Prolyl isomerase Pin1 promotes amyloid precursor protein (APP) turnover by inhibiting glycogen synthase kinase-3β (GSK3β) activity: novel mechanism for Pin1 to protect against Alzheimer disease

Alzheimer disease (AD) is characterized by the presence of senile plaques of amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) and neurofibrillary tangles made of hyperphosphorylated Tau. Increasing APP gene dosage or expression has been shown to cause familial early-onset AD. However, whether and how protein stability of APP is regulated is unclear. The prolyl isomerase Pin1 and glycogen synthase kinase-3β (GSK3β) have been shown to have the opposite effects on APP processing and Tau hyperphosphorylation, relevant to the pathogenesis of AD. However, nothing is known about their relationship. In this study, we found that Pin1 binds to the pT330-P motif in GSK3β to inhibit its kinase activity. Furthermore, Pin1 promotes protein turnover of APP by inhibiting GSK3β activity. A point mutation either at Thr-330, the Pin1-binding site in GSK3β, or at Thr-668, the GSK3β phosphorylation site in APP, abolished the regulation of GSK3β activity, Thr-668 phosphorylation, and APP stability by Pin1, resulting in reduced non-amyloidogenic APP processing and increased APP levels. These results uncover a novel role of Pin1 in inhibiting GSK3β kinase activity to reduce APP protein levels, providing a previously unrecognized mechanism by which Pin1 protects against Alzheimer disease.     

Complex formation and reciprocal regulation between GSK3β and C3G

GSK3β, a ubiquitously expressed Ser/Thr kinase, regulates cell metabolism, proliferation and differentiation. Its activity is spatially and temporally regulated dependent on external stimuli and interacting partners, and its deregulation is associated with various human disorders. In this study, we identify C3G (RapGEF1), a protein essential for mammalian embryonic development as an interacting partner and substrate of GSK3β. In vivo and in vitro interaction assays demonstrated that GSK3β and Akt are present in complex with C3G. Molecular modelling and mutational analysis identified a domain in C3G that aids interaction with GSK3β, and overlaps with its nuclear export sequence. GSK3β phosphorylates C3G on primed as well as unprimed sites, and regulates its subcellular localization. Over-expression of C3G resulted in activation of Akt and inactivation of GSK3β. Huntingtin aggregate formation, dependent on GSK3β inhibition, was enhanced upon C3G overexpression. Stable clones of C2C12 cells generated by CRISPR/Cas9 mediated knockdown of C3G, that cannot differentiate, show reduced Akt activity and S9-GSK3β phosphorylation compared to wild type cells. Co-expression of catalytically active GSK3β inhibited C3G induced myocyte differentiation. C3G mutant defective for GSK3β phosphorylation, does not alter S9-GSK3β phosphorylation and, is compromised for inducing myocyte differentiation. Our results show complex formation and reciprocal regulation between GSK3β and C3G. We have identified a novel function of C3G as a negative regulator of GSK3β, a property important for its ability to induce myogenic differentiation.     

Ca2+/Calmodulin-dependent protein kinase kinase beta is regulated by multisite phosphorylation

Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKβ activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKβ activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKβ, which regulate its Ca(2+)/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKβ regulates its half-life. We find that cellular levels of CaMKKβ correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKβ is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKβ-dependent signaling cascades.     

GRK5 influences the phosphorylation of tau via GSK3β and contributes to Alzheimer's disease

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase whose dysfunction results in cognitive impairment and Alzheimer-like pathology, including tau hyperphosphorylation. However, the mechanisms whereby GRK5 influences tau phosphorylation remain incompletely understood. In the current study, we showed that GRK5 influenced the phosphorylation of tau via glycogen synthase kinase 3β (GSK3β). The activity of both tau and GSK3β in the hippocampus was increased in aged GRK5-knockout mice, which is consistent with what occurs in APP/PS1 transgenic mice. Furthermore, GRK5 regulated the activity of GSK3β and phosphorylated tau in vitro. Regardless of changes of GRK5 protein levels, tau hyperphosphorylation remained reduced after GSK3β activity was inhibited, suggesting that GRK5 may specifically influence tau hyperphosphorylation by modulating GSK3β activity. Taken together, our findings suggest that GRK5 deficiency contributes to the pathogenesis of Alzheimer's disease by influencing the hyperphosphorylation of tau through the activation of GSK3β.     

