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Keywords:

  • Hexokinase 2;
  • Glucose sensing;
  • Glucose repression;
  • Glucose induction;
  • Transcriptional control;
  • Saccharomyces cerevisiae

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

Sugars, predominantly glucose, evoke a variety of responses in Saccharomyces cerevisiae. These responses are elicited through a complex network of regulatory mechanisms that transduce the signal of presence of external glucose to their final intracellular targets. The HXK2 gene, encoding hexokinase 2 (Hxk2), the enzyme that initiates glucose metabolism, is highly expressed during growth in glucose and plays a pivotal role in the control of the expression of numerous genes, including itself. The mechanism of this autocontrol of expression is not completely understood. Hxk2 is found both in the nucleus and in the cytoplasm of S. cerevisiae; the nuclear localization is dependent on the presence of a stretch of amino acids located from lysine-6 to methionine-15. Although serine-14, within this stretch, can be phosphorylated in the absence of glucose, it is still unsettled whether this phosphorylation plays a role in the cellular localization of Hxk2. The elucidation of the mechanism of transport of Hxk2 to and from the nucleus, the influence of the oligomeric state of the protein on the nuclear transport and the fine mechanism of regulation of transcription of HXK2 are among the important unanswered questions in relation with the regulatory role of Hxk2.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

Adaptation to changes in the environment is critical for the survival of all organisms, therefore along evolution, systems of different complexity have been selected to meet this demand. Changes in external medium generate signals that are transduced to the interior of the cell, where they provoke changes in gene expression and protein activity which result in an adequate cellular response to variations in the extracellular environment.

The ability to grow in different media exhibited by the yeast Saccharomyces cerevisiae is due to its capacity to sense and respond to changes in the availability of nutrients. The transduction pathways of the nutritional signals are induced by specific nutrients, and in general these pathways bring about changes in gene expression [1], mRNA stability [2] and post-translational modifications [3–7]. Among sugars, glucose is likely the major signalling nutrient for S. cerevisiae, as well as the carbon and energy source used preferentially. Present knowledge concerning factors required to transmit the glucose signal from the environment to the cell nucleus is mainly derived from studies with mutants affected in different regulatory circuits governed by glucose [8]. These studies have characterized, in different detail, several signal transduction pathways that allow the yeast to perceive the level of glucose in the medium and initiate the appropriate metabolic response [9,10]. Many of these responses involve alterations in gene expression, and the majority of these alterations occur at the level of mRNA transcription, a phenomenon known as glucose or catabolite repression [11–14]. The genes affected by this process include among others those involved in utilization of alternative carbon sources, gluconeogenesis, the glyoxylate and Krebs cycles, respiration, and peroxisomal functions. Glucose also induces expression of genes required for its own utilization, like the genes encoding several glycolytic enzymes and glucose transporters [15]. In addition, glucose acts in yeast as a ‘growth hormone’, regulating several aspects of cell growth, metabolism and development [16,17].

Although several of the genes implicated in the pathways that control glucose repression and induction have been identified [9], a complete mechanistic picture of the phenomenon is not yet available. In particular, the position of each factor in the signalling cascade and the interactions among them are still not well known. However, in the last few years important advances in this field have been made [9,10]. We consider in this review the present knowledge about an important factor in the signal transduction pathway, namely hexokinase 2 (Hxk2), a protein that in addition to its classical metabolic role plays an important role in glucose signalling.

2Hxk2 and glucose signalling

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

2.1The glucose-phosphorylating enzymes of S. cerevisiae

In S. cerevisiae glucose metabolism starts by phosphorylation of the sugar at C6. Three enzymes, namely Hxk1, Hxk2 and glucokinase (Glk1) (encoded respectively by the HXK1, HXK2 and GLK1 genes) can catalyze this first irreversible step in the intracellular metabolism of glucose. However, Northern analysis showed that when glucose is used as carbon source, only the HXK2 gene is highly expressed. In contrast, the expression of the HXK1 and GLK1 genes is only important when the culture medium contains non-fermentable carbon sources or galactose [18]. These facts have been confirmed by exploring the metabolic and genetic control of gene expression on a genomic scale [19]. These findings indicate that in wild-type S. cerevisiae, only HXK2 is important for glucose phosphorylation in vivo. Either Hxk1 or Glk1 support growth of mutants in which Hxk2 is absent [13], indicating that these proteins are able to act in vivo, and that their irrelevance for glucose metabolism is due to the poor expression of the genes which encode them.

