THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 47, Issue of November 25, pp. 29720-29724, 1994 Printed in U.S.A.

Molecular Cloning of the p45 Subunit of Pyruvate Dehydrogenase Kinase*

(Received for publication, June 7, 1994, and in revised form, August 30, 1994)

Kirill M. Popov, Natalia Y. Kedishvili, Yu Zhao, Ramadevi Gudi, and Robert k Harris* From the Department of Biochemistry and Molecular Biology, Zndiana University School of Medicine, Indianapolis, Zndiana 46202-5122

Purified preparations of rat heart pyruvate dehydro- genase kinase have two polypeptides with molecular weights of 48,000 (p48) and 45,000 (p45). Recently, we reported the primary structure of p48 (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and Harris, R. A. (1993) J. BioZ. Chem. 268,26602-26606) and presented evidence that (i) it exhibits kinase activ- ity for pyruvate dehydrogenase and (ii) it belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. Here, we report the molecular cloning and deduced amino acid sequence of p45. The protein sequence of p45 has 70% identity to the protein sequence of p48. Minor differences exist throughout the protein sequences with the greatest dif- ference occurring at the amino termini. Recombinant p45 protein, expressed in Escherichia coZi and purified to homogeneity, catalyzed the phosphorylation and in- activation of kinase-depleted pyruvate dehydrogenase complex, indicating that p45 and p48 correspond to dif- ferent isoforms of pyruvate dehydrogenase kinase. Northern blot analysis revealed a single hybridizing species of 2.5 kilobases. The highest level of p45 message expression was found in heart and skeletal muscle and the lowest in spleen and lung. Liver, kidney, brain, and testis express intermediate amounts of p45 mFtNA. In contrast, p48 mRNA is predominantly expressed in heart, with other tissues expressing only a modest amount of this message. Tissue-specific expression of isoforms of pyruvate dehydrogenase kinase may indi- cate the existence of tissue-specific mechanisms for the regulation of pyruvate dehydrogenase activity.

The mitochondrial multienzyme complex pyruvate dehydro- genase (PDH)l catalyzes an irreversible step in the degradation of glucose. Rapid adjustment of flux through PDH is regulated by end-product inhibition (acetyl-coA, NADH) (reviewed in

from the U.S. Public Health Services, Grant PHS AM 20542 from the * This work was supported by grants PHS DK 19259 and DK 47844

Medicine, the Grace M. Showalter Residual Trust, American Heart Diabetes Research and Training Center of Indiana University School of

Association Grant-in-aid (to K. M. P.), American Heart Association, In- diana AtXliate, Inc. postdoctoral fellowship (to N. Y. K.), and the March of Dimes Predoctoral Fellowship (to Y. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankm/EMBL Data Bank with accession number(s) U10357. The nucleotide sequence(s) reported in this paper has been submitted

Fax: 317-274-4686; E-mail: iqjqlOO@indycmsiupui.edu. $ ‘Ib whom correspondence should be addressed. Tel.: 317-274-1586;

The abbreviations used are: PDH, pyruvate dehydrogenase; PDK, PDH kinase; bp, base pairk); E,, a-ketoacid dehydrogenase component of PDH; p48, subunit of PDK with lowest electrophoretic mobility; p45, subunit of PDK with highest electrophoretic mobility; KAP, kinase ac- tivator protein; PCR, polymerase chain reaction.

Refs. 1 and 21, and this mechanism is likely to be of importance whenever conditions favor rapid rates of fatty acid oxidation (1). However, the more important control mechanism for mam- malian PDH is regulation by reversible phosphorylation (3, 4). PDH kinase (PDK), an integral component of the complex (31, phosphorylates three serines of the a subunit of the E, compo- nent (5). Phosphorylation leads to inactivation of E, (3,4) and, as a consequence, of the holocomplex (4,5). Dephosphorylation and reactivation of PDH is catalyzed by a highly specific pyru- vate dehydrogenase phosphatase (4), loosely associated with the complex (6). It is generally believed that the overall activity of PDH is determined by the activity of an intrinsic PDK, which itself is regulated by the products and substrates of the PDH reaction (4,7-9). In general, the products of the dehydrogenase reaction (acetyl-coA and NADH) stimulate the kinase (4, 81, whereas the substrate (pyruvate) and the coenzyme (thiamine pyrophosphate) are inhibitory (4,7,9). Besides this short-term regulation, stable changes in activity of PDK have been ob- served during starvation and diabetes (10, l l ) . Recent evidence suggests that starvation increases the specific activity of the kinase, implicating the involvement of covalent modification of the kinase as a potential regulatory mechanism (12). However, the exact mechanism of long-term regulation of PDK is still obscure.

