(Received for publication, June 12, 1995; and in revised form, September 12, 1995)
From the
Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Gudi, R., and Harris, R. A.(1994) J. Biol. Chem. 269, 29720-29724). The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family. Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans. In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively. PDK3 cDNA expressed in Escherichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase. PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins.
Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis. The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung. Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver). The tissue distribution of PDK1 mRNA differs markedly from PDK2. The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney). In contrast to PDK1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues. In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.
Mitochondrial multienzyme complex, pyruvate dehydrogenase (PDH), ()catalyzes the oxidative decarboxylation of pyruvate:
pyruvate + CoA + NAD
acetyl-CoA
+ NADH + H
+ CO
and is one
of the major enzymes responsible for the regulation of homeostasis of
carbohydrate fuels in mammals (for review, see (1) ). The
enzymatic activity of PDH is controlled by a
phosphorylation/dephosphorylation
cycle(2, 3, 4, 5) . The
phosphorylation of PDH that leads to the complete inactivation of its
enzymatic activity is catalyzed by a highly specific pyruvate
dehydrogenase kinase (PDK)(6, 7) . The
dephosphorylation with concomitant reactivation is catalyzed by
pyruvate dehydrogenase phosphatase(8, 9) . It is
generally believed that the phosphorylation state of PDH in
mitochondria is determined by the activity of its intrinsic kinase,
which itself is regulated by the products and substrates of the
dehydrogenase reaction(10, 11, 12) . The
products stimulate the kinase activity, whereas the substrates are
inhibitory. The kinase activity is also inhibited by ADP (competitive
with ATP) that acts synergistically with pyruvate(13) .
Therefore, in mitochondria, the steady state activity of the kinase
should depend on intramitochondrial ratios of CoA/acetyl-CoA,
NAD
/NADH, and ADP/ATP, as well as on the
intramitochondrial concentration of pyruvate.
Recent evidence from this laboratory indicates that, at least in rodents, there are two isoenzymic forms of PDK sharing up to 70% of amino acid identity(14) . These isoenzymes of PDK have been designated as PDK1 and PDK2. Both isoenzymes, obtained as the recombinant proteins, were found able to catalyze the phosphorylation and inactivation of PDH(14, 15) . The tissue distribution of isoenzymes appeared to be quite different. By Northern blot analysis, the isoenzyme PDK1 was found to be expressed predominantly in cardiac muscle. The isoenzyme PDK2, in contrast, is highly expressed in all tissues tested thus far, therefore suggesting that PDK2 may be responsible for regulation of the enzymatic activity of PDH complex in most of the tissues(14) . Consistent with this idea, some recent immunological evidence indicates that PDK2 corresponds to one of the subunits of PDK from bovine kidney mitochondria (16) and also corresponds to the free catalytic subunit of PDK identified by Randle's laboratory in rat liver mitochondria(14, 17) .
The physiological significance for existence of isoenzymes of PDK has not been established. Moreover, to date, it has not been shown whether isoenzymes of PDK exist in other mammalian species besides rodents. In the present study, we further explored the diversity of the PDK gene family. Here we report the data on the primary structures of three isoenzymic forms of PDK found in humans, one of which is a new isoenzyme of PDK, along with data on their tissue distribution and catalytic activities.
In order to obtain the full-length cDNAs encoding for human isoenzymes homologous to rat PDK1 and PDK2, human liver cDNA library was screened with corresponding rat cDNAs. Two positive clones were obtained during screening with PDK1 cDNA. One of the clones contained a cDNA of 1,593 bp that encodes for the full-length protein product with a molecular mass of 49,244 Da corresponding to the human PDK1. Screening of human liver cDNA library with cDNA of rat PDK2 yielded eight positive clones. Six of them appeared to be partial clones, whereas two contained cDNAs of 1,330 and 1,422 bp that encode a full-length protein with a molecular mass of 46,181 Da homologous to the rat PDK2.
