Martha J. Kuntz

Serine/threonine protein phosphatases in the control of cell function

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Phosphorylation sites and inactivation of branched-chain α-ketoacid dehydrogenase isolated from rat heart, bovine kidney, and rabbit liver, kidney, heart, brain, and skeletal muscle☆

Branched-chain alpha-ketoacid dehydrogenase complex was isolated from rat heart, bovine kidney, and rabbit liver, heart, kidney, brain, and skeletal muscle. Phosphorylation to approximately 1 mol Pi/mol alpha-subunit of the alpha-ketoacid decarboxylase component was linearly associated with 90-95% inactivation. The complex from some tissues (i.e., from rabbit kidney and heart, and rat heart) showed 30-40% more phosphate incorporation for an additional 5-10% inactivation. Reverse-phase HPLC analysis of tryptic digests of 32P-labeled complexes from all of the above tissues revealed two major (peaks 1 and 2) and one minor (peak 3) phosphopeptide which represent phosphorylation sites 1, 2, and a combination of 1 and 2, respectively. These phosphopeptides, numbered according to the order of elution from reverse-phase HPLC, had the same elution time regardless of the tissue or animal source of the complex. The amino acid sequence of site 1 from rabbit heart branched-chain alpha-ketoacid dehydrogenase was Ile-Gly-His-His-Ser(P)-Thr-Ser-Asp-Asp-Ser-Ser-Ala-Tyr-Arg. Regardless of the source of the complex, both sites were almost equally phosphorylated until total phosphorylation was approximately 1 mol Pi/mol of alpha-subunit and the rate of inactivation was correlated with the rate of total, site 1, or site 2 phosphorylation. Phosphorylation beyond this amount was associated with greater site 2 than site 1 phosphorylation. alpha-Chloroisocaproate, a potent inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase activity, greatly reduced total phosphorylation and inactivation; however, phosphorylation of site 2 was almost abolished and inactivation was directly correlated with phosphorylation of site 1. Thus, the complex isolated from different tissues and mammals had an apparent conservation of amino acid sequence adjacent to the phosphorylation sites. Both sites were phosphorylated to a similar extent temporally although site 1 phosphorylation was directly responsible for inactivation.

Regulation of branched-chain α-ketoacid dehydrogenase complex by covalent modification

Abstract The branched-chain α-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain α-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain α-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain α-ketoacids, α-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain α-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the α-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol α-subunit for 95% inactivation and 1.5 mol P/mol α-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol α-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30–40% additional phosphorylation of the α-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with α-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain α-ketoacid dehydrogenase to oxidize pyruvate ( K m = 0.8 m m , V max = 20% of that of α-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain α-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain α-ketoacid dehydrogenase in chow-fed rats is active (> 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (> 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain α-ketoacid dehydrogenase, i.e. > 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain α-ketoacid dehydrogenase, presumably because of low hepatic levels of branched-chain α-ketoacids, established inhibitors of branched-chain α-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently fasted, inhibition of branched-chain α-ketoacid dehydrogenase kinase by branched-chain α-ketoacids generated from branched-chain amino acid produced by proteolysis of endogenous protein most likely accounts for the greater activity state of the branched-chain α-ketoacid dehydrogenase complex. Hepatocytes isolated from rats fed a chow diet or a low-protein (8%) diet were used to study the activity state and flux through the branched-chain α-ketoacid dehydrogenase complex. Alpha-chloroisocaproate stimulated α-ketoisovalerate decarboxylation with hepatocytes from rats fed a low-protein diet but had no effect with hepatocytes from rats fed chow diet. Activity measurements indicated that branched-chain α-ketoacid dehydrogenase was mainly in the inactive (phosphorylated) state in hepatocytes from low-protein-fed rats but mainly in the active (dephosphorylated) state in hepatocytes from chow-fed rats. Furthermore, α-ketoisocaproate greatly activated ( A 50 = 20 μ m ) α-ketoisovalerate oxidation by hepatocytes isolated from low-protein-fed rats but had no effect with hepatocytes isolated from chow-fed rats. The dietary studies and the hepatocyte experiments, taken together, suggest that portal blood levels of branched-chain α-ketoacids, particularly α-ketoisocaproate,are important determinants of the activity state of the hepatic branched-chain α-ketoacid dehydrogenase complex.

