Carboxy-terminal modulator protein induces Akt phosphorylation and activation, thereby enhancing antiapoptotic, glycogen synthetic, and glucose uptake pathways

2007 ◽  
Vol 293 (5) ◽  
pp. C1576-C1585 ◽  
Author(s):  
Hiraku Ono ◽  
Hideyuki Sakoda ◽  
Midori Fujishiro ◽  
Motonobu Anai ◽  
Akifumi Kushiyama ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Gsk 3Β ◽  
Induced Apoptosis ◽  
Carboxy Terminal ◽  
Interfering Rna

Carboxy-terminal modulator protein (CTMP) was identified as binding to the carboxy terminus of Akt and inhibiting the phosphorylation and activation of Akt. In contrast to a previous study, we found CTMP overexpression to significantly enhance Akt phosphorylation at both Thr308and Ser473as well as the kinase activity of Akt, while phosphatidylinositol 3-kinase (PI3-kinase) activity was unaffected. Translocation of Akt to the membrane fraction was also markedly increased in response to overexpression of CTMP, with no change in the whole cellular content of Akt. Furthermore, the phosphorylations of GSK-3β and Foxo1, well-known substrates of Akt, were increased by CTMP overexpression. On the other hand, suppression of CTMP with small interfering RNA partially but significantly attenuated this Akt phosphorylation. The cellular activities reportedly mediated by Akt activation were also enhanced by CTMP overexpression. UV-B-induced apoptosis of HeLa cells was significantly reversed not only by overexpression of the active mutant of Akt (myr-Akt) but also by that of CTMP. Increases in glucose transport activity and glycogen synthesis were also induced by overexpression of either myr-Akt or CTMP in 3T3-L1 adipocytes. Taking these results into consideration, it can be concluded that CTMP induces translocation of Akt to the membrane and thereby increases the level of Akt phosphorylation. As a result, CTMP enhances various cellular activities that are principally mediated by the PI3-kinase/Akt pathway.

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

2008 ◽  
Vol 295 (5) ◽  
pp. H2079-H2086 ◽  
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.

Role of Phosphatidylinositol 3′- Kinase Pathway in Primary Effusion Lymphoma Survival.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2282-2282
Author(s):  
Azhar R. Hussain ◽  
Shahab Uddin ◽  
Khalid Al-Hussein ◽  
Pulicat S. Manogaran ◽  
Marina I. Gutierrez ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Fas L ◽  
Induced Apoptosis ◽  
Pi3 Kinase Pathway ◽  
Kinase Pathway

Abstract Phosphatidylinositol 3′-kinase (PI3′-kinase) is a key player in cell growth signaling and has been shown to be activated by the K1 protein of Kaposi sarcoma associated herpes virus (KSHV/HHV8). However, the exact role of PI3′-kinase activation in KSHV-associated PEL has not been elucidated. Therefore, we have studied the PI3′-kinase pathway and apoptosis in five PEL cell lines (BC1, BC3, BCBL1, BCP1 and HBL6). Our data show that inhibition of PI3′-kinase by a specific inhibitor, LY294002, induced apoptosis as detected by Annexin V/Propidium Iodide dual staining in the majority of PEL cell lines, including BC1 (43.5+9%), BC3 (62.7+2.4%), BCBL1 (75+5.2%) and HBL6 (36+4.7%). In contrast, BCP1 was resistant to LY294002-induced apoptosis (2%+0.5). We then dissected the PI3′-kinase pathway by analyses of downstream targets of phosphorylation by Western blot. We found that AKT/PKB was constitutively phosphorylated, and thus activated, in all PEL cell lines including BCP1. Interestingly, 24 hours after LY294002 treatment, AKT was completely de-phosphorylated in all cell lines except BCP1, in which a residual phosphorylation level was detected. The downstream elements of AKT, ForkHead (FKHR) and GSK3 were also constitutively phosphorylated in all PEL cell lines. Similarly, treatment with LY294002 prevented this phenomenon in all the cell lines regardless of their final apoptotic endpoint. To confirm specificity of LY294002 treatment on the PI3′-kinase pathway, we tested an unrelated signaling cascade (p38/MAPK) and no changes were observed. Since FKHR was previously shown to upregulate Fas-L in a variety of cells, we analyzed the Fas/Fas-L system in sensitive PEL cell lines following treatment with LY294002. We have previously shown surface expression of CD95 in these cell lines. We now observed that neutralization of Fas/CD95 by the ZB4 antibody did not influence LY294002 apoptosis. Furthermore, co-treatment with LY294002 and CH11 had an additive apoptotic effect. Inhibition of PI3′-kinase activity further downstream induced cleavage of Bid in all PEL cells. However, cytochrome C was only released from mitochondria in LY294002- sensitive BC1 cells and not in the resistant BCP1 cells. The release of cytochrome C in the sensitive BC1 cell line led to activation of Caspase-9 and 3 and cleavage of PARP, none of which occured in the LY294002 resistant BCP1 cell line. Similarly, the expression of the inhibitor of apoptosis, XIAP, which is also a downstream target of AKT, was compromised in the sensitive cell lines following LY294002 treatment. Our data demonstrate that the PI3′-kinase pathway plays a major role in growth and survival of PEL cells since blocking PI3′-kinase activity induces apoptosis. Although this LY294002 induced apoptosis does not appear to involve Fas/Fas-L, it is caspase dependent and compromises XIAP expression. The residual AKT activity in the LY294002 resistant BCP1 cell line may be protecting this cell line from apoptosis. Altogether, these results suggest that blocking the PI3′-kinase pathway may be a potential target for therapeutic intervention in most primary effusion lymphomas.