The first successful purification of PDK from bovine kidney mitochondria was reported by Stepp et al. (13). They reported that the enzyme exists as a dimer of two nonequivalent sub- units of 48 and 45 kDa, designated as a and P. The kinase activity was reported to reside in the a subunit, with the func- tion of the P-subunit unknown, although it was suggested that it may be involved in regulation of kinase activity and/or the attachment of the kinase to the complex (13). Recently, we reported the purification of PDK from rat heart (14). Like the enzyme from bovine kidney, rat heart PDK appears to consist of two subunits that differ in electrophoretic mobility (M, 48 kDa (p48) and 45 kDa (p45), respectively). Amino-terminal sequence analysis of individual subunits electroblotted on polyvinylidene fluoride membrane yielded two different sequences: ASD- SASGSGPASESGV for p48 and KNASLAGAPKYIEFSKFS for p45, indicating that the polypeptides found in highly purified preparations of PDK may be products of different genes or alternative splicing. In an attempt to further characterize the structure of the subunits of PDK, we cloned a cDNA encoding p48 (15). The analysis of the deduced protein sequence revealed high homology to another mitochondrial protein kinase, the branched chain a-ketoacid dehydrogenase kinase (161, an en- zyme functionally similar to PDK (17). Especially high homol- ogy was found within putative kinase domains located near the carboxyl termini of the proteins (15). This finding established p48 as a catalytic subunit of PDK.

In this paper, we report the molecular cloning of the cDNA of p45 of rat heart PDK. An analysis of the deduced protein se- quence of p45 revealed 70% amino acid identity with p48.

29720

Pyruvate Dehydrogenase Kinase 29721

These results, along with evidence that the recombinant p45 protein catalyzes an ATP-dependent inactivation of the activity of the PDH complex, indicate that the p45 cDNA encodes for another isoform of PDK. We also report here the tissue distri- bution of both isoforms based on Northern blot analysis.

EXPERIMENTAL PROCEDURES

cleotide primer (AAA/G TGT/C TCMGPT ATMG TACPT IT1 GGI GC) Polymerase Chain Reaction-The downstream gene-specific oligonu-

was designed according to the amino-terminal sequence of p45 (AP- KYEHF). Inosines were used in the third position of codons with a degeneracy of four. Upstream primers (one for each insert orientation) were synthesized to correspond to bases 4266-4289 (GGTGGCGAC- GACTCCGGAGCCCG) and bases 4323-4352 (TTGACACCAGAC- CAACTGGTAATGGTAGCG) of the Escherichia coli Lac operon flank- ing the EcoRI site in hgtl l phage (18). hgtl l phage DNA from 1 ml of stock (titer 1O'O of plaque-forming unitlml) of an amplified rat heart cDNA library (Clontech) was purified by conventional techniques (18) and used as a template for PCR. Each reaction mixture contained 50 pmol of the gene-specific primer, 50 pmol of one of the A-specific primers, 500 ng of purified hgt l l DNA along with deoxyribonucleic triphos- phates, buffer, and 0.5 units of Taq polymerase added according to the manufacturer's instructions (Perkin-Elmer Cetus). 40 cycles of PCR were performed by using 45 s at 94 "C for denaturation, 1 min at 56 "C for annealing, and 1 min at 72 "C for extension. A PCR product of 119 bp was purified and subcloned in M13 mp18 for sequencing.

5'Stretch k t10 Rat Hear t cDNA Library Screening-A nondegener- ate oligonucleotide probe (CCTGCCAGGGACGCAITCTC) was syn- thesized according to the sequence of the PCR product. It was end- labeled with [Y-~~PIATP using T, kinase (18) to a specific activity lo9 cpm/pg. This probe was used to screen 0.5 x lo6 individual plaque- forming units of a A g t l O rat heart cDNA library (Clontech) essentially as described by Sambrook et al. (18). Hybridization conditions were as follows: 6 x SSC (1 x SSC = 150 mM sodium chloride, 15 mM sodium citrate, pH 7.51, 5 x Denhardt's solution (0.1% (w/v) bovine serum al- bumin, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) Ficoll 4001, 0.1 mg/ml denatured salmon sperm DNA, and the radiolabeled probe (2 x lo6 cpm/ml) at 55 "C for 17 h. The filters were washed with 6 x SSC, 0.1% (w/v) SDS four times at room temperature and one time with 2 x SSC, 0.1% (w/v) SDS at 55 "C for 5 min. l b o positive plaques were purified through four more cycles of plating and screening. cDNAs were cut out of A phage with EcoRI and religated in EcoRI-digested M13 mp18 for sequencing.