In order to clone a
full-length cDNA of PDK3, human liver cDNA library was screened with
the unique partial cDNA obtained by PCR as discussed above. Only one
positive clone was found after screening of approximately 1.0
10
plaque-forming units, indicating that this cDNA is
under-represented in the liver library. The analysis of the nucleotide
sequence of the respective cDNA (914 bp long) revealed that it encodes
for the carboxyl terminus of PDK3. The 5`-end of PDK3 cDNA was obtained
by using a 5`-RACE protocol. Amplification of human heart templates
with primers described under ``Experimental Procedures'' gave
rise to an 821-bp-long PCR product that encodes for the complete
5`-coding region of PDK3 cDNA, as well as for the 5`-noncoding region.
Further attempts to amplify the 5`-noncoding region of PDK3 cDNA with
primers located near the 5`-end failed to produce products longer than
expected from the sequence of 821-bp cDNA, suggesting that it covers
almost the entire 5`-noncoding region. The resulting 1599-bp-long
composite cDNA for PDK3 was constructed by aligning the 821-bp 5`-RACE
product with the 914-bp cDNA obtained during library screening. It
encodes for a protein with molecular mass of 46,938 Da.
Figure 1:
Comparison of the
predicted amino acid sequences of mitochondrial protein kinases. HPDK1, HPDK2, HPDK3, RPDK1, and RPDK2 stand for the respective isoenzymes of human or rat
pyruvate dehydrogenase kinase; ZK370.5, hypothetical protein
ZK370.5 from C. elegans; BCKDK, rat branched chain
-keto acid dehydrogenase kinase; YIL 042C, hypothetical
protein YIL 042C from yeast. The hypothetical phosphoprotein 3 (HPP3)
from T. brucei lacking the subdomain IV and the last glycine
of subdomain III were not included in the alignment. Multiple sequence
alignment was made according to the method described by Higgins and
Sharp(25, 26) . Conserved amino acid residues are
shown by reverse text, amino acids defining the subdomains I
through V are shown by asterisks.
Five regions of an extremely high
conservation that presumably define the putative catalytic domain of
mitochondrial protein kinases were identified in previous
studies(14, 15, 21) : subdomain I defined by
an invariant histidine residue (His, numbering here and
below is in accord with the mature sequence of rat PDK2); subdomain II
defined by an invariant asparagine residue (Asn
);
subdomain III corresponding to the first glycine rich loop
Asp
-X-Gly
-X-Gly
;
subdomain IV defined by aromatic residue (Tyr
); and
subdomain V corresponding to the second glycine rich loop
Gly
-X-Gly
-X-Gly
-Lys
-Pro
.
As expected from these observations, the sequences corresponding to
subdomains I, IV, and V are perfectly conserved among all isoenzymes of
human PDK (Fig. 1). The minor differences were found within the
sequences of subdomains II and III. It appeared that the alanine of
subdomain II (-KNAMRAT-), as well as the arginine of subdomain III
(-DRGGG-) of PDK3, are substituted to serine (-KNSMRAT-) and leucine
(-DLGGG-), respectively. The lower level of conservation was observed
only in rather short stretches of spacers connecting subdomains II and
III, as well as subdomains IV and V (positions 263-276 and
315-321 of human PDK2, respectively). Surprisingly, an extremely
high degree of conservation (up to 81%) was found within the amino
termini of human isoenzymes of PDK (positions 26-73 of human
PDK2), in spite of the fact that the overall level of conservation
within this region of mitochondrial protein kinases is fairly
low(15) . Presumably, it indicates that the amino terminus
serves some specialized function characteristic of PDK, such as docking
of the kinase to the complex. The greatest degree of divergency between
the isoenzymes was found within the sequences immediately flanking
subdomain I (positions 74-119 of PDK2). Taking into account that
the invariant histidine residue of subdomain I (His
of
PDK2) may serve as a catalyst of the phosphotransfer
reaction(21) , it seems reasonable to suggest that the
differences within sequences flanking subdomain I may be directly
responsible for the unique catalytic properties of the isoenzymes of
PDK.