Decrease of GSK3β phosphorylation in the rat nucleus accumbens core enhances cocaine‐induced hyper‐locomotor activity

Glycogen synthase kinase 3β (GSK3β), which is abundantly present in the brain, is known to contribute to psychomotor stimulant‐induced locomotor behaviors. However, most studies have been focused in showing that GSK3β is able to attenuate psychomotor stimulants‐induced hyperactivity by increasing its phosphorylation levels in the nucleus accumbens (NAcc). So, here we examined in the opposite direction about the effects of decreased phosphorylation of GSK3β in the NAcc core on both basal and cocaine‐induced locomotor activity by a bilateral microinjection into this site of an artificially synthesized peptide, S9 (0.5 or 5.0 μg/μL), which contains sequences around N‐terminal serine 9 residue of GSK3β. We found that decreased levels of GSK3β phosphorylation in the NAcc core enhance cocaine‐induced hyper‐locomotor activity, while leaving basal locomotor activity unchanged. This is the first demonstration, to our knowledge, that the selective decrease of GSK3β phosphorylation levels in the NAcc core may contribute positively to cocaine‐induced locomotor activity, while this is not sufficient for the generation of locomotor behavior by itself without cocaine. Taken together, these findings importantly suggest that GSK3β may need other molecular targets which are co‐activated (or deactivated) by psychomotor stimulants like cocaine to contribute to generation of locomotor behaviors.     

GSK3β inactivation in podocytes results in decreased phosphorylation of p70S6K accompanied by cytoskeletal rearrangements and inhibited motility

The inhibition of mTOR kinase after renal transplantation has been associated with podocyte injury and proteinuria; however, the signaling pathways regulating these effects are not well understood. We found that prolonged rapamycin treatment in podocytes leads to an increase in glycogen synthase kinase 3β (GSK3β) phosphorylation, resulting in inactivation of total GSK3β kinase activity. To investigate the cellular consequences of the inactivation of GSK3β, we used two inhibitors reducing kinase activity and studied the cross talk between GSK3 function and the Akt/mammalian target of rapamycin (mTOR) pathway. Both GSK3 inhibitors reduced the phosphorylation of the mTOR downstream target, p70(S6K), indicating that GSK3 inhibition in podocytes is able to cause similar effects as treatment with rapamycin. Moreover, GSK3 inhibition was accompanied by the reduced expression of slit diaphragm-associated proteins and resulted in an altered cytoskeletal structure and reduced motility of podocytes, suggesting that GSK3 kinase can modulate Akt/mTOR-dependent signaling in podocytes.     

In AβPP‐overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AβPP751 and GSK3β activation: effects mitigated by lithium and apparently relevant to sporadic inclusion‐body myositis

Muscle fiber degeneration in sporadic inclusion‐body myositis (s‐IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid‐β (Aβ)‐precursor protein 751 (AβPP751), Aβ, phosphorylated tau, and other ‘Alzheimer‐characteristic’ proteins. Proteasome inhibition is an important component of the s‐IBM pathogenesis. In brains of Alzheimer’s disease (AD) patients and AD transgenic‐mouse models, phosphorylation of neuronal AβPP695 (p‐AβPP) on Thr668 (equivalent to T724 of AβPP751) is considered detrimental because it increases generation of cytotoxic Aβ and induces tau phosphorylation. Activated glycogen synthase kinase3β (GSK3β) is involved in phosphorylation of both AβPP and tau. Lithium, an inhibitor of GSK3β, was reported to reduce levels of both the total AβPP and p‐AβPP in AD animal models. In relation to s‐IBM, we now show for the first time that (1) In AβPP‐overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3β activity and AβPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated‐AβPP, (ii) total amount of AβPP, (iii) Aβ oligomers, and (iv) GSK3β activity; and (c) lithium improves proteasome function. (2) In biopsied s‐IBM muscle fibers, GSK3β is significantly activated and AβPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3β inhibitors, might benefit s‐IBM patients.     

Amyloid beta-induced glycogen synthase kinase 3β phosphorylated VDAC1 in Alzheimer's disease: Implications for synaptic dysfunction and neuronal damage

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase that is involved in the multiple signaling processes of a cell. Increasing evidence suggests that GSK3β plays a key role in multiple cellular processes in the progression of diabetes, obesity, Alzheimer's disease (AD), Parkinson's disease (PD), inflammatory diseases, schizophrenia, bipolar and several mood disorders, and mitochondrial diseases. Recent research has found that increased GSK3β activity is linked to the pathogenesis of AD through amyloid beta (Aβ), phosphorylated tau and mitochondrial dysfunction. Recent research has also revealed that GSK3β is elevated in AD-affected tissues and is critically involved in dissociating the voltage-dependent anion channel 1 (VDAC1) protein from hexokinases, and causing disrupted glucose metabolism, mitochondrial dysfunction and activating apoptotic cell death. The purpose of this article is to review recent research that is elucidating the role of GSK3β in AD pathogenesis. We discuss the involvement of GSK3β in the phosphorylation of VDAC1 and dissociation of VADC1 with hexokinases in AD neurons.     

Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.     

Maintained activity of glycogen synthase kinase-3β despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

Glycogen synthase kinase-3β (GSK3β) is recognized as one of major kinases to phosphorylate tau in Alzheimer’s disease (AD), thus lots of AD drug discoveries target GSK3β. However, the inactive form of GSK3β which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3β substrates, such as β-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3β at serine-9 and other substrates including tau, β-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3β inhibitors such as lithium chloride and 6-bromoindirubin-3′-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3β may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3β inhibitors could be a valuable drug candidate in AD.     

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3β

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.     

DRG2 knockdown induces Golgi fragmentation via GSK3β phosphorylation and microtubule stabilization

The perinuclear stacks of the Golgi apparatus maintained by dynamic microtubules are essential for cell migration. Activation of Akt (protein kinase B, PKB) negatively regulates glycogen synthase kinase 3β (GSK3β)-mediated tau phosphorylation, which enhances tau binding to microtubules and microtubule stability. In this study, experiments were performed on developmentally regulated GTP-binding protein 2 (DRG2)-stably knockdown HeLa cells to determine whether knockdown of DRG2 in HeLa cells treated with epidermal growth factor (EGF) affects microtubule dynamics, perinuclear Golgi stacking, and cell migration. Here, we show that DRG2 plays a key role in regulating microtubule stability, perinuclear Golgi stack formation, and cell migration. DRG2 knockdown prolonged the EGF receptor (EGFR) localization in endosome, enhanced Akt activity and inhibitory phosphorylation of GSK3β. Tau, a target of GSK3β, was hypo-phosphorylated in DRG2-knockdown cells and showed greater association with microtubules, resulting in microtubule stabilization. DRG2-knockdown cells showed defects in microtubule growth and microtubule organizing centers (MTOC), Golgi fragmentation, and loss of directional cell migration. These results reveal a previously unappreciated role for DRG2 in the regulation of perinuclear Golgi stacking and cell migration via its effects on GSK3β phosphorylation, and microtubule stability.     

Glycogen synthase kinase 3β (GSK3β) modulates antiviral activity of zinc-finger antiviral protein (ZAP)

Zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including HIV-1, Ebola virus, and Sindbis virus. ZAP binds directly to specific viral mRNAs and recruits cellular mRNA degradation machinery to degrade the target RNA. ZAP has also been suggested to repress translation of the target mRNA. In this study, we report that ZAP is phosphorylated by glycogen synthase kinase 3β (GSK3β). GSK3β sequentially phosphorylated Ser-270, Ser-266, Ser-262, and Ser-257 of rat ZAP. Inhibition of GSK3β by inhibitor SB216763 or down-regulation of GSK3β by RNAi reduced the antiviral activity of ZAP. These results indicate that phosphorylation of ZAP by GSK3β modulates ZAP activity.     

Brain derived neurotrophic factor is involved in the regulation of glycogen synthase kinase 3β (GSK3β) signalling

Glycogen synthase kinase 3β (GSK3β) is involved in several biochemical processes in neurons regulating cellular survival, gene expression, cell fate determination, metabolism and proliferation. GSK3β activity is inhibited through the phosphorylation of its Ser-9 residue. In this study we sought to investigate the role of BDNF/TrkB signalling in the modulation of GSK3β activity. BDNF/TrkB signalling regulates the GSK3β activity both in vivo in the retinal tissue as well as in the neuronal cells under culture conditions. We report here for the first time that BDNF can also regulate GSK3β activity independent of its effects through the TrkB receptor signalling. Knockdown of BDNF lead to a decline in GSK3β phosphorylation without having a detectable effect on the TrkB activity or its downstream effectors Akt and Erk1/2. Treatment with TrkB receptor agonist had a stimulating effect on the GSK3β phosphorylation, but the effect was significantly less pronounced in the cells in which BDNF was knocked down. The use of TrkB receptor antagonist similarly, manifested itself in the form of downregulation of GSK3β phosphorylation, but a combined TrkB inhibition and BDNF knockdown exhibited a much stronger negative effect. In vivo, we observed reduced levels of GSK3β phosphorylation in the retinal tissues of the BDNF^+/− animals implicating critical role of BDNF in the regulation of the GSK3β activity. Concluding, BDNF/TrkB axis strongly regulates the GSK3β activity and BDNF also exhibits GSK3β regulatory effect independent of its actions through the TrkB receptor signalling.     