Rapid Amplification of 5'-End of p45 cDNA (S'RACE)-Rat heart 5'RACE-ready templates were obtained from Clontech. The templates were amplified with a gene-specific primer (CCTGCCAGGGACGCAT- TCTTC) corresponding to bases 115-136 of the p45 cDNA and the an- chor primer (CTGGITCGGCCCACCTCTGAAGGTTCCAGAATCGA- TAG) obtained from Clontech. Each reaction contained 50 pmol of the gene-specific primer, 50 pmol of the anchor primer, and 100 ng of tem- plates. dNTPs, buffer, and Taq polymerase were added according to the manufacturer's instructions (Clontech). 40 cycles of PCR were per- formed by using 1 min at 94 "C for denaturation and 1 min at 60 "C for annealing. PCR products were separated by electrophoresis and ana- lyzed by Southern blotting with an end-labeled oligonucleotide probe corresponding to bases 93-110 (CGCCCGGAACCAGCGCAT) of p45 cDNA. A hybridizing PCR product of 136 bp was purified and subcloned in M13 mp18 for sequencing.

Expression of p45 cDNA in E. co l iSac I and XhoI restriction sites flanking the coding region of p45 cDNA were constructed by PCR. The resulting 1.2-kilobase SacIIXhoI fragment was ligated in a PET-28a expression vector (Novagen, Madison, WI), cut with Sac1 and XhoI, and dephosphorylated with alkaline phosphatase to produce an in- frame amino-terminal fusion with a consecutive stretch of 6 histidine residues (His-Tag) and polypeptide MAMTGGQQMG (T,-Tag). The resulting plasmid was confirmed by direct sequencing. The p45 cDNA was expressed in E. coli HMS 174 (DE31 (Novagen) as previously described (15). Recombinant protein was purified by metal chelation chromatography (19).

ATP-dependent Inactivation ofKinase-depleted PDH by Recombinant p45-ATP-dependent inactivation of kinase-depleted PDH by recombi- nant p45 was assayed essentially as previously described (15). Phos- phorylation reactions (total volume, 600 pl) contained 20 mM Tris-HC1 (pH 7.4), 5 m~ MgCl,, 50 mM KC1, 5 m~ dithiothreitol, 0.5 mM ATP, 10 pg of kinase-depleted PDH (14) and 0.5 pg of p45. Phosphorylation was initiated by ATP, and incubations were conducted at room temperature. Aliquots (50 pl) were withdrawn at indicated times to measure residual

activity of PDH spectrophotometrically as previously described (14). DNA Sequencing-Single-stranded M13 DNA was prepared and se-

quenced by the dideoxy chain termination method (20) with Sequenase version 2.0 (U. S. Biochemicals Corp.).

Northern Blot Analysis-Multiple rat tissue Northern blots were ob- tained from Clontech. Blots were probed with random primed 32P- labeled p45 or p48 cDNAs. Hybridization conditions were as follows: 5 x SSPE (1 x SSPE = 180 m~ sodium chloride, 10 m~ sodium phosphate, 1 mM EDTA, pH 7.5), 5 x Denhardt's solution, 1.0% (w/v) SDS, 50% (v/v) formamide, 0.1 mg/ml denatured salmon sperm DNA, and radiolabeled probe (1-1.5 x lo6 cpm/ml) at 42 "C for 12 h. Blots were washed four times in 2 x SSC, 0.1% (wh) SDS at room temperature and two times in 0.1 x SSC, 0.1% (w/v) SDS at 55 "C for 5 min.

