β-TrCP-Mediated Ubiquitination and Degradation of PHLPP1 Are Negatively Regulated by Akt

ABSTRACT PHLPP1 belongs to a novel family of Ser/Thr protein phosphatases that serve as tumor suppressors by negatively regulating Akt signaling. Our recent studies have demonstrated that loss of PHLPP expression occurs at high frequency in colorectal cancer. In this study, we identified PHLPP1 as a proteolytic target of a β-TrCP-containing Skp-Cullin 1-F-box protein (SCF) complex (SCFβ-TrCP) E3 ubiquitin ligase in a phosphorylation-dependent manner. Overexpression of wild-type but not ΔF-box mutant β-TrCP leads to decreased expression and increased ubiquitination of PHLPP1, whereas knockdown of endogenous β-TrCP has the opposite effect. In addition, we show that the β-TrCP-mediated degradation requires phosphorylation of PHLPP1 by casein kinase I and glycogen synthase kinase 3β (GSK-3β), and activation of the phosphatidylinositol 3-kinase/Akt pathway suppresses the degradation of PHLPP1 by inhibiting the GSK-3β activity. Furthermore, expression of a degradation-deficient PHLPP1 mutant in colon cancer cells results in a more effective dephosphorylation of Akt and inhibition of cell growth. Taken together, our findings demonstrate a key role for β-TrCP in controlling the level of PHLPP1, and activation of Akt negatively regulates this degradation process.

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Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00092.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. H2079-H2086 ◽

Cited By ~ 47

Author(s):

Katsuhiko Ohori ◽

Tetsuji Miura ◽

Masaya Tanno ◽

Takayuki Miki ◽

Takahiro Sato ◽

...

Keyword(s):

Fluorescent Protein ◽

Oxidant Stress ◽

Dependent Manner ◽

Phosphatidylinositol 3 Kinase ◽

H9c2 Cardiomyocytes ◽

Gsk 3Β ◽

Induced Apoptosis ◽

Green Fluorescent ◽

Interfering Rna

The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.

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Regulation of thymocyte β-selection, development and positive selection by glycogen synthase kinase-3

10.1101/712521 ◽

2019 ◽

Author(s):

Michael J. Parsons ◽

Satish Patel ◽

Bradley W. Doble ◽

Pamela S. Ohashi ◽

James R. Woodgett

Keyword(s):

Positive Selection ◽

Glycogen Synthase ◽

Functional Expression ◽

Glycogen Synthase Kinase ◽

Glycogen Synthase Kinase 3 ◽

Phosphatidylinositol 3 Kinase ◽

Double Positive ◽

Serine Threonine Kinase ◽

Gsk 3Β ◽

Knockout Animals

AbstractGlycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine kinase, that exists as two isoforms in mammals, GSK-3α and GSK-3β, that are key downstream mediators of the phosphatidylinositol 3’ kinase, Wnt, Notch and other pathways. Here, we report that simultaneous inactivation of both GSK-3α and GSK-3β during early thymocyte ontogeny has profound effects on both β-selection and positive selection, key checkpoints essential to producing functionally mature αβ T cells. Conditional GSK-3α/β knockout animals (LckCre+ GSK-3αβfl/fl) possessed pre-double positive (pre-DP) thymocytes (CD4−CD8−CD117−CD25−) with compromised TCRβ chain expression along with elevated levels of β-catenin and reduced Notch activity. β-selection was impaired allowing pre-DP thymocytes to differentiate to DP thymocytes (CD4+CD8+) while bypassing strict requirements for productive TCRβ chain rearrangements and functional expression. Also impaired was the requisite pre-TCR and Notch-mediated expansion that normally precedes differentiation to the DP stage. Consequently, LckCre+ GSK-3αβfl/fl mice initially generated fewer DP thymocytes that expressed significantly reduced levels of mature TCR. The aberrant DP thymocytes expressed high levels of the pro-survival Bcl-2 family member Mcl-1, failed positive selection and accumulated as CD4hiCD8lo positive selection intermediates resulting in loss of both mature CD4 and CD8 lineages. LckCre+ GSK-3αβfl/fl mice succumbed to oligoclonal peripheral lymphomas with high penetrance. These data reveal essential roles for GSK-3 in several checkpoints of early T cell development.