Regulation of the branched-chain α-ketoacid dehydrogenase and elucidation of a molecular basis for maple syrup urine disease

The hepatic branched-chain alpha-ketoacid dehydrogenase complex plays an important role in regulating branched-chain amino acid levels. These compounds are essential for protein synthesis but toxic if present in excess. When dietary protein is deficient, the hepatic enzyme is converted to the inactive, phosphorylated state to conserve branched-chain amino acids for protein synthesis. When dietary protein is excessive, the enzyme is in the active, dephosphorylated state to commit the excess branched-chain amino acids to degradation. Inhibition of protein synthesis by cycloheximide, even when the animal is starving for dietary protein, results in activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex to prevent accumulation of branched-chain amino acids. Likewise, the increase in branched-chain amino acids caused by body wasting during starvation and uncontrolled diabetes is blunted by activation of the hepatic branched-chain alpha-ketoacid dehydrogenase complex. The activity state of the complex is regulated in the short term by the concentration of branched-chain alpha-ketoacids (inhibitors of branched-chain alpha-ketoacid dehydrogenase kinase) and in the long term by alteration in total branched-chain alpha-ketoacid dehydrogenase kinase activity. cDNAs have been cloned and the primary structure of the mature proteins deduced for the E1 alpha subunit of the human and rat liver branched-chain alpha-ketoacid dehydrogenase complex. The cDNA and protein sequences are highly conserved for the two species. Considerable sequence similarity is also apparent between the E1 alpha subunits of the human branched-chain alpha-ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex. Maple syrup urine disease is caused by an inherited deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. The molecular basis of one maple syrup urine disease family has been determined for the first time. The patient was found to be a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father, and an allele which is not expressed from the mother. The only known animal model for the disease (Polled Hereford cattle) has also been characterized. The mutation in these animals introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in premature termination of translation. Two thiamine responsive patients have been studied. The deduced amino acid sequences of the mature E1 alpha subunit and its leader sequence were normal, suggesting that the defect in these patients must exist in some other subunit of the complex. 3-Hydroxyisobutyrate dehydrogenase and methylmalonate-semialdehyde dehydrogenase, two enzymes of the valine catabolic pathway, were purified from liver tissue and characterized.(ABSTRACT TRUNCATED AT 400 WORDS)

Monovalent cations and inorganic phosphate alter branched-chain α-ketoacid dehydrogenase—Kinase activity and inhibitor sensitivity☆

Potassium ion protects the branched-chain alpha-ketoacid dehydrogenase complex against inactivation by thermal denaturation and protease digestion. Rubidium was effective but sodium and lithium were not, suggesting that the ionic size of the cation is important for stabilization of the enzyme. Thiamine pyrophosphate stabilization of the complex [Danner, D. J., Lemmon, S. K., and Elsas, S. J. (1980) Arch. Biochem. Biophys. 202, 23-28] was found dependent on the presence of potassium ion. Studies with resolved components indicate that the thiamine pyrophosphate-dependent enzyme of the complex, i.e., the 2-oxoisovalerate dehydrogenase (lipoamide) (EC 1.2.4.4), is the component stabilized by potassium ion. Branched-chain alpha-ketoacid dehydrogenase-kinase activity measured by inactivation of the branched-chain alpha-ketoacid dehydrogenase complex was maximized at a potassium ion concentration of 100 mM. Stimulation of kinase activity was also found with rubidium ion but not with lithium and sodium ions. All salts tested increased the efficiency of inactivation by phosphorylation, i.e., decreased the degree of enzyme phosphorylation required to cause inactivation of the complex. The effectiveness and efficacy of alpha-chloroisocaproate as an inhibitor of branched-chain alpha-ketoacid dehydrogenase kinase were enhanced by the presence of monovalent cations, and further increased by inorganic phosphate. These findings suggest that monovalent cations and anions, particularly potassium and phosphate, cause structural changes in the dehydrogenase-kinase complex that alter its susceptibility to phosphorylation and responsiveness to kinase inhibitors.

Enzymatic determination of the branched-chain α-keto acids

A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.

Molecular cloning of the E1β subunit of the rat branched chain α-ketoacid dehydrogenase

Abstract The rat liver E1β subunit of the branched-chain α-ketoacid dehydrogenase complex has been cloned and sequenced. The amino acid sequence is highly conserved between rat, human and bovine forms of the protein. The β subunits of the branched-chain α-ketoacid dehydrogenase complex from rat and Pseudomonas putida, and the pyruvate dehydrogenase complex from human and Bacillus subtilis show sequence similarities which are shared with two other thiamine pyrophosphate-dependent enzymes, yeast (Hansenula polymorpha) formaldehyde transketolase and human transketolase. These similarities suggest that the β subunits may be involved in thiamine pyrophosphate binding.

cDNA Cloning of the E1α Subunit of the Branshed-Chain α-Keto Acid Dehydrogenase and Elucidation of a Molecular Basis for Maple Syrup Urine Diseasea

We have cloned cDNAs encoding human and rat liver BCKDH E1 alpha subunits and deduced the primary structure of the mature protein. The sequences of the cDNA and protein are highly conserved between the two species. Significant sequence similarity has also been found between human BCKDH and PDH E1 alpha subunits. We have studied the molecular basis of MSUD by determining the enzyme activity and levels of BCKDH protein and mRNA, and by enzymatic amplification and sequencing of BCKDH E1 alpha-specific mRNA, from an MSUD patient and his parents. Different mutant alleles were identified in the two parents. The patient was a compound heterozygote, inheriting an allele encoding an abnormal E1 alpha from the father and an allele containing a defect in regulation from the mother. Our results demonstrate that a case of MSUD was caused by structural and regulatory mutations involving the E1 alpha subunit.