RESULTS AND DISCUSSION

Amplification of the 5'-End of p45 cDNA-Available amino- terminal sequence of p45 (KNASLAGAPKYIEFSKFS) allowed the design of an oligonucleotide gene-specific primer with fairly low degeneracy. The degeneracy was further decreased by in- corporation of inosine residues in the third positions of codons for alanine and proline, as described under "Experimental Pro- cedures." This primer was used in combinations with primers specific for h g t l l DNA sequences to amplify a rat heart cDNA library. One set of primers gave rise to a PCR product of 119 bp (data not shown), which was subcloned in M13 mp18 and se- quenced. Analysis of the deduced protein sequence of the PCR product yielded a sequence exactly matching the amino-termi- nal sequence of p45 (data not shown). This information was used to design a perfect oligonucleotide probe (see "Experimen- tal Procedures"), corresponding to the amino terminus of p45 (KNASLAG), for library screening.

k t 1 0 Rat Heart cDNA Library Screening-Arat heart cDNA library was screened with the perfect oligonucleotide probe described above. Approximately 0.5 x lo6 clones were screened, and two positive clones were isolated. The first contained an insert of 716 bp, and the second gave two fragments of 286 and 1834 bp, indicating that the p45 cDNA has an internal EcoRI site. All fragments were subcloned in M13 mp18, and both strands were sequenced.

Nucleotide and Predicted Amino Acid Sequences of p45- Sequence analysis of the three cDNAs revealed that the 1834-bp fragment contained the complete 3'-end of the p45 cDNA, while the 286-bp fragment contained a partial sequence for the 5'-end. The 716-bp cDNA contained the complete se- quence of the 286-bp fragment as well as that of the 119-bp product obtained by h g t l l cDNA library amplification. It also contained, however, 330 bp of extraneous sequence at its 5' - end. The latter was found from the DNA data banks available through GenInfo to correspond to a portion of the sequence of the 0-subunit of cytochrome oxidase. To obtain more informa- tion about the sequence of the 5'-end of the p45 cDNA, a partial cDNA was generated by the 5'RACE protocol (see "Experimen- tal Procedures"). Amplification of rat heart templates gave rise to a 136-bp PCR product lacking the 330 bp at the B'-end of the 716-bp cDNA, indicating that the latter sequence is most likely an artifact produced during construction of the library.

The resulting 2216-bp composite cDNA for p45 was con- structed by aligning the 136-bp 5'RACE product with the 286- and 1834-bp cDNA fragments (Fig. 1). The cDNA has one open reading frame defined by an ATG triplet at base 93 and an in-frame stop codon at position 1314. Authenticity of the cloned cDNA was confirmed by perfect matches of the three peptides encoded by the open reading frame to the sequences of two tryptic peptides derived from purified preparations of p45 (14) as well as the amino-terminal sequence of the mature protein (underlined in Fig. 1). The sequence coding for the mature protein begins at nucleotide 117. It is 399 residues in length with a calculated molecular weight of 45,031, in good agree- ment with the estimated molecular weight of the p45 subunit of

29722 Pyruvate Dehydrogenase Kinase

GGGCTGTGCTTGGCCGTGCGGAGGCCCGGTGCCACCACCTCCAGCTCCGGGACA~AGCGGGAGCCMGCCCGACCCGCAOOCGTC GTCGCCATGCCCTGGTTCCGGGCGCTGTTGM~TCCGTCCCTOOCAGGGGCGCCCAAGTACATCGAGCACTTCAGCMGTTCTCCCCG

M R W F R A L L E N A S L A G A P K Y I E H F S K F S P -8 1 10 20

TCCCCGCTGTCCATGMGCAGTTTCTAGACTTCGGATCCAGCMTGCCTGCGAG~CTTCATTCACCTTCCTCCGGCAGGAGCTGCCC S P L S M K Q F L D F G S S N A C E K T S F T F L R Q E L P 21 30 40 50 GTGCCCCTGGCCMCATCATOllAAOAOAPCAACCTGCTTCCTGACCGGGTCCTGAGCACCCCCTCAGTGCMCTGGTGCAGAGCTGGTAT V R L A N I M K E I N L L P D R V L S T P S V Q L V Q S W Y 51 60 70 80

GTCCAGAGTCTGCTGGACATCATGGMTTCCTOOA~GGACCCCGAGGACCACCGGACCCTMCCCAGTTCACTGATCCCCTGGTCACC V Q S L L D I M E F L D K D P E D H R 3 L S O F T D A L V T 81 90 100 110

ATCCGGMCCGGCACAATGACGTAGTGCCCACCATGGCACAG~AGTGTTGGAGTACAAGGACACCTATGGTGATGACCCAGTCTCCAAC I R N R H N D V V P T M A Q G V L E Y K D T Y G D D P V S N

