674Crossref PubMed Scopus (34) Google Scholar, 4Khailova L.S. Korochkina L.G. Severin S.E. Ann. N. Y. Acad. Sci. 1989; 573: 36-54Crossref PubMed Scopus (34) Google Scholar). There are three specific serine residues in the α subunit of E1 that are subject to ATP-dependent phosphorylation and inactivation by PDK (5Yeaman S.J. Hutcheson E.T. Roche T.E. Pettit F.H. Brown J.R. Reed L.J. Watson D.C. Dixon G.H. Biochemistry. 1978; 17: 2364-2370Crossref PubMed Scopus (203) Google Scholar). Phospho-pyruvate dehydrogenase phosphatase dephosphorylates these three serine residues and reactivates PDC (1Reed L.J. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (648) Google Scholar). PDK exists as the multiple isoenzymes in several organisms, four isoenzymes of PDK in humans (PDK1, PDK2, PDK3, PDK4), three in rodents (PDK1, PDK2, PDK4 (PDK3 was not cloned, but its existence is not excluded)), two in plants (two in Zea mays and one inArabidopsis thaliana), one in nematodes (Ascaris suum and Caenorhabditis elegans), and one in fruit fly (6Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. Chem. 1995; 270: 28989-28994Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 7Rowles 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, 8Thelen J.J. Muszynski M.G. Miernyk J.A. Randall D.D. J. Biol. Chem. 1998; 273: 26618-26623Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Thelen J.J. Miernyk J.A. Randall D.D. Biochem. J. 2000; 349: 195-201Crossref PubMed Scopus (53) Google Scholar, 10Katsube T. Nomoto S. Togashi S. Ueda R. Kobayashi M. Takahisa M. DNA Cell Biol. 1997; 16: 335-339Crossref PubMed Scopus (10) Google Scholar, 11Chen W. Huang X. Komuniecki P.R. Komuniecki R. Arch. Biochem. Biophys. 1998; 353: 181-189Crossref PubMed Scopus (15) Google Scholar). Although PDKs phosphorylate specific serine residues of the E1α subunit, they show very little amino acid sequence similarity with eukaryotic Ser/Thr protein kinases. PDKs were suggested to belong to the ATPase/kinase superfamily (composed of bacterial histidine protein kinase, DNA gyrases, and molecular chaperone Hsp90) based on similarity of their catalytic domains (12Bowker-Kinley M. Popov K.M. Biochem. J. 1999; 344: 47-53Crossref PubMed Scopus (51) Google Scholar). PDKs are bound to the PDC core through the lipoyl domains of E2, and this interaction is important for their efficient catalytic function (13Yang 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). PDK activity is enhanced through changes in the status of the lipoyl domains from oxidized to reduced and acetylated forms, which in turn depend on the NADH/NAD+ and acetyl-CoA/CoA ratios (14Ravindran 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). Mammalian PDK isoenzymes differ in their catalytic activity, responsiveness to the modulators such as NADH and acetyl-CoA, and tissue-specific expression (15Bowker-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). PDK1 is present mostly in heart, whereas PDK2 is found in most tissues. PDK3 is predominantly expressed in testis, whereas heart and skeletal muscle have the highest amount of PDK4 (6Gudi R. Bowker-Kinley M.M. Kedishvili N.Y. Zhao Y. Popov K.M. J. Biol. 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