Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase

Klumpp, David J.; Plank, David W.; Bowdin, Linda; Stueland, C S; Chung, Taeowan; LaPorte, David C.

doi:10.1128/jb.170.6.2763-2769.1988

Cited by 49 publications

(25 citation statements)

References 55 publications

(44 reference statements)

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“…The phosphorylation and dephosphorylation of IDH are catalyzed by a bifunctional protein, IDH kinase/phosphatase, expressed from the aceK gene (5,9,10). The IDH kinase and IDH phosphatase reactions appear to occur at the same active site, a conclusion supported by the observation that a mutation at the consensus ATP binding site eliminates both activities (18).…”

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confidence: 59%

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity

1992

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Mutations in aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase, which selectively inhibit phosphatase activity have been isolated. These mutations yield amino acid substitutions within a 113-residue region of this 578-residue protein. These mutations may define a regulatory domain of this protein.In Escherichia coli, the Krebs cycle enzyme isocitrate dehydrogenase (IDH) is regulated by reversible phosphorylation (1, 3). The phosphorylation of IDH controls the partitioning of isocitrate between the Krebs cycle and the glyoxylate bypass. The flow of isocitrate through this bypass is essential for growth on acetate, since it avoids the quantitative loss of the acetate carbons as CO2 in the Krebs cycle (6, 7). During growth on acetate, ca. 70% of the IDH is maintained in the inactive, phosphorylated form, forcing isocitrate through the glyoxylate bypass (2,8,11,13, 14). In most genetic backgrounds, phosphorylation of IDH appears to be essential for use of the glyoxylate bypass, since mutations which eliminate IDH kinase prevent growth on acetate (13).The phosphorylation and dephosphorylation of IDH are catalyzed by a bifunctional protein, IDH kinase/phosphatase, expressed from the aceK gene (5, 9, 10). The IDH kinase and IDH phosphatase reactions appear to occur at the same active site, a conclusion supported by the observation that a mutation at the consensus ATP binding site eliminates both activities (18).One of the approaches which we have taken to examine the structure of IDH kinase/phosphatase has been the random mutagenesis of aceK, the gene which encodes this protein. Our strategy has been to screen for alleles which have lost the ability to complement a null mutation in aceK and then assay the products of these candidate alleles for IDH kinase and IDH phosphatase activities. In a previous report (4), we described the isolation of two alleles of aceK (aceK3 and aceK4) whose products retain IDH kinase activity but have suffered drastic reductions in their IDH phosphatase activities. We have subsequently isolated three additional alleles whose products have similar defects (Table 1). Four of these alleles (aceK3, aceK4, aceKS, and aceK7) were generated by replication of a plasmid bearing aceK+ in the mutator strain W3550 (mutD5) (15). To increase the range of possible mutations, we have also employed chemical mutagenesis with hydroxylamine (16). This method resulted in the generation of an additional allele whose product has selectively lost IDH phosphatase activity, aceK6. In order to ensure that each allele had resulted from a single point mutation, we employed relatively mild conditions for mutagenesis. Replication in strain W3550 yielded 0.3% noncomplementing alleles, while treatment with hy-* Corresponding author. droxylamine generated these alleles in only 0.1% of transformants.The locations of the mutations were approximated by testing the abilities of fragments of aceK+ to direct repair of these mutations (17). In this procedure, each mapping plasmid (which carries a fragment of aceK...

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confidence: 59%

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity

1992

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“…In other words, are all phosphorylation-based signalling systems assembled out of the same basic suite of protein kinase and protein phosphatase prototypes? Yet one exception also exists, the isocitrate dehydrogenase kinase/phosphatase, which has been found solely in bacteria and whose protein kinase domain reflects neither of the aforementioned paradigms (22).…”

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Fancy meeting you here! A fresh look at "prokaryotic" protein phosphorylation

1996

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Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig. 1). As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific. Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes. Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought. Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc. The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.

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“…However, computer analysis of the A6 sequence failed to reveal significant matches with these atypical kinases such as BCR (21), HBx, the hepatitis B virus transactivator protein (28), and AceK, an isocitrate dehydrogenase kinase/phosphatase from Escherichia coli (16). For further comparative purposes, pairwise alignments between the A6 sequence, representatives of the major protein kinase subfamilies (6), and the atypical kinases BCR and HBx were generated by using the FASTA program (23).…”

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Prokaryotic expression cloning of a novel human tyrosine kinase.

et al. 1994

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496-8479. threonine residues when examined in bacterial expression systems or in vitro assays (3,5,22,26). This has led to the classification of a third group of enzymes referred to as dual-specificity protein kinases (19).In the present report, we describe the prokaryotic expression cloning and in vitro characterization of a novel protein kinase from a human embryonic lung fibroblast cDNA library by using an anti-pTyr monoclonal antibody. When expressed as a 0-galactosidase fusion protein, this molecule demonstrates the ability to phosphorylate tyrosine as well as serine residues. Sequence analysis revealed that the predicted protein lacks any of the consensus motifs commonly found in protein kinases, although its in vitro biochemical properties resemble those of previously known members of the protein kinase family. MATERIALS AND METHODSLibrary construction and screening. The vector for prokaryotic expression, XpCEVlacz, was designed as previously described (12

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Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase

Cited by 49 publications

References 55 publications

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity

Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity

Fancy meeting you here! A fresh look at "prokaryotic" protein phosphorylation

Prokaryotic expression cloning of a novel human tyrosine kinase.

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