What makes Hxk2 a special protein is not only that it is the enzyme responsible for the phosphorylation of glucose, but the fact that it is also implicated in glucose repression. It was found that both point [20] and null mutations [21] in the HXK2 gene blocked glucose repression of certain genes. Since the glucose-phosphorylating activity in the corresponding extracts was reduced, the idea that there was a correlation between the glucose-phosphorylating activity of Hxk2 and glucose repression appeared as a very attractive one [22,23]. However, this idea was shaken by the following findings: (i) when the GLK1 gene is overexpressed in a hxk1/hxk2 double-null mutant the transformed strains are still insensitive to glucose repression, even though a three-fold increase of phosphorylating activity is achieved [23]; (ii) glucose repression is not linearly relieved with decreasing kinase activity, indicating that sugar kinase activity and sugar signalling are mediated at least in part through separated domains of Hxk2 [24,25]; (iii) mutant alleles with low catalytic activity were still fully functional in glucose signalling [26]. In this context it is also interesting to point out that early glucose repression of the SUC2 gene (a glucose repression reporter gene) does not require specifically Hxk2 [27] and that Hxk2 is only necessary for the long-term glucose response [28]. The correlation between glucose-phosphorylation activity of Hxk2 and glucose repression appears less likely at present.

The dual role of hexokinase as a metabolic enzyme and as a regulatory protein, may be not so unique in sugar metabolism. Recently, it has been found that yeast galactokinase (Gal1), has also a dual function: it functions as an enzyme in the phosphorylation of galactose and as a transcriptional regulator. It has been found recently that in the presence of galactose and ATP, Gal1 activates the transcriptional factor Gal4 by direct binding to the Gal4 inhibitor Gal80 [29].

2.2Hxk2 can enter the nucleus

If Hxk2 plays a role as a transcriptional regulator, one should expect that under certain conditions, the enzyme will be present in the cell nucleus. Several results using different approaches support such a localization: (i) Hxk2 was localized in isolated nuclei by specific antibodies and concomitantly hexokinase activity was found in the preparation [30]; (ii) expression of a Hxk2–GFP fusion revealed that a fraction of the total Hxk2 was present in the nucleus, making it unlikely that the previous finding was due to cross-contamination during subcellular fractionation [30]; (iii) a determinant for the nuclear localization of Hxk2p has been characterized as an internal sequence located between lysine-6 and methionine-15 (KKPQARKGSM), that has been named nuclear localization sequence (NLS) [31]. Elimination of this sequence abolishes both nuclear localization of Hxk2 and glucose repression of SUC2, HXK1 and GLK1[31,32]. Therefore, Hxk2 complies with the conditions needed to participate directly in the control of the transcription of several genes. In yeast cells grown in the absence of glucose, Hxk2 is phosphorylated at serine-14 [33]. This phosphorylation catalyzed by a still-unknown kinase has several consequences: (i) Hxk2 becomes sensitive to inhibition by free ATP [34,35] thus allowing the cell to integrate the intracellular ATP concentration into the control of glucose phosphorylation; (ii) autophosphorylation at serine-157 is stimulated, a reaction which results in inhibition of the Hxk2 enzymatic activity [33,34,36,37]. How the new conformation of the diphosphoenzyme may affect glucose signalling is unknown at the moment; (iii) the equilibrium between the two isoforms of Hxk2, a monomer and a dimmer [38–40], is shifted to the monomeric form [34,41].

In the presence of glucose, the regulatory protein Reg1 targets the protein phosphatase 1 Glc7, to dephosphorylate serine-14 in Hxk2 [42] and displaces the equilibrium towards the homodimeric form [34,41]. Since serine-14, which can be phosphorylated in vivo, is within the NLS sequence, it was not illogical to think that the state of phosphorylation of this residue could regulate NLS function. However, the available results have not allowed an unequivocal validation of this idea. It has been reported that the expression of a mutant gene HXK2(S14A) in a hxk1/hxk2 double mutant strain allowed nuclear localization of the mutant protein and that this one restored glucose repression of the SUC2 gene [31,43]. However, another report showed that both glucose repression of SUC2 and glucose-induced expression of glucose transporters were impaired in cells which expressed the mutated protein [41]. The experiments were carried out in two different genetic backgrounds and the discrepancy between the results could be related to differences in the amount of hexokinase being produced in the different transformed strains.

