In vivo phosphorylation site of hexokinase 2 in Saccharomyces cerevisiae

Kriegel, Thomas; Rush, A. John; Vojtek, Anne B.; Clifton, D; Fraenkel, D G

doi:10.1021/bi00167a019

Cited by 59 publications

(88 citation statements)

References 24 publications

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“…In the case of ScHxk2, the phosphorylation of a single amino acid in the dimer interface (Ser-15) is likely to represent an efficient mechanism enabling the cell to induce the dissociation of the hexokinase homodimer, even at the high enzyme concentrations expected to exist in situ (20,21), and to facilitate ScHxk2 interaction with the major nuclear export receptor Xpo1 (9). The physiological signal that initiates the covalent modification of residue Ser-15 by a hitherto unidentified protein kinase in S. cerevisiae is glucose limitation (19). Interestingly, it was found only recently that KlHxk1 may be phosphorylated in vivo at the equivalent position Ser-15 and that this modification efficiently stimulates the dissociation of the homodimeric enzyme in vitro, 5 as observed similarly for ScHxk2 (20).…”

Section: Discussionmentioning

confidence: 99%

“…Because of the observation that glucose limitation stimulates both the phosphorylation of ScHxk2 at Ser-15 in vivo (18,19) (which in vitro causes the dissociation of the homodimeric enzyme (20)) and its export from the nucleus (9), the covalent modification at Ser-15 and subsequent changes of the oligomeric state have been presumed to be involved in the control of ScHxk2 regulatory functions (9,21). In contrast, no data are available on the covalent modification and/or intracellular distribution of KlHxk1 in the nonfermentative yeast K. lactis.…”

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confidence: 99%

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Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

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Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ringshaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. The enzymes of the hexokinase family catalyze the intracellular trapping and the initiation of metabolism of glucose, fructose, and mannose. In addition to their role in glycolysis, an increasing number of yeast, plant, and mammalian hexokinases have been found to represent multifunctional proteins that are implicated in glucose sensing and signaling (1-4), whereas their glycolytic sugar substrate plays a dual role as a carbon source and hormone-like regulator (4, 5). The molecular basis underlying the involvement of hexokinases in the transcriptional control of glucose metabolism and in glucose homeostasis is their ability to interact with mitochondria and to reversibly translocate to nuclei (3, 6 -9).In the Crabtree-positive yeast Saccharomyces cerevisiae, glucose abundance is accompanied by the translocation of the cytosolic hexokinase isoenzyme 2 (ScHxk2) 4 and the transcriptional repressor Mig1 (ScMig1) into the nucleus, where both proteins participate in the formation of a hetero-oligomeric complex that suppresses the transcription of ScMig1 target genes like SUC2 encoding invertase (9). The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).Contrary to the situation in bakers' yeast, glucose and fructose limitation causes the translocation of the mammalian hexokinase isoenzyme IV (also referred to as "glucokinase" or hexokinase D) to the nucleus of the liver parenchymal cell where it binds to its regulatory protein, GKRP, and retranslocates when glucose is abundantly av...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

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“…The exact opposite result (intact glucose signaling and low catalytic activity), however, was obtained by others upon deletion of an overlapping and slightly larger Hxk2 segment (residues 1 through 15) (231). Likewise, the phosphorylatable Ser14 residue in Hxk2 (193) is reported either to mediate (305) or not to mediate (146,231) glucose repression. Further analysis is needed to resolve these discrepancies.…”

Section: Involvement Of the Hexokinase Hxk2 In The Glucose Responsementioning

confidence: 99%

Glucose Signaling in Saccharomyces cerevisiae

Santangelo

2006

Microbiol Mol Biol Rev

481

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SUMMARY Eukaryotic cells possess an exquisitely interwoven and fine-tuned series of signal transduction mechanisms with which to sense and respond to the ubiquitous fermentable carbon source glucose. The budding yeast Saccharomyces cerevisiae has proven to be a fertile model system with which to identify glucose signaling factors, determine the relevant functional and physical interrelationships, and characterize the corresponding metabolic, transcriptomic, and proteomic readouts. The early events in glucose signaling appear to require both extracellular sensing by transmembrane proteins and intracellular sensing by G proteins. Intermediate steps involve cAMP-dependent stimulation of protein kinase A (PKA) as well as one or more redundant PKA-independent pathways. The final steps are mediated by a relatively small collection of transcriptional regulators that collaborate closely to maximize the cellular rates of energy generation and growth. Understanding the nuclear events in this process may necessitate the further elaboration of a new model for eukaryotic gene regulation, called “reverse recruitment.” An essential feature of this idea is that fine-structure mapping of nuclear architecture will be required to understand the reception of regulatory signals that emanate from the plasma membrane and cytoplasm. Completion of this task should result in a much improved understanding of eukaryotic growth, differentiation, and carcinogenesis.

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“…This enzyme is located in both the nucleus and the cytoplasm, consistent with dual roles in signaling and catalysis (20,39), but its actual role in glucose repression is still poorly understood. When glucose is limiting, Hxk2 is a phosphoprotein (27,52), and the Reg1-Glc7 phos-phatase complex dephosphorylates Hxk2 in response to glucose (1,40).…”

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Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase

Sanz

Alms

Haystead

et al. 2000

Molecular and Cellular Biology

218

280

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Protein phosphatase 1, comprising the regulatory subunit Reg1 and the catalytic subunit Glc7, has a role in glucose repression in Saccharomyces cerevisiae. Previous studies showed that Reg1 regulates the Snf1 protein kinase in response to glucose. Here, we explore the functional relationships between Reg1, Glc7, and Snf1. We show that different sequences of Reg1 interact with Glc7 and Snf1. We use a mutant Reg1 altered in the Glc7-binding motif to demonstrate that Reg1 facilitates the return of the activated Snf1 kinase complex to the autoinhibited state by targeting Glc7 to the complex. Genetic evidence indicated that the catalytic activity of Snf1 negatively regulates its interaction with Reg1. We show that Reg1 is phosphorylated in response to glucose limitation and that this phosphorylation requires Snf1; moreover, Reg1 is dephosphorylated by Glc7 when glucose is added. Finally, we show that hexokinase PII (Hxk2) has a role in regulating the phosphorylation state of Reg1, which may account for the effect of Hxk2 on Snf1 function. These findings suggest that the phosphorylation of Reg1 by Snf1 is required for the release of Reg1-Glc7 from the kinase complex and also stimulates the activity of Glc7 in promoting closure of the complex.

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In vivo phosphorylation site of hexokinase 2 in Saccharomyces cerevisiae

Cited by 59 publications

References 24 publications

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Glucose Signaling in Saccharomyces cerevisiae

Regulatory Interactions between the Reg1-Glc7 Protein Phosphatase and the Snf1 Protein Kinase

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