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Xuebijing Protects Against Septic Acute Liver Injury Based on Regulation of GSK-3β Pathway

Frontiers in Pharmacology ◽

10.3389/fphar.2021.627716 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liping Cao ◽

Zhenghong Li ◽

Yi Ren ◽

Mengmeng Wang ◽

Zhizhou Yang ◽

...

Keyword(s):

Liver Injury ◽

Acute Liver Injury ◽

Glycogen Synthase ◽

Binding Protein ◽

Cecal Ligation ◽

Inflammatory Factors ◽

Dependent Manner ◽

Protective Effects ◽

Creb Binding Protein ◽

Gsk 3Β

Xuebijing (XBJ), the only drug approved for the sepsis and multiple organ dysfunction, and its protective effects against acute liver injury (ALI) and its mechanism. The aim of this study was to evaluate the protective effect of XBJ on cecal ligation and perforation (CLP)-induced mouse ALI model and LPS-induced RAW264.7 cell ALI model. Mice were pretreated with XBJ before the CLP model was established, and serum and liver tissues were collected at the end of the experiment to assess the levels of inflammatory factors and liver injury. Results showed that XBJ pretreatment reduced liver/body weight, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in serum, and inhibited levels of pro-inflammatory factors in serum. Cells were treatment with XBJ and modeled by LPS modeling increased cell viability in the XBJ-treated group compared to the model group and XBJ also decreased serum pro-inflammatory factors in a dose-dependent manner. Western blot detected that XBJ also up-regulated the phosphorylated levels of glycogen synthase kinase-3β (p-GSK-3β) and cAMP-response element-binding protein (p-CREB) and down-regulated the phosphorylated level of nuclear factor kappa-B (p-NF-κB) in liver and cell. After overexpression of GSK-3β in cells, the mechanism was further investigated using CO-IP analysis. The binding of p-NF-κB and p-CREB to CREB-binding protein (CBP) was increased and decreased, respectively, indicating that GSK-3β regulated inflammation by regulating the binding of p-NF-κB and p-CREB to CBP. The present studies suggested that the hepatoprotective effect of XBJ may be through up-regulation of GSK-3β (Ser9) and increasing the binding of p-CREB to CBP, thereby alleviating the inflammatory response.

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Characterization and Hypoglycemic Activity of a Rhamnan-Type Sulfated Polysaccharide Derivative

Marine Drugs ◽

10.3390/md17010021 ◽

2019 ◽

Vol 17 (1) ◽

pp. 21 ◽

Cited By ~ 6

Author(s):

Jie-Fen Cui ◽

Han Ye ◽

Yu-Jie Zhu ◽

Yin-Ping Li ◽

Jing-Feng Wang ◽

...

Keyword(s):

Glycogen Synthase ◽

Lethal Dose ◽

Insulin Receptors ◽

Sulfated Polysaccharide ◽

Phosphatidylinositol 3 Kinase ◽

Pharmaceutical Development ◽

Hypoglycemic Agent ◽

Gsk 3Β ◽

Polysaccharide Derivative

Polysaccharide chromium (III) derivatives are gaining increasing attention in improving type 2 diabetes. In this study, the sulfated polysaccharide from Enteromorpha prolifera (SPE) with 4.8 kDa was prepared by specific enzymatic hydrolysis. The obtained SPE was used to prepare a rhamnan-type sulfated polysaccharide derivative (SPED). Results indicated that O-H, C=O, and S=O were effectively involved in the chelation of SPED (chromium content 20.26%). Acute (half lethal dose > 2.38 g/kg) and sub-acute toxicity showed that SPED had no damaging effects on mice. Anti-diabetic experiment demonstrated that SPED improved glucose metabolism. Moreover, SPED promoted the PI3K/PKB/GSK-3β signaling pathway by regulating mRNA expression of insulin receptors (IR), insulin receptor substrate 2 (IRS-2), phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3β (GSK-3β). In conclusion, the SPED might represent a novel marine-derived candidate against hyperglycemia, which may undergo further pharmaceutical development as a hypoglycemic agent.

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Central and Peripheral Mechanisms in ApoE4-Driven Diabetic Pathology

International Journal of Molecular Sciences ◽

10.3390/ijms21041289 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1289

Author(s):

Amit Koren-Iton ◽

Shiran Salomon-Zimri ◽

Alex Smolar ◽

Efrat Shavit-Stein ◽

Amir Dori ◽

...

