Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos

Glycogen synthase kinase 3 (GSK-3) is homologous to the product of the Drosophila gene shaggy (zeste-white 3), which is required for signalling by wingless during Drosophila development. To test whether GSK-3 is also involved in vertebrate pattern formation, its role was investigated during early Xenopus development. It was found that dominant-negative GSK-3 mutants induced dorsal differentiation, whereas wild-type GSK-3 induced ventralization. These results indicate that GSK-3 is required for ventral differentiation, and suggest that dorsal differentiation may involve the suppression of GSK-3 activity by a wingless/wnt-related signal.     

The effects of noise in transient EOAE newborn hearing screening

Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha-amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205-217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.     

Evaluation of hypothalamic-pituitary-adrenocortical hormones and inflammatory cytokines in patients with persistent vegetative state

Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication. In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA. The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin. Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus. In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.     

A novel role for glycogen synthase kinase-3 in Xenopus development: maintenance of oocyte cell cycle arrest by a beta-catenin-independent mechanism

We have examined the expression of glycogen synthase kinase-3beta in oocytes and early embryos of Xenopus and found that the protein is developmentally regulated. In resting oocytes, GSK-3beta is active and it is inactivated on maturation in response to progesterone. GSK-3beta inactivation is necessary and rate limiting for the cell cycle response to this hormone and the subsequent accumulation of beta-catenin. Overexpression of a dominant negative form of the kinase accelerates maturation, as does inactivation by expression of Xenopus Dishevelled or microinjection of an inactivating antibody. Cell cycle inhibition by GSK-3beta is not mediated by the level of beta-catenin or by a direct effect on either the MAP kinase pathway or translation of mos and cyclin B1. These data indicate a novel role for GSK-3beta in Xenopus development: in addition to controlling specification of the dorsoventral axis in embryos, it mediates cell cycle arrest in oocytes.     

Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a)

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin.     

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.     

Modification of Ran GTPase-activating protein by the small ubiquitin-related modifier SUMO-1 requires Ubc9, an E2-type ubiquitin-conjugating enzyme homologue

Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1,2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.     

Purification and mass spectrometric analysis of ADP-ribosylation factor proteins from Xenopus egg cytosol

The GTP analog GTP gamma S potently inhibits nuclear envelope assembly in cell-free Xenopus egg extracts. GTP gamma S does not affect vesicle binding to chromatin but blocks vesicle fusion. Fusion inhibition by GTP gamma S is mediated by a soluble factor, initially named GSF (GTP gamma S-dependent soluble factor). We previously showed that vesicles pretreated with GTP gamma S plus recombinant mammalian ARF1 were inhibited for fusion, suggesting that "GSF activity" was due to the ARF (ADP-ribosylation factor) family of small GTP-binding proteins. To ask if any soluble proteins other than ARF also inhibited vesicle fusion in the pretreatment assay, we purified GSF activity from Xenopus egg cytosol. At all steps in the purification, fractions containing ARF, but no other fractions, showed GSF activity. The purified GSF was identified as Xenopus ARF by immunoblotting and peptide sequence analysis. Reverse phase HPLC and mass spectrometry revealed that GSF contained at least three distinct ARF proteins, all of which copurified through three chromatography steps. The most abundant isoform was identified as ARF1 (62% of the total GSF), because its experimentally determined mass of 20 791 Da matched within experimental error that predicted by the sequence of the Xenopus ARF1 cDNA, which is reported here. The second-most abundant isoform (25% of GSF activity) was identified as ARF3. We concluded that ARF is most likely the only soluble protein that inhibits nuclear vesicle fusion after pretreatment with GTP gamma S.     

A new evaluation system to predict the sequelae of late obstetric brachial plexus palsy

Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.     

TPEN, a Zn2+/Fe2+ chelator with low affinity for Ca2+, inhibits lamin assembly, destabilizes nuclear architecture and may independently protect nuclei from apoptosis in vitro