The structural motifs of mitochondrial protein
kinases were identified based on the analysis of the amino acid
sequences deduced from the nucleotide sequences of five genes available
at that time: rat PDK1 and PDK2, rat branched chain -keto acid
dehydrogenase kinase, hypothetical phosphoprotein 3 (HPP3) from Trypanosoma brucei and ZK370.5 from Caenorhabditis elegans (22, 23). However, with four more sequences available now for
comparison (human isoenzymes of PDK and ORF of hypothetical protein
YIL042C from yeast, GenBank
accession number Z47047),
additional structural motifs of the mitochondrial protein kinases
become apparent (Fig. 1). The first motif defined by consensus
sequence
Ala
-X-Gly
-(Val/Lys)-X-Glu
is localized immediately downstream from subdomain I. The second motif,
corresponding to consensus
Arg
-(Ile/Leu)-X-(Ile/Met)-(Arg/Lys)-(Met/Lys)-Leu
,
is positioned in the middle of the spacer connecting the subdomains I
and II. Two other consensus sequences
Tyr
-(Ala/Leu)-X-(Tyr/Leu)-(Phe/Leu)-X-Gly
and
Gly
-X-Gly
-Thr
-Asp
,
are situated within the carboxyl terminus of the kinase molecule and
flank the catalytic domain on its carboxyl terminus. It is generally
believed that regions of high conservation are important for catalytic
function, either directly as components of the active site or
indirectly by contributing structurally to the formation of the active
site. The functional significance of subdomains of mitochondrial
protein kinases identified through the comparison of their primary
structures will be determined during future structure-functional
analysis.
To facilitate the purification of recombinant PDK1, PDK2,
and PDK3, stretches of six consecutive histidine residues
(HisTag) were constructed at the amino termini of the respective
proteins. Corresponding His
Tagged kinases were purified from E.
coli extracts in one step by using metal chelate chromatography. The
recombinant kinases, purified in this way, were found to be more than
90% pure as judged by SDS-polyacrylamide gel electrophoresis analysis (Fig. 2). The electrophoretic mobility of purified PDK1 was
found to be somewhat slower than that of PDK2, whereas PDK3 had
electrophoretic mobility comparable to that of PDK2 in good agreement
with previous estimates of the molecular weights of the respective
isoenzymes based on the deduced amino acid sequences (in this
manuscript and in (14) and (15) ). Preparations of
isoenzyme PDK1 appear to consist of several closely positioned bands on
SDS gel (Fig. 2). This microheterogeneity was found to be due to
phosphorylation occurring during expression. (
)
Figure 2:
SDS-PAGE analysis of the recombinant
isoenzymes of pyruvate dehydrogenase kinase purified by metal chelate
chromatography. Lane 1 contains molecular size markers
(Bio-Rad): lysozyme (18.1 kDa), soybean trypsin inhibitor (26.9 kDa),
carbonic anhydrase (36.0 kDa), ovalbumin (45.0 kDa), bovine serum
albumin (45.0 kDa), and -galactosidase (112.0 kDa). Lanes
2-4 contain approximately 1 µg of the respective PDK
isoenzyme.
The
recombinant isoenzymes of PDK expressed and purified in the present
study appeared to have enzymatic activity toward the kinase-depleted
PDH complex (Fig. 3). Based on an ATP-dependent inactivation
assay that measures the rate of ATP-dependent inactivation of PDH
activity due to phosphorylation, PDK3 was found to be the most active
isoenzyme. The enzymatic activity of PDK1 was approximately
30-50% lower than that of PDK3 in three independent experiments.