2.3Hxk2 participates both in glucose repression and in induction of gene expression

The amount of glucose-phosphorylating enzymes in S. cerevisiae is mainly regulated at the transcriptional level by mechanisms not completely elucidated [18]. In the case of HXK2, DNA–protein complexes have been described [44,45] that involve two nucleotide sequences (downstream repressing sequences, DRSs) located within the coding region of the gene. The regulatory factors that operate through these DRSs repress HXK2 transcription under conditions of sugar limitation or when ethanol is used as a carbon source [18]. Transcription of the GLK1 gene is repressed in media with glucose but is high upon glucose depletion [46]. This regulation is achieved through the combinatorial effect of three regulatory sequences of the GLK1 promoter: a STRE sequence (stress-responsive element) [47,48], an ethanol repression autoregulation (ERA)/TA box) element [49], and a Gcr1-binding site [50–52]. The transcription of HXK1 is also repressed in the presence of glucose by regulatory factors that operate through an ERA element; upon glucose depletion, a quick induction of transcription, through several STRE elements, has been observed (F. Moreno, unpublished results). Hxk2 is involved both in the glucose-induced repression of the HXK1 and GLK1 genes and in the glucose-induced expression of the HXK2 gene [32]. Hxk1 also acts in this regulatory system as an inhibiting factor for the expression of the GLK1 and HXK2 genes [32]. Further experimental evidences, derived from a hxk2 mutant expressing a truncated version of Hxk2 unable to enter the nucleus, showed that a nuclear localization of Hxk2 is necessary for glucose-induced repression of the HXK1 and GLK1 genes, for glucose-induced expression of HXK2 gene and for glucose repression of the SUC2 gene [31,32]. Hxk2 participates in DNA–protein complexes with cis-acting regulatory elements of the SUC2 gene, which contain the heptameric motif (C/A)(G/A)GAAAT [31]. Interestingly, the sequences of both DRSs in the HXK2 gene include the stretches CGGAAAT and AAGAAAT, which are also found in the sequence of an UAS element of SUC2 gene [45]. These sequences are also found in the promoters of the HXK1 and GLK1 genes overlapping with ERA motifs that participate in the Hxk2-regulated transcription [31,53]. Thus, as can be seen in Table 1, the HXK1, GLK1, HXK2 and SUC2 genes have regulatory elements with the consensus sequence (C/A)(G/A)(G/A)AAAT.

Table 1.  DNA sequence of regulatory regions in the HXK1, GLK1, HXK2 and SUC2 promoters
inline image
Important sequence elements are indicated. They include: for all the genes, UASSUC-like regulatory sequences for HXK1, GLK1 and HXK2 (DRS1), ERA regulatory sequences, and for SUC2 promoter, one Mig1p-binding site overlapping the first UASSUC regulatory sequence.

These findings suggest a mechanism of gene regulation whereby in the presence of glucose the product of the HXK2 gene, normally resident in the cytosol, is translocated to the nucleus where it impairs the activation of transcription by UASSUC-like heptameric motif contained in the SUC2, HXK1 and GLK1 promoters and hinders the blocking of transcription by DRSHXK2.

The induction by low levels of glucose of the expression of HXT2–4 genes that encode certain glucose transporters is significantly reduced in hxk2 mutants [54]. This may partially account for the observation that a mutant unable to phosphorylate glucose (hxk1 hxk2 glk1), lacks high-affinity glucose transport [55,56], and makes less likely the conclusions of previous kinetic analyses of glucose transport in glucose kinase mutants (hxk1 hxk2 glk1), namely that the hexose kinases interact directly with hexose transporter proteins and modulate their affinity for glucose [57,58]. Control of the transporters at the level of transcription is also supported by the fact that the sole hexokinase of Kluyveromyces lactis (encoded by RAG5) is essential for glucose-induced transcription of the RAG1 gene, which encodes a low-affinity glucose transporter [59]. Hxk2 is also required for full induction of HXT1 expression by high levels of glucose, suggesting that the protein is involved in the Rgt1-independent glucose induction mechanism that operates on HXT1[54].

3Integration of the glucose induction and repression pathways

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

The facts described indicate that Hxk2 is a factor both of the repression and induction pathways triggered by glucose in yeast. Although these pathways seem to share some components and may respond to the same primary signal(s) derived from glucose, the connection between the induction and the repression pathways is not completely understood at present.

We describe in the following paragraphs some of the elements of these pathways.