scholarly journals NF-κB-Inducing Kinase (NIK) Mediates Skeletal Muscle Insulin Resistance: Blockade by Adiponectin

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3622-3627 ◽  
Author(s):  
Sanjeev Choudhary ◽  
Sandeep Sinha ◽  
Yanhua Zhao ◽  
Srijita Banerjee ◽  
Padma Sathyanarayana ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Muscle Insulin Resistance ◽  
Akt Phosphorylation ◽  
Skeletal Muscle Insulin Resistance ◽  
Obese Subjects

Enhanced levels of nuclear factor (NF)-κB-inducing kinase (NIK), an upstream kinase in the NF-κB pathway, have been implicated in the pathogenesis of chronic inflammation in diabetes. We investigated whether increased levels of NIK could induce skeletal muscle insulin resistance. Six obese subjects with metabolic syndrome underwent skeletal muscle biopsies before and six months after gastric bypass surgery to quantitate NIK protein levels. L6 skeletal myotubes, transfected with NIK wild-type or NIK kinase-dead dominant negative plasmids, were treated with insulin alone or with adiponectin and insulin. Effects of NIK overexpression on insulin-stimulated glucose uptake were estimated using tritiated 2-deoxyglucose uptake. NF-κB activation (EMSA), phosphatidylinositol 3 (PI3) kinase activity, and phosphorylation of inhibitor κB kinase β and serine-threonine kinase (Akt) were measured. After weight loss, skeletal muscle NIK protein was significantly reduced in association with increased plasma adiponectin and enhanced AMP kinase phosphorylation and insulin sensitivity in obese subjects. Enhanced NIK expression in cultured L6 myotubes induced a dose-dependent decrease in insulin-stimulated glucose uptake. The decrease in insulin-stimulated glucose uptake was associated with a significant decrease in PI3 kinase activity and protein kinase B/Akt phosphorylation. Overexpression of NIK kinase-dead dominant negative did not affect insulin-stimulated glucose uptake. Adiponectin treatment inhibited NIK-induced NF-κB activation and restored insulin sensitivity by restoring PI3 kinase activation and subsequent Akt phosphorylation. These results indicate that NIK induces insulin resistance and further indicate that adiponectin exerts its insulin-sensitizing effect by suppressing NIK-induced skeletal muscle inflammation. These observations suggest that NIK could be an important therapeutic target for the treatment of insulin resistance associated with inflammation in obesity and type 2 diabetes.