The second isoform of PDK had the lowest basal activity of all three
isoenzymes. However, it is generally believed that in mitochondria the
rate of kinase activity depends upon the relative concentrations of
products and substrates of the dehydrogenase reaction. The products,
NADH and acetyl-CoA, activate PDK, whereas the substrates, pyruvate,
NAD, and CoA inhibit the kinase(1) .
Therefore, direct comparison of the basal activities of isoenzymes of
PDK may be somewhat misleading. Accordingly, it was found that the
enzymatic activity of isoenzyme PDK2, when it is tested in the presence
of high ratios of NADH/NAD
and acetyl-CoA/CoA,
increases almost five times and approaches the activity of isoenzyme
PDK1,
thus indicating that isoenzymes of PDK are different
not only in terms of their basal activities, but also in terms of
regulation of their enzymatic activities by the compounds known to
affect kinase activity in vivo.
Figure 3:
Time courses of the ATP-dependent
inactivation of kinase-depleted PDH complex reconstituted with
different isoenzymes of pyruvate dehydrogenase kinase. 40 milliunits of
kinase-depleted PDH was reconstituted with 0.5 µg of the
recombinant PDK1 (), PDK2 (
), or PDK3 (
),
respectively, in 570 µl of the phosphorylation mixture containing
20 mM Tris
HCl (pH 7.4), 5 mM MgCl
,
50 mM KCl, 2 mM dithiothreitol, and 0.1 mg/ml bovine
serum albumin. Phosphorylation reactions were initiated with ATP (final
concentration 100 µM) after a 5-min preincubation at room
temperature. Aliquots of the phosphorylation mixture were withdrawn at
indicated times for determination of the PDH residual activity. Each
point represents an average from three independent expression
experiments. Under comparable conditions of incubation, the
kinase-depleted PDH complex itself did not show any
inactivation.
Figure 4:
Northern blot analysis of tissue
distribution of the isoenzymes of human pyruvate dehydrogenase kinase.
Each line contains approximately 2 µg of purified mRNA. Blots were
probed with random-primed P-labeled cDNAs of PDK1 (upper panel), PDK2 (middle panel), and PDK3 (lower panel).
The existence of
multiple isoenzymes of PDK in mammalian tissues suggests that the
differences in the properties of kinase reported in previous studies
may reflect the repertoire of isoenzymes of PDK expressed in a
particular tissue being studied. For example, the isoenzymic
composition of PDK in heart and liver is different (see Fig. 4of this manuscript, as well as (14) ).
Accordingly, in heart muscle, -oxidation of fatty acids is
necessary for the effect of starvation on the level of the enzymatic
activity of PDH complex and may be reversed easily by
tetradecylglycidate, an inhibitor of
-oxidation(24) . In
contrast, in liver, tetradecylglycidate does not reverse the effect of
starvation on the percent of active PDH complex (24) . These
observations are suggestive that the different isoenzymes expressed in
heart and liver determine the mechanism of the adaptive response of PDK
during starvation. In this respect, further analysis of the enzymatic
and regulatory properties of the isoenzymes of PDK will bring new
insight with respect to the fine control mechanisms that regulate the
phosphorylation and activity states of PDH complex in mammalian
tissues.
At least at the level of mRNAs, rat PDK1 and PDK2 appear to have a similar tissue distribution and relative abundance as the mRNAs of the homologous isoenzymes in humans(14) . This makes the rodent model pertinent to humans and therefore important for studies on regulation of PDH activity in different pathologic states like diabetes, cardiac myopathy, sepsis, and cancer. It remains to be established, however, whether isoenzyme PDK3 exists in rodents.
Figure 5: Phylogenetic tree of mitochondrial protein kinases. The individual protein kinases are indicated by abbreviated names used in the legend to the Fig. 1. The phylogenetic tree was built from the pairwise similarity scores obtained during multiple sequence alignment according to Higgins and Sharp (25, 26) by using average linkage cluster analysis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L42450[GenBank], L42451[GenBank], and L42452[GenBank].