111 120 130 140 CAGMCATCCAGTACTTTTTGGACCGCTTCTACCTCAGCCGCATCTCTATCCGCATGCTCATTMCCAGCACACCCTCATCTTTGATGGC Q N I Q Y F L D R F Y L S R I S I R M L I N Q H T L I F D G 14 1 150 160 170 AGCACCMCCCACCCCACCCCAAACACATTGGCAGCATTGATCC~CTGCACCGTGTCTGATGTGGTGAAAGATGCCTATGACATGGCT S T N P A H P K H I G S I D P N C S V S D V V K D A Y D M A 171 180 190 200 MGCTCCTGTGTGACAAGTATTACATGGCTTCCCCTOACCTGGAOATCCAGGMGTCAATGCCACCMCGCCACCCAGCCCATTCACATG K L L C D K Y Y M A S P D L E I Q E V N A T N A T Q P I H M 201 210 220 230 GTCTACGTCCCCTCCCACCTCTACCACATGCTCTTTGMCTCTT~GMTGC~TCCGGGCCACAGTGGAAAGCCACGAGTCCAGCCTC V Y V P S H L Y H M L F E L F K N A M R A T V E S H E S S L 231 240 250 260 ACTCTCCCTCCCATCAAAATCATGGTOOCCCTCGGT~GMGATCTGTCCATC~TGAGTGACCGAGGCGGGGGTGTCCCCTTGAGG T L P P I K I M V A L G E E D L S I K M S D R G G G V P L R 261 270 280 290 MGATCGAGAGGCTCTTCAGCTACATGTACTCTACAGCTCCTACACCCCAGCCTGGCACTGGGGGTACCCCGCTGGCTGGCTTTGGGTAT K I E R L F S Y M Y S T A P T P Q P G T G G T P L A G F G Y 291 300 310 320 GGACTCCCCATTTCCCGCCTCTACCC~GTACTTCCAGGOOGACTTGCACTOCGCTCTTCTCTATGGAGGGCTTT~GACAGATGCTGTCATC G L P I S R L Y A K Y F Q G D L Q L F S M B G F G T D A V I 321 330 340 350 TATCTGMGGCCCTGTCCACGGACTCAGTGGACCCCCTCCCTGTCTACM~GTCTGCCTGGCGCCACTACCAGACCATCCAGGAGGCC Y L K A L S T D S V E R L P V Y N K S A W R H Y Q T I Q E A 351 360 370 380 GGTGACTGGTCCGTGCCCAGCACAGAGCCCMGMCACATCGACGTATCGGGTCAGCTAGGGCCCTTCTCTTCCTGGCACCTGGGAGGAT G D W C V P S T E P K N T S T Y R V S 381 390 399 GCTGCCACCTCTGMTCCAGCCACCACAGGGACTTCCCTATCTATCCCCTGGGGTACGGGGGTG~CTGOOTCTCCCCGATGGCCAGAT CTGTCTTTGTAGAAATCGCAGTGGCCCATCTGTGCCGATCCCTMGTGCCMTCTGTCTCTATGGAGAAACCT~OOGGGTTTCCCTGGAG CCTGGTCTCCATGGTGATOATGCTTGAGGGTTGGOOAC~CTCTACCTGGTGOOGTGGCCCCAGAGACACTTCTCC~GACCAGAGCTG TCTGTTTTCTACCAGAAACCCTGGGTCCCCCTCACTOCCTGCCTGCATAGTCCTGGTCTCCCACGTGGCTGCCTCGCTTGCCTTATGCCCACAC CCTGTACAGGCACATTOOGCTGGTTTCTTCGTCAGTAGTMGAAAGATGGAGAGAGACTGOO~CGOOGGCCMCCTTGTCTCTGGTC CTCCAGCCTCTCTCCATCTCCACTCTGGACACT~GTTGCCACTGOOMCTTGAGMTGOOTGGCCGTTCTCACCCAAGCCCCACCGAG MGGCCTMGAGTMCCTGTCCCCMGGCGATCTTAGCMTGTTTCTGCCGCTTCCTGGCCTGGCATGTCCTCACGTGTATACCTCCCCT GCCCAGTGTACGCTCACCCTATCCCTCCTTGACCTTTAGACCCCAGACTTCCTATGCCCACTATGTGTGCACAGACGACTCAAACCCAGG ATGCCCCATGTACATAGCCAGTTTTGTMTCTCA~TGCCTCACCCTTGCCCTCCGCACACAGGGGTTAAAGCCGTGTCCCCCTCCCAGT G G C T G G G A T G G T G A C A G T G A C A T C C A C A G T A A A T A G A T G A A A T G ~

FIG. 1. Nucleotide and deduced protein sequences of the p45 subunit of PDK. The amino-terminal and internal tryptic peptide E

determined for the purified protein are underlined.