3.1The glucose sensors of the pathway

Glucose is sensed by the receptors proteins Snf3 and Rgt2, members of a family of hexose transport proteins that in S. cerevisiae consist of Hxt1 to Hxt17, Snf3, Rgt2 and Gal2 [15,60]. Snf3 and Rgt2 are structurally distinct from the other 18 members of this family in yeast by the presence of a large, hydrophilic C-terminal domain [61]. Several lines of evidence indicate that these C-terminal tails are essential for glucose sensing and signal transduction: (i) deletion of the tail domain reduces Snf3 function [62,63]; (ii) fusion of the tail domain to Hxt1 or Hxt2 confers glucose-sensing ability to those proteins [63]; and (iii) expression of the Snf3 tail domain by itself can suppress defects in glucose transport observed in a snf3 strain [64]. Snf3 and Rgt2 proteins do not actually transport hexoses themselves [65] but control hexose transport by regulating the expression of high- and low-affinity transporters [61]. Thus, the yeast glucose sensors may have evolved from a glucose transporter that changed the transporter domain into a glucose-binding domain able to transmit information about extracellular glucose concentration to the interior of the cell [66,67].

Glucose transport and metabolism are not absolutely required for glucose signalling, as shown by the fact that a dominant mutation in RGT2 could produce changes in gene expression in the absence of glucose [61].

3.2The central repressors of the transduction pathway

Two major repressors specific for the glucose signal transduction pathway have been identified: Mig1 and Rgt1. Mig1 is a Cys2His2 zinc-finger protein that represses the transcription of several genes in the presence of high levels of glucose by recruiting the co-repressors Ssn6–Tup1 to glucose-repressed genes [68,69]. Rgt1, a Cys6 zinc-cluster protein, represses the transcription of several HXT genes when glucose is absent by direct binding to the corresponding promoters and recruitment of the co-repressors Ssn6–Tup1 [70].

Mig1 binds to the promoters of numerous glucose-repressed genes through the consensus sequence T(C/G)(C/T)GGGG, but it also requires an AT-rich region 5′ of the GC box [71–73]. The subcellular localization of Mig1 is regulated by glucose: Mig1 is imported into the nucleus when glucose is present and transported to the cytoplasm when cells are glucose-limited [74]. This regulated movement of Mig1 appears to be due to phosphorylation: in derepressed cells Mig1 is both phosphorylated and translocated to the cytosol [74]. Mig1 contains a nuclear export signal that is phosphorylated by Snf1 upon glucose exhaustion, causing it to be recognized by the nuclear exportin Msn5 and carried out of the nucleus into the cytoplasm [75]. The protein phosphatase that acts on the phosphorylated Mig1 protein has been identified as the protein phosphatase 1 encoded by the essential gene GLC7. Like its mammalian counterpart [76], Glc7 is regulated by interaction with many distinct regulatory targeting subunits [77]. In S. cerevisiae, genetic studies together with two-hybrid analyses have implicated the Glc7-binding protein Reg1 in the regulation of the glucose repression pathway [78,79] and demonstrated a direct interaction of Reg1 with Hxk2 and Snf1, suggesting that Reg1 targets Glc7 to dephosphorylate both Hxk2 and Snf1 in vivo [42,78,80,81].

Rgt1 plays two central roles in glucose induction of gene expression. It is required for repression in the absence of glucose and for maximal induction of the HXT1 gene at high glucose concentrations. Grr1p is required both for activation of Rgt1 repressor function in response to low levels of glucose and for conversion of Rgt1 from a repressor to an activator by high levels of glucose. Hxk2 also appears to be involved in the process because in hxk2 mutants, a reduction or increase in glucose-induction of HXT genes expression was observed [70,82].

3.3Med8, a further regulatory element connected with Hxk2

In a search to identify new factors required for expression of SUC2 gene, a protein, Med8, was identified which specifically binds both to the DRSs of the HXK2 gene and to the upstream activating sequences of the SUC2 gene [83,84]. Because Med8 has been described as a subunit of the Srb/mediator complex interacting with the carboxy terminal domain of the RNA polymerase II [85,86], its role could be to act as a coupling factor that links activating and repressing transcription complexes to the RNA polymerase II holoenzyme transcriptional machinery. Co-precipitation experiments, two-hybrid assays and gel-mobility analyses with purified proteins have been used to initiate the generation of protein-interaction maps of factors involved in the glucose signalling pathway of S. cerevisiae. This approach has resulted in the identification of interactions of Hxk2 with Med8 and Mig1 (F. Moreno, unpublished results); the three proteins found interacting together in a cluster, may be part of a protein–DNA complex involved in the regulation of glucose repression. A possible model of how Hxk2 and Mig1 repress transcription of the SUC2 gene is shown in Fig. 1.

