Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.2 (PDK1)
2,238 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The highly purfied pyruvate dehydrogenase complex (EC 1.2.4.1) and uncomplexed pyruvate dehydrogenase from bovine kidney and heart mitochondria were phosphorylated and inactivated with pyruvate dehydrogenase kinase and [gamma-32P]ATP. Tryptic digestion of the phosphorylated pyruvate dehydrogenase yielded three phosphopeptides, a mono- (site 1) and a di- (sites 1 and 2) phosphorylated tetradecapeptide and a monophosphorylated nonapeptide (site 3). The amino acid sequences of the three phosphopeptides were established to be Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Tyr-Arg, and Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg. Phosphorylation proceeded markedly faster at site 1 than at sites 2 and 3, and phosphorylation at site 1 correlated closely with inactivation of pyruvate dehydrogenase. Complete inactivation of pyruvate dehydrogenase was associated with incorporation at site 1 of 1.0--1.6 mol of phosphoryl groups per mol of enzyme. Since pyruvate dehydrogenase is a tetramer (alpha2beta2) and since phosphorylation occurs only on the alpha subunit, the possibility of half-site reactivity is considered.
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PMID:Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. 67 13

Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of alpha-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site 1 was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the alpha-pyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono- and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.
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PMID:Phosphorylation and inactivation of the pyruvate dehydrogenase from the anaerobic parasitic nematode, Ascaris suum. Stoichiometry and amino acid sequence around the phosphorylation sites. 319 13

The pyruvate dehydrogenase complex was purified to homogeneity from bakers' yeast (Saccharomyces cerevisiae). No pyruvate dehydrogenase kinase activity was detected at any stage of the purification. However, the purified pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. The protein-bound radioactivity was localized in the pyruvate dehydrogenase alpha subunit. The phosphorylated, inactive pyruvate dehydrogenase complex was dephosphorylated and reactivated with purified pyruvate dehydrogenase phosphatase from bovine heart. Tryptic digestion of the 32P-labeled complex yielded a single phosphopeptide, which was purified to homogeneity. The sequence of the phosphopeptide was established to be Tyr-Gly-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Thr-Tyr-Arg. This sequence is very similar to the sequence of a tryptic phosphotetradecapeptide derived from the alpha subunit of bovine kidney and heart pyruvate dehydrogenase: Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser-Tyr-Arg.
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PMID:Phosphorylation-dephosphorylation of pyruvate dehydrogenase from bakers' yeast. 353 83

Tyrphostins inhibit tyrosine kinases and have little effect on the activity of serine/threonine kinases. Pyruvate dehydrogenase kinase inactivates pyruvate dehydrogenase by phosphorylating serine residues within the multienzyme complex. This serine/theronine kinase represents a new family of protein kinases, and one (tyrphostin 47) of two tyrphostins tested appeared to activate the pyruvate dehydrogenase kinase as determined by [1-14C]-lactate oxidation to 14CO2. Experiments designed to determine if the tyrphostins altered pyruvate dehydrogenase activity in mitochondria prepared from rat epididymal adipocytes using [1-14C]-pyruvate as the substrate demonstrated a dose dependent increase in enzyme activity in the presence of tyrphostin 47, but not in tyrphostin 23. This apparent stimulation of pyruvate dehydrogenase activity was attributed to tyrphostin 47's ability to nonenzymatically decarboxylate [1-14C]-pyruvate, the substrate for the pyruvate dehydrogenase assay. Neither tyrphostin directly altered pyruvate dehydrogenase kinase activity. Therefore, assays utilizing [1-14C]-pyruvate and tyrphostin 47 are subject to analytical interference.
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PMID:Tyrphostin 47 nonenzymatically decarboxylates [1-14C]-pyruvate. 781 37