GSK‐3β activation index is a potential indicator for recurrent inflammation of chronic rhinosinusitis without nasal polyps

Chronic rhinosinusitis without nasal polyps (CRSsNP) is one of the most common otorhinolaryngologic diseases worldwide. However, the underlying mechanism remains unclear. In this study, the expression of glycogen synthase kinase 3 (GSK‐3) was quantitatively evaluated in patients with CRSsNP (n = 20) and healthy controls (n = 20). The mRNA levels of GSK‐3α and GSK‐3β were examined by qPCR, the immunoreactivities of GSK‐3β and nuclear factor‐κB (NF‐κB) were examined by immunohistochemistry (IHC) staining, and the protein levels of GSK‐3β, phospho‐GSK‐3β (p‐GSK‐3β, s9) and NF‐κB were examined using Western blot analysis. We found that GSK‐3 was highly expressed in both CRSsNP and control groups without significant difference in both GSK‐3β mRNA and protein levels. However, when compared with healthy control group, the GSK‐3β activation index, defined as the ratio of GSK‐3β over p‐GSK‐3β, was significantly decreased, whereas the NF‐κB protein abundance was significantly increased in CRSsNP group (P < 0.05). Strikingly, the GSK‐3β activation index, was highly correlated with NF‐κB protein level, as well as CT scores in CRSsNP group (P < 0.05). It was also highly correlated with the mRNA expressions of inflammation‐related genes, including T‐bet, IFN‐γ and IL‐4 in CRSsNP group (P < 0.05). Our findings suggest that GSK‐3β activation index, reflecting the inhibitory levels of GSK‐3β through phosphorylation, may be a potential indicator for recurrent inflammation of CRSsNP, and that the insufficient inhibitory phosphorylation of GSK‐3β may play a pivotal role in the pathogenesis of CRSsNP.     

Structural basis for the complete loss of GSK3β catalytic activity due to R96 mutation investigated by molecular dynamics study

Many Ser/Thr protein kinases, to be fully activated, are obligated to introduce a phospho‐Ser/Thr in their activation loop. Presently, the similarity of activation loop between two crystal complexes, i.e. glycogen synthase kinase 3β (GSK3β)‐AMPNP and GSK3β‐sulfate ion complex, indicates that the activation segment of GSK3β is preformed requiring neither a phosphorylation event nor conformational changes. GSK3β, when participated in glycogen synthesis and Wnt signaling pathways, possesses a unique feature with the preference of such substrate with a priming phosphate. Experimental mutagenesis proved that the residue arginine at amino acid 96 mutations to lysine (R96K) or alanine (R96A) selectively abolish activity on the substrates involved in glycogen synthesis signaling pathway. Based on two solved crystal structures, wild type (WT) and two mutants (R96K and R96A) GSK3β‐ATP‐phospho‐Serine (pSer) complexes were modeled. Molecular dynamics simulations and energy analysis were employed to investigate the effect of pSer involvement on the GSK3β structure in WT, and the mechanisms of GSK3β deactivation due to R96K and R96A mutations. The results indicate that the introduction of pSer to WT GSK3β generates a slight lobe closure on GSK3β without any remarkable changes, which may illuminate the experimental conclusion, whereas the conformations of GSK3β and ATP undergo significant changes in two mutants. As to GSK3β, the affected positions distribute over activation loop, α‐helix, and glycine‐rich loop. Based on coupling among the mentioned positions, the allosteric mechanisms for distorted ATP were proposed. Energy decomposition on the residues of activation loop identified the important residues Arg96 and Arg180 in anchoring the phosphate group. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Advanced glycation end-product-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 are dependent on glycogen synthase kinase 3beta in LLC-PK1 cells

Glycogen synthase kinase 3β (GSK3β) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3β in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 μg/ml) time-dependently (8-48 h) increased phospho-GSK3β-Tyr216 (active GSK3β) and time-dependently (4-24 h) decreased phospho-GSK3β-Ser21/9 (inactive GSK3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK3β kinase at 8-48 h. AGE (100 μg/ml) dose-dependently (75-100 μg/ml) decreased β-catenin protein expression but AGE did not decrease β-catenin protein expression until 48 h. SB216763 (a GSK3β inhibitor) and GSK3β shRNA attenuated AGE (100 μg/ml)-inhibited cell proliferation and protein expression of β-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3β in LLC-PK1 cells. AGE-inhibited β-catenin and cyclin D1 protein expression are dependent on GSK3β. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3β.     

Attenuation of ischemia–reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts

Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.