-86 -176

-266

-356

-446

-536

-626

-716

-806

-896

-986

-1076

-1166

-1256

-1346

-1436 -1526 -1616 -1706 -1796 -1886 -1976 -2'066 -2156 -2215

Iequences

PDH kinase (13, 14). The ATG codon at nucleotides 93-95 cor- responds to the first methionine in an open reading and there- fore is the most probable start of the protein sequence. If this is case, the protein has an 8-residue-long mitochondrial entry sequence.

Alignment of the Deduced Protein Sequence ofp48 and p45- Alignment of the deduced protein sequences of p48 and p45 revealed 70% amino acid identity between the two sequences, indicating that the p45 cDNA encodes for another isoform of PDK (Fig. 2). The similarity extends through the entire se- quence, but differences occur throughout, particularly within the amino terminus.

Like other members of the mitochondrial protein kinase fam- ily (151, p45 has a putative kinase domain of approximately 150 amino acid residues, located in the carboxyl-terminal part of

the kinase. In the amino terminus, the catalytic domain is defined by an invariant asparagine residue occurring within a highly conserved region with the sequence KNAMRAT (subdomain 11) (Fig. 2). At the carboxyl terminus, the catalytic domain is defined by a glycine-rich loop Gly317-X-Gly319-X-Gly3z1 (subdomain V). The central core of the catalytic domain con- sists of subdomains I11 (consensus sequence Asp282-X-Gly284-X- G1yZB6) and IV (defined by invariant PheZg6). Invariant residues of subdomains 11,111, W, and V are presumably involved in ATP binding and catalysis, but their exact functions are currently unknown. As proposed earlier (15), a phosphohistidine inter- mediate may be involved in the catalysis by mitochondrial pro- tein kinases. The only histidine residue conserved among all members of the family (15) occurs in subdomain I, located within the amino terminus (His115 of p45) and separated by a

Pyruvate Dehydrogenase Kinase

KN--------ASLAGAPKIEHI?SKZ’SPSPLSMXQFLDFGSSNACEXTSF - 42 ASDSASGSGPASESC;VPGQViU’YARFSSPSPLSMXQFLDFGSSF - 50 T P L R ~ P ~ ~ P D R n S T P S V P L V Q S W W Q S L L D ~ - 92 ~QELPvRLAN~ISLLPDNLLRTPSVQLVQSWYIQSLQELLDPPCD - 100 ~ P E o H R T L S Q ~ ~ ~ I ~ ~ Q ~ ~ ~ G D D P V ~ ~ - 142 l C S A E O A K T I Y Z F T D T V I X f P ~ Q ~ ~ ~ S F ~ P ~ S Q N - 150 IQYFLDRFYLSRISIRBLINQHTLIFDGSTNPAP~IGSIDPNCSVSDV - 192 VQYPLDRFYMSRISIRMLLNQHSLLFGGXGSPSHRXHIGSINPNCDVVEV - 200 V K D A Y D % A K L L C D ~ P D L E I Q E V N A T N A T Q P I ~ S R L Y I B % F - 242

. . . . ........................ .................................

.................................... ............................................ . . sab&B&u

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........................................

..................... .......................... : :.: :::::: ::: : .:

. . . . . . . . . . . . . . . . .............. ....................... ................ IKooYENARXLCDLYYVNSPELELEELNAKSPGQPIQVVYVPSHL~ - 250 - - ~ T V E S H E S S L T L P P I X ~ ~ G ~ D L S 1 ~ S D R ~ C ; V P ~ I - 292 ............ ........................ ............. ............................ E I g ~ ~ K ~ P I Q V H V T L G E Z D L ~ S D R G G G V P L R X I - 3 0 0 Eubd-ln IV sl&m&c? ERLFSYMYSTAPTPQPGTGGT-PLAGFGYGLPISRLYAFYFQGDLQLFSM - 341 . . . . . . . . . . . . ........................ ........................................

