Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae (original) (raw)
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Molecular and Cellular Biology, 1998
InSaccharomyces cerevisiae,PHO85encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion ofPHO85causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption ofPCL8andPCL10caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85mutants,pcl8 pcl10cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In co...
Molecular & Cellular Proteomics, 2002
At the onset of nutrient limitation, the yeast Saccharomyces cerevisiae synthesizes glycogen to serve as a carbon and energy reserve. We undertook a systematic survey for the genes that affect glycogen accumulation by taking advantage of the strain deletion set generated by the Saccharomyces Genome Deletion Project. The strain collection analyzed contained some 4600 diploid homozygous null deletants, representing ϳ88% of all viable haploid disruptants. We identified 324 strains with low and 242 with elevated glycogen stores, accounting for 12.4% of the genes analyzed. The screen was validated by the identification of many of the genes known already to influence glycogen accumulation. Many of the mutants could be placed into coherent families. For example, 195 or 60% of the hypoaccumulators carry mutations linked to respiratory function, a class of mutants well known to be defective in glycogen storage. The second largest group consists of ϳ60 genes involved in vesicular trafficking and vacuolar function, including genes encoding 13 of 17 proteins involved in the structure or assembly of the vacuolar ATPase. These data are consistent with our recent findings that the process of autophagy has a significant impact on glycogen storage (Wang, Z., Wilson, W. A., Fujino, M. A., and Roach, P. J. (2001) Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol. Cell. Biol. 21, 5742-5752). Autophagy delivers glycogen to the vacuole, and we propose that the impaired vacuolar function associated with ATPase mutants (vma10 or vma22) results in reduced degradation and subsequent hyperaccumulation of glycogen. Molecular & Cellular Proteomics 1: 232-242, 2002.
Glucose-6-P control of glycogen synthase phosphorylation in yeast
Journal of Biological Chemistry, 1997
The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the -subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the ␣-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2 cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at ϳ2 mM. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase.
Control of mammalian glycogen synthase by PAS kinase
Proceedings of the National Academy of Sciences of the United States of America, 2005
The regulation of glycogen metabolism is critical for the maintenance of glucose and energy homeostasis in mammals. Glycogen synthase, the enzyme responsible for glycogen production, is regulated by multisite phosphorylation in yeast and mammals. We have previously identified PAS kinase as a physiological regulator of glycogen synthase in Saccharomyces cerevisiae. We provide evidence here that PAS kinase is an important regulator of mammalian glycogen synthase. Glycogen synthase is efficiently phosphorylated by PAS kinase in vitro at Ser-640, a known regulatory phosphosite. Efficient phosphorylation requires a region of PAS kinase outside the catalytic domain. This region appears to mediate a direct interaction between glycogen synthase and PAS kinase, thereby targeting kinase activity to this substrate specifically. This interaction is regulated by the PAS kinase PAS domain, raising the possibility that this interaction (and phosphorylation event) is modulated by the cellular metabolic state. This mode of regulation provides a mechanism for metabolic status to impinge directly on the cellular decision of whether to store or use available energy. phosphorylation
Nuclear Glycogen and Glycogen Synthase Kinase 3
Biochemical and Biophysical Research Communications, 1998
tion originated in the study of glycogen metabolism. Glycogen is the principal storage form of glucose in The interconversion of key regulatory enzymes beanimal cells. It accumulates in electron-dense cytotween phosphorylated and dephosphorylated forms is plasmic granules and is synthesized by glycogen synan extremely versatile mechanism for reversibly alterthase (GS), the rate-limiting enzyme of glycogen deposiing their activities and, in mammalian cells, may be tion. Glycogen synthase kinase-3 (GSK-3) is a protein as common as allosteric regulation. Glycogen synthase kinase that phosphorylates GS. Two nearly identical (GS), the rate-limiting enzyme of glycogen deposiforms of GSK-3 exist: GSK-3 a and GSK-3 b. Both are tion, is phosphorylated by glycogen synthase kinases constitutively active in resting cells and their activity (GSK's). Five GSK's were described initially (GSK-1 can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological to -5) (1) although additional kinases that phosphoryresponses in mammalian cells by phosphorylating sublate GS in vitro have subsequently been identified (1strates including neuronal cell adhesion molecule, neu-3). Direct evidence showing that GSK-3 regulates GS rofilaments, synapsin I, and tau. Recent observations comes from experiments showing that insulin activates point to functions for glycogen and glycogen metabolism GS by site-specific dephosphorylation of GSK-3 sites in the nucleus. GSK-3 phosphorylates several transcrip-(4). Elevated cellular levels of GSK-3 activity also suption factors, and we have recently shown that it modifies presses endogenous GS activity. Under certain circumthe major nuclear pore protein p62. It also regulates stances, GSK-3 inactivation can cause a stimulation of PK1, a protein kinase required for maintaining the in-GS (5).