The family of protein kinases includes many oncogenes and growth-factor receptors, as well as genes that are involved in cell-cycle regulation. We have identified protein kinases expressed in a human breast-cancer cell line, 600PEI, and a primary human breast carcinoma, using PCR cloning techniques based on consensus sequences in the kinase domain. Twenty-five different protein kinases were isolated, including 3 novel putative tyrosine kinases (designated TK1, TK2, and TK5), and 2 novel putative cell-cycle-associated serine/threonine kinases (designated STK1 and STK2). TK1 is a new member of the src family of kinases that is expressed predominantly in epithelial cells. TK2 is homologous to the receptor kinase, HEK, and TK5 appears to be another member of the JAK family of kinases. The novel serine/threonine kinases, designated STK1 and STK2, were homologous to the human cdc2 and the Aspergillus nimA genes. We subsequently analyzed the levels of expression of all of these protein kinases in a panel of human breast carcinomas, using PCR-based methods. This analysis revealed different expression profiles in different primary breast carcinomas and, therefore, may determine new molecular sub-sets of human breast cancer.
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PMID:Novel protein kinases expressed in human breast cancer. 809

Using polymerase chain reaction (PCR)-based methods, we have isolated cDNA clones of two new members of serine/threonine kinases, STK1 and STK2, from a cDNA library constructed from the BT-20 human breast cancer cell line. STK1 is transcribed as a 1.4 kilobase (kb) mRNA encoding for a protein of 346 amino acids. Based on amino acid sequence analysis, STK1 is 86% identical to the Xenopus p40mo15, a cdc2-related serine/threonine kinase recently found to be the activating kinase for p34cdc2 and p33cdk2. Thus, STK1 is most likely the human homologue of MO15. An alternatively spliced STK1 message expressed variably in cell lines and in primary carcinomas generates a predicted 58 amino acid protein that lacks the kinase domain. STK2 is transcribed into a 4.0 kb mRNA encoding for an 841 residue protein which exhibits 50% identity in the kinase domain with the mouse nek1 gene product, the relative of the fungal G2-M regulator, nimA. STK1 and STK2 display a variable pattern of expression among a series of primary carcinomas as well as cancer cell lines. Both STK1 and STK2 were expressed at the highest levels in the heart but were also detected in all other organs tested. In embryonal tissues, lower levels of expression were noted. Using cell cycle inhibitors, we have shown that both STK1 and STK2 mRNA levels remain relatively invariant through the cell cycle. Chromosomal assignment has localized STK1 on chromosome 2pcen-2p15, a region implicated in hereditary non-polyposis colorectal carcinoma, and STK2 on chromosome 3p21.1, a region frequently showing chromosomal alterations in renal cells carcinomas.
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PMID:Two novel human serine/threonine kinases with homologies to the cell cycle regulating Xenopus MO15, and NIMA kinases: cloning and characterization of their expression pattern. 820 44

We recently reported molecular cloning of the branched chain alpha-ketoacid dehydrogenase kinase, the first mitochondrial protein kinase to be cloned (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). From a search for proteins related to the branched chain alpha-ketoacid dehydrogenase kinase, a cDNA encoding the 434 amino acid residues corresponding to pyruvate dehydrogenase kinase has been cloned from a rat heart cDNA library. Evidence that the clone codes for pyruvate dehydrogenase kinase includes: (a) the deduced amino acid sequence is identical to the partial sequence of the kinase determined by direct sequencing; (b) expression of the cDNA in Escherichia coli resulted in synthesis of a protein that phosphorylated and inactivated the pyruvate dehydrogenase complex; (c) kinase activity of the recombinant protein is sensitive to inhibition by a specific inhibitor of pyruvate dehydrogenase kinase; and (d) antiserum raised against the recombinant protein recognized the protein subunit known to correspond to pyruvate dehydrogenase kinase in a highly purified preparation of the pyruvate dehydrogenase complex. Like the branched chain alpha-ketoacid dehydrogenase kinase, pyruvate dehydrogenase kinase lacks motifs usually associated with eukaryotic Ser/Thr-protein kinases. Considerable sequence similarity exists between these mitochondrial protein kinases and members of the prokaryotic histidine kinase family, a diverse set of sensing and response systems important in the regulation of bacterial processes. Thus, molecular cloning of these proteins establishes a new eukaryotic family of protein kinases that is related to a prokaryotic family of protein kinases.
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PMID:Primary structure of pyruvate dehydrogenase kinase establishes a new family of eukaryotic protein kinases. 825 90

