Control of cell cycle in response to osmostress: lessons from yeast.

To maximize the probability of survival and proliferation, cells coordinate various intracellular activities in response to changes in the extracellular environment. Eukaryotic cells transduce diverse cellular stimuli by multiple mitogen-activated protein kinase (MAPK) cascades. Exposure of cells to stress results in rapid activation of a highly conserved family of MAPKs, known as stress-activated protein kinases (SAPKs). Activation of SAPKs results in the generation of a set of adaptive responses that leads to the modulation of several aspects of cell physiology essential for cell survival, such as gene expression, translation, and morphogenesis. This chapter proposes that regulation of cell cycle progression is another general stress response critical for cell survival. Studies from yeast, both Schizosaccharomyces pombe and Saccharomyces cerevisiae, have served to start understanding how SAPKs control cell cycle progression in response to stress.

Okadaic acid-sensitive activation of Maxi Cl(-) channels by triphenylethylene antioestrogens in C1300 mouse neuroblastoma cells.

1. The regulation of Maxi Cl(-) channels by 17beta-oestradiol and non-steroidal triphenylethylene antioestrogens represents a rapid, non-classical effect of these compounds. In the present study we have investigated the signalling pathways used for the regulation of Maxi Cl(-) channel activity by oestrogens and antioestrogens in C1300 neuroblastoma cells. 2. Whole-cell Maxi Cl(-) currents were readily and reversibly activated by tamoxifen, toremifene and the membrane-impermeant ethyl-bromide tamoxifen, only when applied to the extracellular medium. 3. Pre-treatment of C1300 cells with oestrogen or cAMP prevented the antioestrogen-induced activation of Maxi Cl(-) channels. The inhibitory effect of 17beta-oestradiol and cAMP was abolished by the kinase inhibitor staurosporine. 4. Current activation was unaffected by the removal of intracellular Ca(2+) and Mg(2+), but was completely abolished in the presence of okadaic acid. These results are consistent with the participation of an okadaic acid-sensitive serine/threonine protein phosphatase in the activation of Maxi Cl(-) channels. However, neither oestrogen or antioestrogen treatment modified the total activity of the two major serine/threonine phosphatases, PP1 and PP2A, in C1300 cells. 5. Although the role of these Maxi Cl(-) channels remains unknown, our findings suggest strongly that their modulation by oestrogens and antioestrogens is linked to intracellular signalling pathways.

Multiple levels of control regulate the yeast cAMP-response element-binding protein repressor Sko1p in response to stress.

The Sko1p transcriptional repressor regulates a subset of osmoinducible stress defense genes in Saccharomyces cerevisiae by binding to cAMP-responsive elements. We have reported previously that in response to stress Sko1p is phosphorylated by the stress-activated Hog1p mitogen-activated protein kinase, which disrupts its interaction with the Ssn6p x Tup1p corepressor. Here we report that other mechanisms are essential for the regulation of the Sko1p repressor activity upon stress. The nuclear localization of Sko1p depends on the stress-inhibited protein kinase A (PKA). Sko1p is localized in the nucleus of unstressed cells, and it redistributes to the cytosol upon severe salt stress (1 m NaCl). Yeast mutants with low PKA activity localize Sko1p to the cytoplasm in the absence of stress and exhibit deregulated expression of cAMP-responsive element-regulated genes. The central part (315) of Sko1p, containing the PKA phosphorylation sites and the basic domain-leucine zipper domain, is essential for its nuclear localization. Salt-induced export of Sko1p from the nucleus is independent of Hog1p and of the Bcy1p regulatory subunit of PKA. Furthermore, phosphorylation by PKA slightly enhanced DNA binding affinity of Sko1p in vitro, whereas Sko1p dimerization in vivo is not regulated by stress. Sko1p repressor activity is associated to its binding to the Ssn6p x Tup1p complex. Interestingly, the Sko1p NH(2) terminus (1), containing the Hog1p phosphorylation sites, associates in vivo with Tup1p in the absence of Ssn6p, suggesting that Sko1p represses gene transcription by interacting directly with the Tup1p subunit of the Ssn6p x Tup1p complex.

Regulation of the Sko1 transcriptional repressor by the Hog1 MAP kinase in response to osmotic stress.

Exposure of yeast to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which is essential for the induction of gene expression required for cell survival upon osmotic stress. Several genes are regulated in response to osmotic stress by Sko1, a transcriptional repressor of the ATF/CREB family. We show by in vivo coprecipitation and phosphorylation studies that Sko1 and Hog1 interact and that Sko1 is phosphorylated upon osmotic stress in a Hog1-dependent manner. Hog1 phosphorylates Sko1 in vitro at multiple sites within the N-terminal region. Phosphorylation of Sko1 disrupts the Sko1-Ssn6-Tup1 repressor complex, and consistently, a mutant allele of Sko1, unphosphorylatable by Hog1, exhibits less derepression than the wild type. Interestingly, Sko1 repressor activity is further enhanced in strains with high protein kinase A (PKA) activity. PKA phosphorylates Sko1 near the bZIP domain and mutation of these sites eliminates modulation of Sko1 responses to high PKA activity. Thus, Sko1 transcriptional repression is controlled directly by the Hog1 MAPK in response to stress, and this effect is further modulated by an independent signaling mechanism through the PKA pathway.

Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway.

The adaptive response to hyperosmotic stress in yeast, termed the high osmolarity glycerol (HOG) response, is mediated by two independent upstream pathways that converge on the Pbs2 MAP kinase kinase (MAPKK), leading to the activation of the Hog1 MAP kinase. One branch is dependent on the Sho1 transmembrane protein, whose primary role was found to be the binding and translocation of the Pbs2 MAPKK to the plasma membrane, and specifically to sites of polarized growth. The yeast PAK homolog Ste20 is essential for the Sho1-dependent activation of the Hog1 MAP kinase in response to severe osmotic stress. This function of Ste20 in the HOG pathway requires binding of the small GTPase Cdc42. Overexpression of Cdc42 partially complements the osmosensitivity of ste20Delta mutants, perhaps by activating another PAK-like kinase, while a dominant-negative Cdc42 mutant inhibited signaling through the SHO1 branch of the HOG pathway. Since activated Cdc42 translocates Ste20 to sites of polarized growth, the upstream and downstream elements of the HOG pathway are brought together through the membrane targeting function of Sho1 and Cdc42.

Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1.

Exposure of yeast cells to increases in extracellular osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK). Activation of Hog1 MAPK results in induction of a set of osmoadaptive responses, which allow cells to survive in high-osmolarity environments. Little is known about how the MAPK activation results in induction of these responses, mainly because no direct substrates for Hog1 have been reported. We conducted a two-hybrid screening using Hog1 as a bait to identify substrates for the MAPK, and the Rck2 protein kinase was identified as an interactor for Hog1. Both two-hybrid analyses and coprecipitation assays demonstrated that Hog1 binds strongly to the C-terminal region of Rck2. Upon osmotic stress, Rck2 was phosphorylated in vivo in a Hog1-dependent manner. Furthermore, purified Hog1 was able to phosphorylate Rck2 when activated both in vivo and in vitro. Rck2 phosphorylation occurred specifically at Ser519, a residue located within the C-terminal putative autoinhibitory domain. Interestingly, phosphorylation at Ser519 by Hog1 resulted in an increase of Rck2 kinase activity. Overexpression of Rck2 partially suppressed the osmosensitive phenotype of hog1Delta and pbs2Delta cells, suggesting that Rck2 is acting downstream of Hog1. Consistently, growth arrest caused by hyperactivation of the Hog1 MAPK was abolished by deletion of the RCK2 gene. Furthermore, overexpression of a catalytically impaired (presumably dominant inhibitory) Rck2 kinase resulted in a decrease of osmotolerance in wild-type cells but not in hog1Delta cells. Taken together, our data suggest that Rck2 acts downstream of Hog1, controlling a subset of the responses induced by the MAPK upon osmotic stress.

The transcriptional response of yeast to saline stress.

Adaptation to changes in extracellular salinity is a critical event for cell survival. Genome-wide DNA chip analysis has been used to analyze the transcriptional response of yeast cells to saline stress. About 7% of the genes encoded in the yeast genome are induced more than 5-fold after a mild and brief saline shock (0.4 m NaCl, 10 min). Interestingly, most responsive genes showed a very transient expression pattern, as mRNA levels dramatically declined after 20 min in the presence of stress. A quite similar set of genes increased expression in cells subjected to higher saline concentrations (0.8 m NaCl), although in this case the response was delayed. Therefore, our data show that cells respond to saline stress by inducing the expression of a very large number of genes and suggest that stress adaptation requires regulation of many cellular aspects. The transcriptional induction of most genes that are strongly responsive to salt stress was highly or fully dependent on the presence of the stress-activated mitogen-activated protein kinase Hog1, indicating that the Hog1-mediated signaling pathway plays a key role in global gene regulation under saline stress conditions.

Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.

Requirement of STE50 for osmostress-induced activation of the STE11 mitogen-activated protein kinase kinase kinase in the high-osmolarity glycerol response pathway.

Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 mitogen-activated protein (MAP) kinase cascade, which is composed of three tiers of protein kinases: (i) the SSK2, SSK22, and STE11 MAP kinase kinase kinases (MAPKKKs), (ii) the PBS2 MAPKK, and (iii) the HOG1 MAP kinase. Activation of the MAP kinase cascade is mediated by two upstream mechanisms. The SLN1-YPD1-SSK1 two-component osmosensor activates the SSK2 and SSK22 MAPKKKs by direct interaction of the SSK1 response regulator with these MAPKKKs. The second mechanism of HOG1 MAP kinase activation is independent of the two-component osmosensor and involves the SHO1 transmembrane protein and the STE11 MAPKKK. Only PBS2 and HOG1 are common to the two mechanisms. We conducted an exhaustive mutant screening to identify additional elements required for activation of STE11 by osmotic stress. We found that strains with mutations in the STE50 gene, in combination with ssk2Delta ssk22Delta mutations, were unable to induce HOG1 phosphorylation after osmotic stress. Both two-hybrid analyses and coprecipitation assays demonstrated that the N-terminal domain of STE50 binds strongly to the N-terminal domain of STE11. The binding of STE50 to STE11 is constitutive and is not affected by osmotic stress. Furthermore, the two proteins relocalize similarly after osmotic shock. It was concluded that STE50 fulfills an essential role in the activation of the high-osmolarity glycerol response pathway by acting as an integral subunit of the STE11 MAPKKK.

The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase.

Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes. Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity. These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells. Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway. Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts. In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed. In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p. Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1). Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator.

Exposure of yeast cells to increased extracellular osmolarity induces the HOG1 mitogen-activated protein kinase (MAPK) cascade, which is composed of SSK2, SSK22 and STE11 MAPKKKs, PBS2 MAPKK and HOG1 MAPK. The SSK2/SSK22 MAPKKKs are activated by a 'two-component' osmosensor composed of SLN1, YPD1 and SSK1. The SSK1 C-terminal receiver domain interacts with an N-terminal segment of SSK2. Upon hyperosmotic treatment, SSK2 is autophosphorylated rapidly, and this reaction requires the interaction of SSK1 with SSK2. Autophosphorylation of SSK2 is an intramolecular reaction, suggesting similarity to the mammalian MEKK1 kinase. Dephosphorylation of SSK2 renders the kinase inactive, but it can be re-activated by addition of SSK1 in vitro. A conserved threonine residue (Thr1460) in the activation loop of SSK2 is important for kinase activity. Based on these observations, we propose the following two-step activation mechanism of SSK2 MAPKKK. In the first step, the binding of SSK1 to the SSK1-binding site in the N-terminal domain of SSK2 causes a conformational change in SSK2 and induces its latent kinase activity. In the second step, autophosphorylation of SSK2 renders its activity independent of the presence of SSK1. A similar mechanism might be applicable to other MAPKKKs from both yeast and higher eukaryotes.

Signal transduction by MAP kinase cascades in budding yeast.

Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.

A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways.

A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant. This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs. In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway. MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK. Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro. Overexpression of a dominant-negative MTK1 mutant [MTK1(K/R)] strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha. The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway. These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway.

Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK.

Exposure of the yeast Saccharomyces cerevisiae to high extracellular osmolarity induces the Sln1p-Ypd1p-Ssk1p two-component osmosensor to activate a mitogen-activated protein (MAP) kinase cascade composed of the Ssk2p and Ssk22p MAP kinase kinase kinases (MAPKKKs), the Pbs2p MAPKK, and the Hog1p MAPK. A second osmosensor, Sho1p, also activated Pbs2p and Hog1p, but did so through the Ste11p MAPKKK. Although Ste11p also participates in the mating pheromone-responsive MAPK cascade, there was no detectable cross talk between these two pathways. The MAPKK Pbs2p bound to the Sho1p osmosensor, the MAPKKK Ste11p, and the MAPK Hog1p. Thus, Pbs2p may serve as a scaffold protein.

The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role.

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.

Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor.

An osmosensing mechanism in the budding yeast (Saccharomyces cerevisiae) involves both a two-component signal transducer (Sln1p, Ypd1p and Ssk1p) and a MAP kinase cascade (Ssk2p/Ssk22p, Pbs2p, and Hog1p). The transmembrane protein Sln1p contains an extracellular sensor domain and cytoplasmic histidine kinase and receiver domains, whereas the cytoplasmic protein Ssk1p contains a receiver domain. Ypd1p binds to both Sln1p and Ssk1p and mediates the multistep phosphotransfer reaction (phosphorelay). This phosphorelay system is initiated by the autophosphorylation of Sln1p at His576. This phosphate is then sequentially transferred to Sln1p-Asp-1144, then to Ypd1p-His64, and finally to Ssk1p-Asp554. We propose that the multistep phosphorelay mechanism is a universal signal transduction apparatus utilized both in prokaryotes and eukaryotes.

Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance.

The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.

The PPZ protein phosphatases are important determinants of salt tolerance in yeast cells.

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li+, the intracellular content of the cation was markedly decreased in ppz1 delta ppz2 delta mutants when compared to wild type cells. No significant differences were observed between both strains when the Li+ influx was measured, but ppz1 delta ppz2 delta mutants eliminated Li+ more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 delta ppz2 delta cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na+ and Li+ of a ena1 delta strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.

Role of protein phosphatase 2A in the control of glycogen metabolism in yeast.

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, Northern-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.