Role of protein phosphatase 2A in the control of glycogen metabolism in yeast
European Journal of …, 1995
The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild-type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2-3-fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A-depleted cells, as determined by both enzymic and immunochemical techniques. However, Northem-blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.
Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgi1) disruption mutant
FEBS Letters, 1992
Disruption of the gene pgi1 of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures. The level or glucose 6‐phosphate was much higher in mutant than in wild‐type cells. Phosphorylase a activity and the state of activation of glycogen synthase were also investigated. Phosphorylase a activity was rather low along the culture in wild‐type cells, whereas it was consistently higher in mutants. Glycogen synthase was mostly in the active form in early‐medium exponential cultures in wild‐type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild‐type cells at later stages. The fact that the intracellular levels of UDP‐glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activati...
Molecular Genetics and Genomics, 2004
Combinatorial control by the protein kinases PKA, PHO85 and SNF1 of transcriptional induction of the Saccharomyces cerevisiae GSY2 gene at the diauxic shift Abstract Genes involved in storage carbohydrate metabolism are coordinately induced when yeast cells are subjected to conditions of stress, or when they exit the exponential growth phase on glucose. We show that the STress Responsive Elements (STREs) present in the promoter of GSY2 are essential for gene activation under conditions of stress, but dispensable for gene induction and glycogen accumulation at the diauxic shift on glucose. Using serial promoter deletion, we found that the latter induction could not be attributed to a single cis-regulatory sequence, and present evidence that this mechanism depends on combinatorial transcriptional control by signalling pathways involving the protein kinases Pho85, Snf1 and PKA. Two contiguous regions upstream of the GSY2 coding region are necessary for negative control by the cyclin-dependent protein kinase Pho85, one of which is a 14-bp G/C-rich sequence. Positive control by Snf1 is mediated by Mig1p, which acts indirectly on the distal part of the GSY2 promoter. The PKA pathway has the most pronounced effect on GSY2, since transcription of this gene is almost completely abolished in an ira1ira2 mutant strain in which PKA is hyperactive. The potent negative effect of PKA is dependent upon a branched pathway involving the transcription factors Msn2/Msn4p and Sok2p. The SOK2 branch was found to be effective only under conditions of high PKA activity, as in a ira1ira2 mutant, and this effect was independent of Msn2/4p. The Msn2/ 4p branch, on the other hand, positively controls GSY2 expression directly through the STREs, and indirectly via a factor that still remains to be discovered. In summary, this study shows that the transcription of GSY2 is regulated by several different signalling pathways which reflect the numerous factors that influence glycogen synthesis in yeast, and suggests that the PKA pathway must be deactivated to allow gene induction at the diauxic shift.
Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae
Molecular and cellular biology, 1989
In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme. We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae. To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed. GPH1 was found not to be an essential gene in yeast cells. Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells. Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores. Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose. Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid. Northern (RNA) analy...