Keyword(s):

Glycogen Synthase ◽

Control Diet ◽

Fasting Blood Glucose ◽

Apoe Genotype ◽

Dependent Manner ◽

Fiber Density ◽

Akt Activation ◽

Glucose Levels ◽

Insulin Tolerance ◽

Gsk 3Β

Apolipoprotein E (APOE) ε4 gene allele and type 2 diabetes mellitus (T2DM) are prime risk factors for Alzheimer’s disease (AD). Despite evidence linking T2DM and apoE4, the mechanism underlying their interaction is yet to be determined. In the present study, we employed a model of APOE-targeted replacement mice and high-fat diet (HFD)-induced insulin resistance to investigate diabetic mechanisms associated with apoE4 pathology and the extent to which they are driven by peripheral and central processes. Results obtained revealed an intriguing pattern, in which under basal conditions, apoE4 mice display impaired glucose and insulin tolerance and decreased insulin secretion, as well as cognitive and sensorimotor characteristics relative to apoE3 mice, while the HFD impairs apoE3 mice without significantly affecting apoE4 mice. Measurements of weight and fasting blood glucose levels increased in a time-dependent manner following the HFD, though no effect of genotype was observed. Interestingly, sciatic electrophysiological and skin intra-epidermal nerve fiber density (IENFD) peripheral measurements were not affected by the APOE genotype or HFD, suggesting that the observed sensorimotor and cognitive phenotypes are related to central nervous system processes. Indeed, measurements of hippocampal insulin receptor and glycogen synthase kinase-3β (GSK-3β) activation revealed a pattern similar to that obtained in the behavioral measurements while Akt activation presented a dominant effect of diet. HFD manipulation induced genotype-independent hyperlipidation of apoE, and reduced levels of brain apoE in apoE3 mice, rendering them similar to apoE4 mice, whose brain apoE levels were not affected by the diet. No such effect was observed in the peripheral plasma levels of apoE, suggesting that the pathological effects of apoE4 under the control diet and apoE3 under HFD conditions are related to the decreased levels of brain apoE. Taken together, our data suggests that diabetic mechanisms play an important role in mediating the pathological effects of apoE4 and that consequently, diabetic-related therapy may be useful in treating apoE4 pathology in AD.

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In vivo regulation of protein-serine kinases by insulin in skeletal muscle of fructose-hypertensive rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.277.2.e299 ◽

1999 ◽

Vol 277 (2) ◽

pp. E299-E307 ◽

Cited By ~ 2

Author(s):

Sanjay Bhanot ◽

Baljinder S. Salh ◽

Subodh Verma ◽

John H. McNeill ◽

Steven L. Pelech

Keyword(s):

Skeletal Muscle ◽

Glycogen Synthase ◽

Insulin Injection ◽

Glycogen Synthase Kinase 3 ◽

Phosphatidylinositol 3 Kinase ◽

S6 Kinase ◽

Serine Kinases ◽

Gsk 3Β

The effects of tail-vein insulin injection (2 U/kg) on the regulation of protein-serine kinases in hindlimb skeletal muscle were investigated in hyperinsulinemic hypertensive fructose-fed (FF) animals that had been fasted overnight. Basal protein kinase B (PKB) activity was elevated about twofold in FF rats and was not further stimulated by insulin. Phosphatidylinositol 3-kinase (PI3K), which lies upstream of PKB, was increased ∼3.5-fold within 2–5 min by insulin in control rats. Basal and insulin-activated PI3K activities were further enhanced up to 2-fold and 1.3-fold, respectively, in FF rats. The 70-kDa S6 kinase (S6K) was stimulated about twofold by insulin in control rats. Both basal and insulin-stimulated S6K activity was further enhanced up to 1.5-fold and 3.5-fold, respectively, in FF rats. In control rats, insulin caused a 40–50% reduction of the phosphotransferase activity of the β-isoform of glycogen synthase kinase 3 (GSK-3β), which is a PKB target in vitro. Basal GSK-3β activity was decreased by ∼40% in FF rats and remained unchanged after insulin treatment. In summary, 1) the PI3K → PKB → S6K pathway was upregulated under basal conditions, and 2) insulin stimulation of PI3K and S6K activities was enhanced, but both PKB and GSK-3 were refractory to the effects of insulin in FF rats.