We used Xenopus egg extracts to examine the effects of TPEN, a chelator with strong affinities for Zn2+, Fe2+, and Mn2+, on nuclear assembly in vitro. At concentrations above 1 mM, TPEN blocked the assembly of the nuclear lamina and produced nuclei that were profoundly sensitive to stress-induced balloon-like 'shedding' of nuclear membranes away from chromatin-associated membranes. TPEN-arrested nuclei were also defective for DNA replication, which could be explained as secondary to the lack of a lamina. Imaging of TPEN-arrested nuclei by field emission in-lens scanning electron microscopy (FEISEM) revealed clustered, structurally-perturbed nuclear pore complexes. TPEN-arrested nuclei were defective in the accumulation of fluorescent karyophilic proteins. All detectable effects caused by TPEN were downstream of the effects of BAPTA, a Ca2+/Zn2+ chelator that blocks pore complex assembly at two distinct early stages. Surprisingly, TPEN-arrested nuclei, but not control nuclei, remained active for replication in apoptotic extracts, as assayed by [32P]-dCTP incorporation into high molecular weight DNA, suggesting that TPEN blocks a metal-binding protein(s) required for nuclear destruction during programmed cell death.     

The activation of glycogen synthase by insulin switches from kinase inhibition to phosphatase activation during adipogenesis in 3T3-L1 cells

The effects of insulin and platelet-derived growth factor (PDGF) on glycogen synthase activation were compared in 3T3-L1 fibroblasts and adipocytes. In the fibroblasts, PDGF elicited a stronger phosphorylation of mitogen-activated protein kinase (MAPK) and AKT than did insulin. Both agents caused a comparable stimulation of receptor autophosphorylation, MAPK, and phosphatidylinositol 3-kinase (PI3-K) activation in the adipocytes. However, adipogenesis resulted in the uncoupling of PI3-K activation by PDGF from subsequent AKT phosphorylation. The relative contributions of glycogen synthase kinase-3 (GSK-3) inactivation and protein phosphatase-1 (PP1) activation in the regulation of glycogen synthase in both cell types were evaluated. Insulin and PDGF caused a small increase in glycogen synthase a activity in the fibroblasts. Additionally, both agents caused a similar inhibition of GSK-3, while having no effect on PP1 activity. Following differentiation, insulin treatment resulted in a 5-fold stimulation of glycogen synthase, whereas PDGF was without effect. Both agents caused a comparable inhibition of GSK-3 activity in the adipocytes, whereas only insulin activated PP1. Finally, wortmannin completely blocked the stimulation of PP1 by insulin in 3T3-L1 adipocytes, indicating that PI3-K inhibition can impinge on PP1 activation. Cumulatively these results suggest that the weak activation of glycogen synthase in 3T3-L1 fibroblasts is mediated by GSK-3 inactivation, whereas in the more metabolically active adipocytes, the insulin-specific activation of glycogen synthase is mediated by PP1 activation.     

An indirect role for cyclin B-Cdc2 in inducing chromosome condensation in Xenopus egg extracts

We have studied the cytoplasmic mechanism that induces metaphase chromosome condensation in cell-free Xenopus egg extracts. To analyze the mechanism responsible for inducing chromosome condensation separately from those responsible for sperm chromatin remodeling and nuclear envelope disassembly, we used Xenopus sperm chromatin that had already been remodeled to nucleosomal chromatin by incubating demembranated sperm with egg extracts added with lysolecithin. We found that inhibition of cyclin B-Cdc2 with butyrolactone I abolished chromosome condensation of the remodeled sperm chromatin by M-phase egg extracts, but incubation of the chromatin with active cyclin B-Cdc2 alone did not induce chromosome condensation, indicating a requirement for cytoplasmic factor(s) in addition to cyclin B-Cdc2 for the induction of chromosome condensation. We further demonstrated that if the cyclin B-Cdc2-dependent phosphorylation state was protected against dephosphorylation by a preincubation of M-phase extracts with ATP-gamma-S, chromosome condensation and phosphorylation of chromosomal histone H1 occurred even when extracts were depleted of cyclin B-Cdc2 activity. The chromosome condensation seen in the absence of cyclin B-Cdc2 was completely inhibited with another protein kinase inhibitor, 6-dimethylaminopurine, implying that a protein kinase other than cyclin B-Cdc2 was involved in the induction of chromosome condensation. These results strongly suggest that a cyclin B-Cdc2-dependent protein kinase cascade is involved in inducing chromosome condensation and the phosphorylation of chromosomal histone H1.     

Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts

Ran is a small GTPase that is required for protein import, mRNA export, and the maintenance of nuclear structures. To gain a better understanding of Ran's role in the nucleus, we have sought to use Xenopus egg extracts for the purification and characterization of proteins from egg extracts bound with a high affinity to a glutathione-S-transferase-Ran fusion protein (GST-Ran). We found that GST-Ran associates specifically with at least 10 extract proteins. We determined the identifies of six Ran-interacting proteins (Rips), and found that they include RanBP2/Nup358, Nup153, Importin beta, hsc70, RCC1, and RanBP1. On the basis of peptide sequence, a seventh Rip (p88) seems to be similar but not identical to Fug1/RanGAP1, the mammalian Ran-GTPase-activating protein. Gel filtration analysis of endogenous extract proteins suggests that Importin beta acts as a primary GTP-Ran effector. Both Ran and Importin beta are coimmunoprecipitated by anti-p340RanBP2 antibodies in the presence of nonhydrolyzable GTP analogues, suggesting that Ran-Importin beta complexes interact with p340RanBP2. Two other Rips, p18 and p88, are coprecipitated with p340RanBP2 in a nucleotide-independent manner. Analysis of the Ran-GTPase pathway in Xenopus extracts allows the examination of interactions between Ran-associated proteins under conditions that resemble in vivo conditions more closely than in assays with purified components, and it thereby allows additional insights into the molecular mechanism of nuclear transport.     

Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest

The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK), and p38. Although MAPK is essential for meiotic processes in Xenopus oocytes and the spindle assembly checkpoint in Xenopus egg extracts, the role of members of the MAPK superfamily in M phase or the spindle assembly checkpoint during somatic cell cycles has not been elucidated. The kinase p38, but not MAPK or JNK, was activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole. Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles.     

The place of laparoscopy in pediatric surgery]

Histopathological features of Alzheimer's disease (AD) include extracellular deposits of amyloid beta (A beta) fibrils in the cores of senile plaques, intracellular neurofibrillary tangles (NFT) which are composed of paired helical filaments (PHF), and neuronal cell loss. The main component of PHF is highly phosphorylated tau protein. We identified a protein kinase converting normal tau into a PHF-like state. The kinase is tau protein kinase (TPK) I/glycogen synthase kinase (GSK)-3 beta. Using a neuronal cell culture system as an AD model, it was recognized that TPK I/GSK-3 beta plays a central role in AD pathology. We hypothesize that A beta-induced neuronal cell death occurs by the following mechanism. A beta inactivates PI3-kinase and activates TPK I/GSK-3 beta, which in turn phosphorylates and inactivates both tau and pyruvate dehydrogenase (PDH). After the ability of tau to promote microtubule assembly is diminished by phosphorylation, soluble tau molecules aggregate into PHF by an unknown mechanism. Destabilization of microtubule arrays causes inhibition of axonal transport and accumulation of amyloid precursor protein (APP). Phosphorylation of PDH inhibits the reaction converting pyruvate to acetyl-CoA, resulting in inhibition of energy metabolism and a decrease in acetylcholine, both of which are also characteristics of AD. These changes may lead to neuronal cell death.     

XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts

We used a peptide antibody to a conserved sequence in the motor domain of kinesins to screen a Xenopus ovary cDNA expression library. Among the clones isolated were two that encoded a protein we named XCTK2 for Xenopus COOH-terminal kinesin 2. XCTK2 contains an NH2-terminal globular domain, a central alpha-helical stalk, and a COOH-terminal motor domain. XCTK2 is similar to CTKs in other organisms and is most homologous to CHO2. Antibodies raised against XCTK2 recognize a 75-kD protein in Xenopus egg extracts that cosediments with microtubules. In Xenopus tissue culture cells, the anti-XCTK2 antibodies stain mitotic spindles as well as a subset of interphase nuclei. To probe the function of XCTK2, we have used an in vitro assay for spindle assembly in Xenopus egg extracts. Addition of antibodies to cytostatic factor-arrested extracts causes a 70% reduction in the percentage of bipolar spindles formed. XCTK2 is not required for maintenance of bipolar spindles, as antibody addition to preformed spindles has no effect. To further evaluate the function of XCTK2, we expressed XCTK2 in insect Sf-9 cells using the baculovirus expression system. When purified (recombinant XCTK2 is added to Xenopus egg extracts at a fivefold excess over endogenous levels) there is a stimulation in both the rate and extent of bipolar spindle formation. XCTK2 exists in a large complex in extracts and can be coimmunoprecipitated with two other proteins from extracts. XCTK2 likely plays an important role in the establishment and structural integrity of mitotic spindles.     

Glucose-6-P control of glycogen synthase phosphorylation in yeast

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 beta-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 alpha-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 approximately 2 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.