29723

PDK 11 - PDK I - PDK 21 - PDK I - PDK I1 - PDK I - PDK 11 - PDK I - PDK I1 - PDX I - PDK I1 - PDX I - PDK I1 - PDK I - DRLFNYMYSTAPRPRVETSRAVPLAGFGYGLPISRLYAQYFQGDLXLYSL - 350 PDK I1 - EGFGTDAVIYLXALSTDSVEXLPTfNXSAWRHYQTIQEAGK - 391 ....................................... ............................................. PDK I - EGYGTDAVIYIXALSTZSIERLPVYNKAAWKHYRTNHEADDWCVPSXEPX - 400

PDK 11 - NTSTYRVS - 399 . . . . . . . . PDK I - DMTTPRSS - 4 0 8

FIG. 2. Alignment of predicted amino acid sequence of the mature polypeptide of PDK I1 (~45) with the predicted sequence of the mature polypeptide of PDK I (p48). Amino acid residues are shown in single letter code. Identity is shown by (:), conserved substitutions (A,S,T; V,L,I,M; F,Y D,E; K,R) are shown by (.). Similarity was established by using the PALIGN routine of PCGENE.

spacer of 131 amino acids from the invariant Enzymatic Activity ofp45 Expressed in E. coli-Expression of

the cDNA encoding p45 in E. coli produced a recombinant pro- tein of an apparent molecular weight of 46,000 that was recog- nized by anti-polypeptide W T G G Q Q M G (T,-Tag) antibodies (data not shown). Recombinant p45 was purified to homogene- ity by metal chelation chromatography (19). Incubation of the recombinant p45 with kinase-depleted PDH in the presence of ATP resulted in a time-dependent inactivation of dehydrogen- ase activity (Fig. 3) with concomitant phosphorylation of the a subunit of E, (data not shown). This observation, together with the above analysis of the p45 sequence relative to that of p48, provides convincing evidence that the cloned cDNA for p45 also encodes an isoform of PDH kinase. Taking into account the demonstration that both p45 and p48 are catalytic subunits of PDH kinase, we propose that the first discovered isoform of PDK (15) be designated PDH kinase I (or PDK I) and the second isoform discovered in this study be designated pyruvate dehydrogenase kinase I1 or (PDK 11).

Northern Blot Analysis-Tissue distribution of both forms of PDK was characterized by Northern blot analysis (Fig. 4). The mRNA of PDK I1 corresponded to a single hybridizing species of approximately 2.5 kilobases in all tissues tested (Fig. 4A). The highest amount of message was found in heart and skeletal muscle and the lowest amount in spleen and lung. Testis, liver, brain, and kidney expressed an intermediate amount of PDK I1 (testis > brain = liver > kidney). The tissue distribution of PDK I mRNA markedly differs from PDK 11. The message for PDK I was predominantly expressed in heart with only modest levels of expression in other tissues (Fig. 4B). This observation is in accord with previous studies, indicating that regulation of PDH in cardiac muscle during starvation is different from other tis- sues (21, 22). Starvation for 48 h decreases the proportion of

0 . 0 1 . o 2 . 0 3 . 0

Time (min)

depleted PDH by recombinant p45 (PDK 11). ATP-dependent inac- FIG. 3. Time course of ATP-dependent inactivation of kinase-

tivation reactions were carried out as described under “Experimental Procedures.” Kinase-depleted PDH was incubated with recombinant PDK I1 and ATP (O), without ATP (01, or without PDK I1 (A) at room temperature for the times indicated.

PDH in the active form in all tissues tested. In heart, this inhibition occurs in part as a result of activation of P-oxidation of long-chain fatty acids (22). The inhibitor of fatty acid P-oxi- dation, 2-tetradecylglycidic acid, reverses the effect of 48-h starvation in heart but has no effect in most other tissues (22), suggesting that in contrast to other tissues, the activity of heart

29724 Pyruvate Dehydr

A. - -. . . . ". . - - .- .- -

- 9.5 - 7.5

- 4.4

- 2.4

- 1.35

I

B. - 9.5 - 7.5

- 4.4

- 2.4

- 1.35

FIG. 4. Northern blot analysis. '"P-labeled cDNAs of PDK I1 (A) and PDK I ( B ) were used to detect the corresponding mRNAs on a rat multiple tissue Northern blot obtained from Clontech (lanes correspond to approximately 2 pg of poly(A)' RNA electrophoresed and blotted from the designated tissues). Positions of RNA ladder are indicated on the right.

PDK may be tightly regulated by metabolites of fatty acid ox- idation.