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PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers

Cell Death and Disease ◽

10.1038/s41419-020-02998-6 ◽

2020 ◽

Vol 11 (9) ◽

Cited By ~ 1

Author(s):

Rui Liu ◽

Youwen Chen ◽

Guangzhi Liu ◽

Chenxi Li ◽

Yurong Song ◽

...

Keyword(s):

Multidrug Resistance ◽

Glycogen Synthase ◽

Mammalian Target Of Rapamycin ◽

Akt Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Cellular Processes ◽

Nuclear Factor Kappa Beta ◽

Apoptosis Related Protein ◽

Gsk 3Β

Abstract Multidrug resistance (MDR) is the dominant challenge in the failure of chemotherapy in cancers. Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that spreads intracellular signal cascades and regulates a variety of cellular processes. PI3Ks are considered significant causes of chemoresistance in cancer therapy. Protein kinase B (AKT) is also a significant downstream effecter of PI3K signaling, and it modulates several pathways, including inhibition of apoptosis, stimulation of cell growth, and modulation of cellular metabolism. This review highlights the aberrant activation of PI3K/AKT as a key link that modulates MDR. We summarize the regulation of numerous major targets correlated with the PI3K/AKT pathway, which is further related to MDR, including the expression of apoptosis-related protein, ABC transport and glycogen synthase kinase-3 beta (GSK-3β), synergism with nuclear factor kappa beta (NF-κB) and mammalian target of rapamycin (mTOR), and the regulation of glycolysis.

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Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen–glucose deprivation-induced apoptosis in primary rat cortical neuronal cells

Journal of Endocrinology ◽

10.1677/joe-08-0160 ◽

2008 ◽

Vol 198 (3) ◽

pp. 511-521 ◽

Cited By ~ 105

Author(s):

Hyunju Chung ◽

Sanghee Seo ◽

Minho Moon ◽

Seungjoon Park

Keyword(s):

Glycogen Synthase ◽

Glucose Deprivation ◽

Glycogen Synthase Kinase ◽

Protective Effects ◽

Glycogen Synthase Kinase 3Β ◽

Phosphatidylinositol 3 Kinase ◽

Unacylated Ghrelin ◽

Gsk 3Β ◽

Induced Apoptosis ◽

Apoptotic Effects

Only acylated ghrelin (AG) binds GH secretagog receptor 1a (GHS-R1a) and has central endocrine activities. An anti-apoptotic effect of AG in neuronal cells has recently been reported. However, whether there is a neuroprotective effect of unacylated ghrelin (UAG), the most abundant form of ghrelin in plasma, is still unknown. Therefore, we investigated whether UAG was neuroprotective against ischemic neuronal injury using primary cultured rat cortical neurons exposed to oxygen and glucose deprivation (OGD). Both AG and UAG inhibited OGD-induced apoptosis. Exposure of cells to the receptor-specific antagonist d-Lys-3-GHRH-6 abolished the protective effects of AG against OGD, whereas those of UAG were preserved, suggesting the involvement of a receptor that is distinct from GHS-R1a. Chemical inhibition of MAPK and phosphatidylinositol-3-kinase (PI3K) blocked the anti-apoptotic effects of AG and UAG. Ghrelin siRNA enhanced apoptosis either during OGD or even in normoxic conditions. The protective effects of AG and UAG were accompanied by an increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt, and glycogen synthase kinase-3β (GSK-3β). Furthermore, treatment of cells with AG or UAG resulted in nuclear translocation of β-catenin. In addition, both AG and UAG increased the Bcl-2/Bax ratio, prevented cytochrome c release, and inhibited caspase-3 activation. The data indicate that, independent of acylation, ghrelin can function as a neuroprotective agent that inhibits apoptotic pathways. These effects may be mediated via activation of the MAPK and PI3K/Akt pathways. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β and stabilization of β-catenin contribute to the anti-apoptotic effects of ghrelin.

