Glucose metabolism attenuates p53 and Puma-dependent cell death upon growth factor deprivation

Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation, DNA fragmentation, and loss of clonogenic survival. Importantly, although p53 levels did not change or were slightly reduced, p53 activity was suppressed by elevated glucose metabolism to inhibit Puma induction after growth factor withdrawal. These data show that p53 is metabolically regulated and that glucose metabolism initiates a signaling mechanism to inhibit p53 activation and suppress Puma induction, thus promoting an anti-apoptotic balance to Bcl-2 family protein expression that supports cell survival.     

Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization

Apoptosis is critical for embryonic development, tissue homeostasis, and tumorigenesis and is determined largely by the Bcl-2 family of antiapoptotic and prosurvival regulators. Here, we report that glycogen synthase kinase 3 (GSK-3) was required for Mcl-1 degradation, and we identified a novel mechanism for proteasome-mediated Mcl-1 turnover in which GSK-3beta associates with and phosphorylates Mcl-1 at one consensus motif ((155)STDG(159)SLPS(163)T; phosphorylation sites are in italics), which will lead to the association of Mcl-1 with the E3 ligase beta-TrCP, and beta-TrCP then facilitates the ubiquitination and degradation of phosphorylated Mcl-1. A variant of Mcl-1 (Mcl-1-3A), which abolishes the phosphorylations by GSK-3beta and then cannot be ubiquitinated by beta-TrCP, is much more stable than wild-type Mcl-1 and able to block the proapoptotic function of GSK-3beta and enhance chemoresistance. Our results indicate that the turnover of Mcl-1 by beta-TrCP is an essential mechanism for GSK-3beta-induced apoptosis and contributes to GSK-3beta-mediated tumor suppression and chemosensitization.     

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.     

Human intestinal epithelial crypt cell survival and death: Complex modulations of Bcl-2 homologs by Fak, PI3-K/Akt-1, MEK/Erk, and p38 signaling pathways

To investigate the mechanisms responsible for survival and apoptosis/anoikis in normal human intestinal epithelial crypt cells, we analyzed the roles of various signaling pathways and cell adhesion on the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad) in the well established HIEC-6 cell model. Pharmacological inhibitors and/or dominant-negative constructs were used to inhibit focal adhesion kinase (Fak) and p38 isoforms, as well as the phosphatidylinositol 3'-kinase (PI3-K)/Akt-1 and mitogen-activated protein kinase [MAPK] kinase (MEK)/extracellular regulated kinases (Erk) pathways. Cell adhesion was disrupted by antibody-inhibition of integrin binding or forced cell suspension. The activation levels of studied kinase pathways were also analyzed. Herein, we report that beta1 integrins, Fak, and the PI3-K/Akt-1 pathway, but not beta4 integrins or the MEK/Erk pathway, are crucial for the survival of HIEC-6 cells. Conversely, p38beta, but not p38alpha or gamma, is required for the induction of apoptosis/anoikis in HIEC-6 cells. However, each of the signaling molecules/pathways analyzed were found to affect distinctively the individual expression of the Bcl-2 homologs studied. For example, the inhibition of the PI3-K/Akt-1 pathway down-regulated Bcl-XL, Mcl-1, and Bad, while at the same time up-regulating Bax, whereas the inhibition of Fak up-regulated both Bax and Bak, down-regulated Bad, and did not affect the other Bcl-2 homologs analyzed. These results indicate that integrins, Fak, PI3-K/Akt-1, MEK/Erk, and p38 isoforms perform distinct roles in the regulation of HIEC-6 cell survival and/or death. In addition, our data show that the functions performed by these molecules/pathways in promoting cell survival or apoptosis/anoikis translate into complex, differential modulations of individual Bcl-2 homologs.     

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.     