Relationship of PDK 11 to the Kinase Activator Protein and the PDH Kinase p Subunit-Recently, Priestman et al. (12) reported purification and characterization of rat liver pyruvate dehydrogenase kinase activator protein (KAP), which appears to be a free catalytic subunit of PDK (23, 24). Amino-terminal sequencing of KAP (12) revealed two overlapping sequences: KAP I, KNASLAGAIE; and KAP 11, SLXGAPKY. These se- quences demonstrate remarkable similarity to the sequence of the amino terminus of p45 (KNASLAGAPKYIE) reported in the present study, suggesting that KAF' is probably identical to p45. KAP was purified as a soluble PDK (12,23), whereas p45 was found to be an integral component of the complex (14), indicating that p45 may exist in bound as well as free forms within the mitochondrial matrix space. This observation sug- gests that the translocation of free kinase from the mitochon- drial matrix space to the PDH complex bound form should be investigated as a possible factor regulating the activity state of PDH. Different hormonal and nutritional conditions known to affect the proportion of PDH in its active dephosphorylated

nogenase Kinase

state may affect the distribution of PDK I1 without changing the amount of the expressed kinase.

We are not certain how the p45 subunit of the rat heart PDK purified (14) and now cloned by this laboratory relates to the 45-kDa regulatory P-subunit of PDK purified from bovine kid- ney by Stepp et al. (13). It does not appear that our rat heart preparations of PDK contain the P-subunit described by Stepp et al. (131, unless it has a blocked amino terminus. The proce- dure we used to purify heart PDK is quite different from the one described by Stepp et al. (13). Perhaps only catalytic sub- units of PDK are released from the PDH complex by our pro- cedure. Abetter appreciation of the structureifunction relation- ships of PDK will likely result from successful cloning of the p-subunit of PDK.

Acknowledgment-We thank Patricia A. Jenkins for help in prepa- ration of this manuscript.

REFERENCES 1. Randle, P. J. (1986) Biochem. Soc. Duns. 14,799-806 2. Yeaman, S. J. (1989) Biochem. J. 257,625-632 3. Linn, T. C., Pettit, F. H., and Reed, L. J. (1969) Proc. Natl. Acad. Sci. U. S. A.

4. Linn, T. C., Pettit, F. H., Hucho, F., and Reed, L. J. (1969) Proc. Natl. Acad. Sci.

5. Walsh, D. A,, Cooper, R. H., Denton, R. M., Bridges, B. J., and Randle, P. J.

6. Teague, W. M., Pettit, F. H., Wu, T.-L., Silbennan, S. R., and Reed, L. J. (1982)

7. Pratt, M. L., and Roche, T. E. (1979) J. Biol. Chem. 254,7191-7196 8. Rahmatullah, M., and Roche, T. E. (1985) J. Biol. Chem. 260, 10146-10152 9. Cooper, R. H., Randle, P. J., and Denton, R. M. (1974) Biochem. J. 143,625-641

10. Kerbey, A. L., Radcliffe, P. M., and Randle, P. J. (1977) Biochem. J. 164,

11. Denyer, G. S., Kerbey, A. L., and Randle, P. J. (1986) Biochern. J. 39,347-354 12. Priestman, D. A,, Mistry, S. C., Kerbey, A. L., and Randle, P. J. (1992) FEBS

13. Stepp, L. R., Pettit, F. H., Yeaman, S. J., and Reed, L. J. (1983) J. Biol. Chem.

14. Popov, K. M., Shimomura,Y., and Hams, R.A. (1991)Protein Expression Purif.

15. Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and

16. Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Hams, R. A. (1992)

17. Fatania, H., Lau, K. S., and Randle, P. J. (1981) FEBS Lett. 132,285-288 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A

Laboratory Manual, pp. 8.46-8.47, 17.3-17.41, 18.40-18.41, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

62,234-241

U. S. A. 62,227-234

(1976) Biochem. J. 157,41-67

Biochemistry 21,5585-5592

509-519

Lett. 308, 83-86

258,9454-9458

2,278-286

Hams, R. A. (1993) J. Biol. Chem. 268,26602-26606

J. Biol. Chem. 267, 13129-13130

19. Hoffman, A,, and Roeder, R. G . (1991) Nucleic Acids Res. 19,6337-6338 20. SanEer, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A.

21. Kerbey, A. L., Randle, P. J., Cooper, R. H., Whitehouse, S., Pask, H. T., and

22. Caterson, I. D., Fuller, S. J., and Randle, P. J. (1982) Biochem. J. 208,53-60 23. Mistry, S. C., Priestman, D. A., Kerbey,A. L., and Randle, P. J. (1991) Biochem.

24. Jones, B. S., and Yeaman, S. J. (1991) Biochem. J. 275,781-784

74, 5463-5467

Denton, R. M. (1976) Biochem. J. 154,327-348

J. 275,775-779