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Ubiquitin Carboxyl-Terminal Hydrolase L3 Promotes Insulin Signaling and Adipogenesis

Endocrinology ◽

10.1210/en.2009-0332 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5230-5239 ◽

Cited By ~ 17

Author(s):

Mari Suzuki ◽

Rieko Setsuie ◽

Keiji Wada

Keyword(s):

Insulin Signaling ◽

Glycogen Synthase ◽

Ectopic Expression ◽

Hydrolase Activity ◽

Pcr Analysis ◽

Dependent Manner ◽

Wild Type ◽

Igf I ◽

Carboxyl Terminal

Abstract Insulin is a potent adipogenic hormone that triggers the induction of a series of transcription factors and specific proteins governing the differentiation of preadipocytes into mature adipocytes. Here we report that ubiquitin carboxyl-terminal hydrolase (UCH)-L3, a deubiquitinating enzyme, promotes insulin signaling and adipogenesis. Uchl3−/− mice had less visceral white adipose tissue compared with wild-type mice. In vitro adipogenesis experiments revealed that mouse embryonic fibroblasts (MEFs) and preadipocytes from Uchl3−/− mice had impaired ability to differentiate into mature adipocytes than those from wild-type mice. This difference was diminished by removing insulin from the medium. RT-PCR analysis showed that insulin-regulated expression of srebp1c, fas, glut4, and adiponectin is impaired in Uchl3−/− cells. The phosphorylation of insulin/IGF-I receptor, Akt, glycogen synthase kinase-3β, and FoxO1 was decreased in Uchl3−/− MEFs treated with insulin. Moreover, ectopic expression of wild-type UCH-L3 restored the phosphorylation of insulin/IGF-I receptor and adipocyte differentiation in Uchl3−/− MEFs. In contrast, hydrolase activity-deficient UCH-L3 did not enhance insulin signaling and the expression of glut4, fabp4, and adiponectin, resulting in impaired formation of large lipid droplets. These results suggest that UCH-L3 promotes adipogenesis by enhancing insulin signaling in a hydrolase activity-dependent manner.

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MicroRNA-126 enhances the biological function of endothelial progenitor cells under oxidative stress via PI3K/Akt/GSK-3β and ERK1/2 signaling pathways

Bosnian Journal of Basic Medical Sciences ◽

10.17305/bjbms.2019.4493 ◽

2020 ◽

Cited By ~ 1

Author(s):

Qinqin Wu ◽

Benling Qi ◽

Xiaoyu Duan ◽

Xiaoyan Ming ◽

Fengqin Yan ◽

...

Keyword(s):

Oxidative Stress ◽

Biological Function ◽

Glycogen Synthase ◽

Early Stage ◽

Tube Formation ◽

Dependent Manner ◽

Endothelial Progenitor ◽

H2o2 Treatment ◽

Reverse Transcription Pcr ◽

Gsk 3Β

Endothelial progenitor cell (EPC) transplantation is a safe and effective method to treat acute myocardial infarction (AMI). However, oxidative stress leads to the death of a large number of EPCs in the early stage of transplantation, severely weakening the therapeutic effect. Previous studies demonstrated that microRNAs (miRNAs) regulate the biological function of EPCs. The aim of the current study was to investigate the effect of miRNA on the biological function of EPCs under oxidative stress. Quantitative reverse transcription PCR was performed to detect the expression of miR-126, miR-508-5p, miR-150, and miR-16 in EPCs from rats, among which miR-126 showed a relatively higher expression. Treatment with H2O2 decreased miR-126 expression in EPCs in a dose-dependent manner. EPCs were further transfected with miR-126 mimics or inhibitors, followed by H2O2 treatment. Overexpression of miR-126 enhanced the proliferation, migration, and tube formation of H2O2-treated EPCs. MiR-126 overexpression also inhibited reactive oxygen species and malondialdehyde levels and enhanced superoxide dismutase levels, as well as increased angiopoietin (Ang)1 expression and decreased Ang2 expression in H2O2-treated EPCs. Moreover, miR-126 participated in the regulation of phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt)/ glycogen synthase kinase-3β (GSK-3β) and extracellular signal-regulated kinase (ERK)1/2 signaling in EPCs, where both pathways were activated after miR-126 overexpression in H2O2-treated EPCs. Overall, we showed that miR-126 promoted the biological function of EPCs under H2O2-induced oxidative stress by activating the PI3K/Akt/GSK-3β and ERK1/2 signaling pathway, which may serve as a new therapeutic approach to treat AMI.

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