image

Figure 1. A schematic model of the participation of Hxk2 in transcriptional regulation. The case of the regulation of SUC2 is shown. In high-glucose media Mig1 is present in the nucleus and binds to a Mig1-binding site that overlaps with one of the two Med8-binding sequences in the SUC2 promoter. Nuclear Hxk2 may interact through different protein domains both with Mig1 and Med8, impairing Med8 function and facilitating the repression of transcription by Mig1. In the absence of glucose, Mig1 is phosphorylated by the Snf1 protein kinase complex and translocated to the cytosol. HXK2 is expressed at low levels or not at all; Med8 binds to both Med8-binding sequences in the promoter, recruits the basal transcription machinery and transcription occurs. RNA polymerase II (RNAPII) represents in the picture the transcription machinery.

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4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

The glucose-sensing pathway of S. cerevisiae has certain similarities with the mammalian pathway controlling the glucose-induced expression of insulin in the pancreatic β-cell.

In most mammalian glucose-sensitive tissues, glucose entry is mediated through specific glucose transporters, such as Glut2p, in the liver and β-cells, and Glut4p, an insulin-sensitive transporter, in adipocytes and muscle. It has been recently suggested that the large intracytoplasmic loop of Glut2p in the liver and β-cells could also play a role in the transmission of a signal [87,88], and it can be thus considered that the Glut2p function in β-cells has similarities with the function of the Snf3/Rgt2-sensor system of yeast.

Glucose regulates insulin production in pancreatic β-cells by stimulating the transcription of the insulin gene. This effect is mediated through a transcription factor (PDX1), which binds to specific regulatory elements within the human insulin gene promoter and to the promoter of several genes expressed preferentially in the β-cell, including those encoding Glut2p and glucokinase (GlkBp) [89]. Glucose activates PDX1 by facilitating the phosphorylation of a cytoplasmic form of the factor that translocates to the nucleus [90] through a mechanism reminiscent of that described for Mig1 in yeast cells. Glucokinase from β-cells and liver that has also been proposed to act as a glucose-sensor molecule [91,92] has a double cytosolic-nuclear localization that is regulated by the nuclear protein Gkrp and by the glucose concentration [93–95]. Thus, mammalian glucokinase and yeast Hxk2 have a similar subcellular distribution and both are necessary for the appropriate regulation of a network of glucose-responsive genes. Interestingly, the expression of the GlkBp gene encoding glucokinase in a hxk2 yeast mutant strain restores both the processes of glucose induction and glucose repression, showing that it acts similarly to its yeast counterpart [96].

5Conclusions and perspectives

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

The results available at this moment clearly show the Hxk2 as an important participant in the complex regulatory system that mediates glucose repression and induction in yeast. However, a series of questions concerning this system are still unanswered and some of them specifically pertain to Hxk2. We summarize briefly some of these. Concerning the dual cytoplasmic-nuclear localization of Hxk2, it has not been established if the protein is in the nucleus in the phosphorylated or unphosphorylated form; also, it remains to be established how the transport to the nucleus is achieved. Moreover, the fate of the protein after glucose withdrawal has not been studied. Another important unsolved question is that of the possible influence of the monomer–dimer equilibrium of the protein on the transport. Up to now there are no results available concerning this point.

Another question that needs study is the analysis of the transcriptional regulation of the HXK2 gene. The fact that Hxk2 itself participates in this regulation raises the question of how the autoactivation of the transcription is controlled.

A final question is that of the eventual participation of some metabolites in the processes regulated by Hxk2. Although up to now largely ignored, it may well be quite important and deserves consideration.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References

We are grateful to Juana M. Gancedo for many scientific comments and to C. Gancedo for critical reading of the manuscript. The work from this laboratory was supported by Grants PB97-1213-C02-02 and BMC2001-1690-C0202 from the Dirección General de Investigación (DGI), Ministerio de Ciencia y Tecnología.

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  2. Abstract
  3. 1Introduction
  4. 2Hxk2 and glucose signalling
  5. 3Integration of the glucose induction and repression pathways
  6. 4A glucose-phosphorylating enzyme is also involved in mammalian glucose signalling pathways
  7. 5Conclusions and perspectives
  8. Acknowledgements
  9. References
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