Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase.

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-A crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic alpha-helices, whereas the C-terminal half is folded into an alpha/beta sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.

Gene structure and expression of the targeting subunit, RGL, of the muscle-specific glycogen-associated type 1 protein phosphatase, PP1G.

The type I phosphatase associated with glycogen, PP1G, plays an important role in glycogen metabolism. PP1G is targeted to glycogen by the R(GL) subunit, which regulates the function of the enzyme. We report the cloning and characterization of the gene as well as the pattern of expression of the R(GL) subunit from mouse. The gene covers more than 37 kb, is composed of four exons and three introns, and codes for a 1089 residue polypeptide with a calculated molecular weight of 121,000. The amino acid sequence has 60% identity with the human and rabbit R(GL). The 5' flanking region of the gene contains a TATA box, c-Myc sites, and a potential cAMP-responsive element. Muscle specific motifs, such as MyoD and MEF-2, were also found. The A-T rich 3'-UTR contained several polyadenylation signals, two associated with poly(A) down-stream consensus motifs. ARE elements, which regulate mRNA stability, were dispersed throughout the 3'-UTR. Northern analysis of poly(A) mRNA from various murine tissues indicates a major transcript of 7.5 kb in skeletal muscle and heart. Western analysis demonstrates that R(GL) protein is present in skeletal and cardiac muscle from mouse, rat, and rabbit but not in L6 myoblasts, L6 myotubes, 3T3 L1 fibroblasts, 3T3 L1 or rat primary adipocytes, confirming that expression of the gene is specific to striated muscle. Analysis of skeletal muscle from rats made diabetic by streptozotocin treatment reveals that the level of R(GL) protein is the same as in control animals, indicating that expression is not regulated by insulin.

Mechanism responsible for inactivation of skeletal muscle pyruvate dehydrogenase complex in starvation and diabetes.

Regulation of the activity of the pyruvate dehydrogenase complex in skeletal muscle plays an important role in fuel selection and glucose homeostasis. Activation of the complex promotes disposal of glucose, whereas inactivation conserves substrates for hepatic glucose production. Starvation and diabetes induce a stable increase in pyruvate dehydrogenase kinase activity in skeletal muscle mitochondria that promotes phosphorylation and inactivation of the complex. The present study shows that these metabolic conditions induce a large increase in the expression of PDK4, one of four pyruvate dehydrogenase kinase isoenzymes expressed in mammalian tissues, in the mitochondria of gastrocnemius muscle. Refeeding starved rats and insulin treatment of diabetic rats decreased pyruvate dehydrogenase kinase activity and also reversed the increase in PDK4 protein in gastrocnemius muscle mitochondria. Starvation and diabetes also increased the abundance of PDK4 mRNA in gastrocnemius muscle, and refeeding and insulin treatment again reversed the effects of starvation and diabetes. These findings suggest that an increase in amount of this enzyme contributes to hyperphosphorylation and inactivation of the pyruvate dehydrogenase complex in these metabolic conditions. It was further found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, and PDK4 mRNA in gastrocnemius muscle. Since long-chain fatty acids activate PPAR-alpha endogenously, increased levels of these compounds in starvation and diabetes may signal increased expression of PDK4 in skeletal muscle.

Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex.

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50nmol/min per mg for PDK2 to 1250nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

Dihydrolipoamide dehydrogenase-binding protein of the human pyruvate dehydrogenase complex. DNA-derived amino acid sequence, expression, and reconstitution of the pyruvate dehydrogenase complex.

Protein X, recently renamed dihydrolipoamide dehydrogenase-binding protein (E3BP), is required for anchoring dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide transacetylase (E2) core of the pyruvate dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E3BP of the human pyruvate dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E2 component of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E2, 38% identity in its central region with the putative peripheral subunit-binding domain of E2, and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E2. The similarity in the latter domain stands in contrast to E3BP of Saccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E3BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E3BP and E2 resulted in the formation of an E2.E3BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E3 and recombinant pyruvate decarboxylase (E1).

Diversity of the pyruvate dehydrogenase kinase gene family in humans.

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 Eschierichia 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.