实时监测高通量低能量激光照射诱导GSK-3β的核质穿梭

糖原合成酶激酶-3(glycogen synthase kinase-3, GSK-3)是一种丝/苏氨酸蛋白激酶.哺乳动物细胞中主要存在GSK-3α与GSK-3β两种亚型.以前的研究认为GSK-3是一种单一的磷酸化糖原合成酶的激酶,可以抑制糖原的合成.最近的研究表明GSK-3可以磷酸化50多种底物,进而调节细胞的多种生理过程,包括细胞结构的改变、代谢,基因表达及细胞凋亡.本文主要研究在高通量低能量激光(high fluence low-power laser irradiation,HF-LPLI)照射下,GSK-3β在活细胞中的动态分布变化.应用荧光蛋白融合蛋白GFP-GSK-3β,在人神经胶质母细胞瘤细胞(U-87)中实时监测高通量低能量激光照射下GSK-3β的动态行为.实验结果显示,120 J/cm~2 的氦氖激光照射后,GSK-3β在9 h时进入细胞核,并维持在核内近2 h, 随后GSK-3β又从细胞核转位到细胞质中. 这表明高通量低能量激光照射激活了GSK-3β.同时,实验结果预示了高通量低能量激光照射可能激活GSK-3β并且参与调控了p53、β-catenin、Myc等相关的转录因子.进一步的研究将探讨在高通量低能量激光照射下,GSK-3β具体调控的转录因子以及调控机制.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

Glycogen synthase kinase-3alpha reduces cardiac growth and pressure overload-induced cardiac hypertrophy by inhibition of extracellular signal-regulated kinases

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3beta. Pressure overload increases expression of GSK-3alpha but not GSK-3beta. Despite our wealth of knowledge about GSK-3beta, the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3beta, inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK.     

mTOR Complex 2 Stabilizes Mcl-1 Protein by Suppressing Its Glycogen Synthase Kinase 3-Dependent and SCF-FBXW7-Mediated Degradation

mTOR complex 2 (mTORC2) regulates cell survival and growth through undefined mechanisms. Mcl-1, a Bcl-2 family protein, functions as an oncogenic protein. The connection between mTORC2 and Mcl-1 stability has not been established and was thus the focus of this study. Mcl-1 levels in cancer cells were decreased by mTOR kinase inhibitors (TORKinibs), which inhibit both mTORCs, by knocking down rictor and by knocking out rictor or Sin1 but not by silencing raptor. TORKinib treatment and rictor knockdown did not alter Mcl-1 mRNA levels but rather decreased its protein stability. Moreover, TORKinib-induced Mcl-1 reduction was rescued by proteasome inhibition. Consistently, TORKinib increased Mcl-1 ubiquitination. Hence, it is clear that inhibition of mTORC2 enhances Mcl-1 degradation, resulting in Mcl-1 reduction. Suppression of glycogen synthase kinase 3 (GSK3) or FBXW7 rescued Mcl-1 reduction induced by TORKinibs or rictor knockdown. Thus, mTORC2 inhibition apparently induces Mcl-1 degradation through a GSK3-dependent and SCF-FBXW7-mediated mechanism. Intriguingly, we detected a direct association between mTORC2 and SCF-FBXW7; this association could be inhibited by TORKinib treatment, suggesting that mTORC2 may directly associate with and inhibit the SCF-FBXW7 complex, resulting in delayed Mcl-1 degradation. Collectively, our findings highlight a novel mechanism by which mTORC2 regulates cell survival and growth by stabilizing Mcl-1.     

Differential mechanisms involved in RG-7388 and Nutlin-3 induced cell death in SJSA-1 osteosarcoma cells

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.     

Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3beta signaling pathways and changes in protein expression

Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin-(IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3beta (GSK-3beta) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3beta may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of "candidate" proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3beta signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases.     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

Increased expression of Mcl-1 is responsible for the blockage of TRAIL-induced apoptosis mediated by EGF/ErbB1 signaling pathway

Epidermal growth factor (EGF) protects against death receptor induced apoptosis in epithelial cells. Herein, we demonstrate that EGF protection against tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis is mediated by increased expression of the Bcl-2 family member myeloid cell leukemia 1 (Mcl-1). EGF increased the mRNA and protein levels of Mcl-1. Furthermore, expression of ErbB1 alone or in combination with ErbB2 in NIH3T3 cells up-regulates Mcl-1 following EGF treatment. In addition, up-regulation of Mcl-1 by EGF is mediated through AKT and NFkappaB activation since kinase inactive AKT and DeltaIkappaB effectively blocks this up-regulation. NFkappaB was also critical for the ability of EGF to prevent TRAIL induced apoptosis as a dominant negative IkappaB (DeltaIkappaB) blocked NFkappaB activation, and relieved EGF protection against TRAIL mediated mitochondrial cytochrome-c release and apoptosis. Finally, anti-sense oligonucleotides directed against Mcl-1 effectively reduced the protein levels of Mcl-1 and blocked EGF protection against TRAIL induced mitochondrial cytochrome-c release and apoptosis. Taken together, EGF signaling leads to increased Mcl-1 expression that is required for blockage of TRAIL induced apoptosis.     