Eukaryotic polyamine transport systems have not yet been characterized at the molecular level. We have used transposon mutagenesis to identify genes controlling polyamine transport in Saccharomyces cerevisiae. A haploid yeast strain was transformed with a genomic minitransposon- and lacZ-tagged library, and positive clones were selected for growth resistance to methylglyoxal bis(guanylhydrazone) (MGBG), a toxic polyamine analog. A 747-bp DNA fragment adjacent to the lacZ fusion gene rescued from one MGBG-resistant clone mapped to chromosome X within the coding region of a putative Ser/Thr protein kinase gene of previously unknown function (YJR059w, or STK2). A 304-amino-acid stretch comprising 11 of the 12 catalytic subdomains of Stk2p is approximately 83% homologous to the putative Pot1p/Kkt8p (Stk1p) protein kinase, a recently described activator of low-affinity spermine uptake in yeast. Saturable spermidine transport in stk2::lacZ mutants had an approximately fivefold-lower affinity and twofold-lower Vmax than in the parental strain. Transformation of stk2::lacZ cells with the STK2 gene cloned into a single-copy expression vector restored spermidine transport to wild-type levels. Single mutants lacking the catalytic kinase subdomains of STK1 exhibited normal parameters for the initial rate of spermidine transport but showed a time-dependent decrease in total polyamine accumulation and a low-level resistance to toxic polyamine analogs. Spermidine transport was repressed by prior incubation with exogenous spermidine. Exogenous polyamine deprivation also derepressed residual spermidine transport in stk2::lacZ mutants, but simultaneous disruption of STK1 and STK2 virtually abolished high-affinity spermidine transport under both repressed and derepressed conditions. On the other hand, putrescine uptake was also deficient in stk2::lacZ mutants but was not repressed by exogenous spermidine. Interestingly, stk2::lacZ mutants showed increased growth resistance to Li+ and Na+, suggesting a regulatory relationship between polyamine and monovalent inorganic cation transport. These results indicate that the putative STK2 Ser/Thr kinase gene is an essential determinant of high-affinity polyamine transport in yeast whereas its close homolog STK1 mostly affects a lower-affinity, low-capacity polyamine transport activity.
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PMID:The STK2 gene, which encodes a putative Ser/Thr protein kinase, is required for high-affinity spermidine transport in Saccharomyces cerevisiae. 915 97

293 cells were transfected with wild-type GSK3beta (WT-GSK3beta) or a mutant in which the PKB phosphorylation site (Ser-9) was altered to Ala (A9-GSK3beta). Upon stimulation with IGF-1 or insulin, WT-GSK3beta was inhibited 75% or 60%, respectively, whereas the activity of the A9-GSK3beta mutant was unaffected. Incubation of WT-GSK3beta with PP2A1 (a Ser/Thr-specific phosphatase) completely reversed the IGF-1- or insulin-induced inhibition. IGF-1 stimulation did not induce any tyrosine dephosphorylation of WT-GSK3beta or A9-GSK3beta. Coexpression of WT-GSK3beta in 293 cells with either PKB alpha (also known as AKT) or PDK1 (the 'upstream' activator of PKB) mimicked the IGF-1- or insulin-induced phosphorylation of Ser-9 and inactivation of GSK3beta.
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PMID:Further evidence that the inhibition of glycogen synthase kinase-3beta by IGF-1 is mediated by PDK1/PKB-induced phosphorylation of Ser-9 and not by dephosphorylation of Tyr-216. 937 75

Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.
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PMID:Phosphorylation and activation of p70s6k by PDK1. 947 28


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