Dual regulation of glycogen synthase kinase-3beta by the alpha1A-adrenergic receptor

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and G? 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.     

The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737

Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.     

Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism

Glycogen synthase kinase 3 comprises two isoforms (GSK-3alpha and GSK-3beta) that are implicated in type II diabetes, neurodegeneration, and cancer. GSK-3 activity is elevated in human and rodent models of diabetes, and various GSK-3 inhibitors improve glucose tolerance and insulin sensitivity in rodent models of obesity and diabetes. Here, we report the generation of mice lacking GSK-3alpha. Unlike GSK-3beta mutants, which die before birth, GSK-3alpha knockout (GSK-3alpha KO) animals are viable but display enhanced glucose and insulin sensitivity accompanied by reduced fat mass. Fasted and glucose-stimulated hepatic glycogen content was enhanced in GSK-3alpha KO mice, whereas muscle glycogen was unaltered. Insulin-stimulated protein kinase B (PKB/Akt) and GSK-3beta phosphorylation was higher in GSK-3alpha KO livers compared to wild-type littermates, and IRS-1 expression was markedly increased. We conclude that GSK-3 isoforms exhibit tissue-specific physiological functions and that GSK-3alpha KO mice are insulin sensitive, reinforcing the potential of GSK-3 as a therapeutic target for type II diabetes.     

Endogenous IGF-I protects human intestinal smooth muscle cells from apoptosis by regulation of GSK-3 beta activity

We have previously shown that endogenous IGF-I regulates human intestinal smooth muscle cell proliferation by activation of phosphatidylinositol 3 (PI3)-kinase- and Erk1/2-dependent pathways that jointly regulate cell cycle progression and cell division. Whereas insulin-like growth factor-I (IGF-I) stimulates PI3-kinase-dependent activation of Akt, expression of a kinase-inactive Akt did not alter IGF-I-stimulated proliferation. In other cell types, Akt-dependent phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) inhibits its activity and its ability to stimulate apoptosis. The aim of the present study was to determine whether endogenous IGF-I regulates Akt-dependent GSK-3 beta phosphorylation and activity and whether it regulates apoptosis in human intestinal muscle cells. IGF-I elicited time- and concentration-dependent GSK-3 beta phosphorylation (inactivation) that was measured by Western blot analysis using a phospho-specific GSK-3beta antibody. Endogenous IGF-I stimulated GSK-3 beta phosphorylation and inhibited GSK-3 beta activity (measured by in vitro kinase assay) in these cells. IGF-I-dependent GSK-3 beta phosphorylation and the resulting GSK-3 beta inactivation were mediated by activation of a PI3-kinase-dependent, phosphoinositide-dependent kinase-1 (PDK-1)-dependent, and Akt-dependent mechanism. Deprivation of serum induced beta-catenin phosphorylation, increased in caspase 3 activity, and induced apoptosis of muscle cells, which was inhibited by either IGF-I or a GSK-3 beta inhibitor. Endogenous IGF-I inhibited beta-catenin phosphorylation, caspase 3 activation, and apoptosis induced by serum deprivation. IGF-I-dependent inhibition of apoptosis, similar to GSK-3 beta activity, was mediated by a PI3-kinase-, PDK-1-, and Akt-dependent mechanism. We conclude that endogenous IGF-I exerts two distinct but complementary effects on intestinal smooth muscle cell growth: it stimulates proliferation and inhibits apoptosis. The growth of intestinal smooth muscle cells is regulated jointly by the net effect of these two processes.     

Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis

The elimination of Mcl-1, an anti-apoptotic Bcl-2 family member, is an early and required step for DNA damage-induced apoptosis. The degradation of Mcl-1 can be blocked by proteasome inhibitors, suggesting a role for the ubiquitin proteasome pathway in apoptosis. Here, we demonstrate that Mcl-1 is ubiquinated at five lysines. Biochemical fractionation of cell extracts allowed us to identify a 482 kDa HECT-domain-containing ubiquitin ligase named Mule (Mcl-1 ubiquitin ligase E3) that is both required and sufficient for the polyubiquitination of Mcl-1. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) domain that allows Mule to specifically interact with Mcl-1. Elimination of Mule expression by RNA interference stabilizes Mcl-1 protein, resulting in an attenuation of the apoptosis induced by DNA-damage agents. Thus, Mule is a unique BH3-containing E3 ubiquitin ligase apical to Bcl-2 family proteins during DNA damage-induced apoptosis.