Clenbuterol prevents epinephrine from antagonizing insulin-stimulated muscle glucose uptake

2002 ◽  
Vol 92 (3) ◽  
pp. 1285-1292 ◽  
Author(s):  
Desmond G. Hunt ◽  
Zhenping Ding ◽  
John L. Ivy
Keyword(s):  
Glucose Uptake ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Intracellular Concentration ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Rat Skeletal Muscle ◽  
Slow Twitch ◽  
Chronic Clenbuterol ◽  
Fast Twitch

In the present study, we investigated the effects of chronic clenbuterol treatment on insulin-stimulated glucose uptake in the presence of epinephrine in isolated rat skeletal muscle. Insulin (50 μU/ml) increased glucose uptake in both fast-twitch (epitrochlearis) and slow-twitch (soleus) muscles. In the presence of 24 nM epinephrine, insulin-stimulated glucose uptake was completely suppressed. This suppression of glucose uptake by epinephrine was accompanied by an increase in the intracellular concentration of glucose 6-phosphate and a decrease in insulin-receptor substrate-1-associated phosphatidylinositol 3-kinase (IRS-1/PI3-kinase) activity. Clenbuterol treatment had no direct effect on insulin-stimulated glucose uptake. However, after clenbuterol treatment, epinephrine was ineffective in attenuating insulin-stimulated muscle glucose uptake. This ineffectiveness of epinephrine to suppress insulin-stimulated glucose uptake occurred in conjunction with its inability to increase the intracellular concentration of glucose 6-phosphate and attenuate IRS-1/PI3-kinase activity. Results of this study indicate that the effectiveness of epinephrine to inhibit insulin-stimulated glucose uptake is severely diminished in muscle from rats pretreated with clenbuterol.

MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3350-3350 ◽  
Author(s):  
Yijiang Shi ◽  
HuaJun Yan ◽  
Patrick Frost ◽  
Bao Hoang ◽  
Joseph Gera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
G1 Arrest ◽  
Akt Activation ◽  
Induced Apoptosis

Abstract MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.

Integrin Linked Kinase Is Expressed in Acute Myeloid Leukemia Blasts and Its Inhibition Is Cytotoxic to Leukemic Progenitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 154-154
Author(s):  
Andrew Muranyi ◽  
Shokat Dedhar ◽  
Donna E. Hogge
Keyword(s):  
Acute Myeloid Leukemia ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Akt Phosphorylation ◽  
Downstream Effector ◽  
Integrin Linked Kinase ◽  
Dose Dependent Decrease ◽  
Gsk 3Β ◽  
Acute Myeloid

Abstract Constitutive activation of the phosphatidylinositol-3-kinase (PI-3K) pathway is frequent in acute myeloid leukemia (AML) blasts and down-regulation of this pathway results in apoptotic death of these cells from many patient samples. Integrin linked kinase (ILK) is stimulated by activated PI-3K and ILK, in turn, activates AKT, a key downstream effector of the PI-3K pathway. The expression and activity of ILK are increased in a range of solid tumors. Small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumour growth, invasion and angiogenesis. We investigated the possible role of ILK in AML blast and colony forming cell (AML-CFC) survival. Using Western blotting, ILK protein was detected in 30 of 35 primary AML blast samples although the levels seen were variable. ILK kinase activity correlated strongly with ILK protein expression as quantitated by densitometry (r = 0.91) in 6 samples where both were studied. Activation of the PI-3K pathway, as measured by detection of AKT phosphorylation on serine 473 (ser473), was also found in 27 of 30 samples expressing ILK protein. QLT0267 is a potent second generation small molecule inhibitor (from QLT Inc., Vancouver, Canada) which selectively inhibits the kinase activity of ILK but not a variety of other kinases. Treatment of AML blasts with QLT0267 resulted in a time and dose dependent decrease in p-AKT ser473 expression as well as downstream targets of AKT (p-S6, p-GSK-3β). 27 AML and 5 normal bone marrow (NBM) samples were incubated for 48h with QLT0267 or 10 μM of the PI-3K inhibitor, LY294002, and then plated in CFC assay. There was a direct correlation between the % AML-CFC kill seen with LY204002 and QLT0267 (r= 0.70 and 0.60 comparing % kill with LY294002 and QLT0267 at 3 and 10 μM, respectively). The IC50 of QLT0267 was ≤ 3μM for 6 of 23 AML samples and 0 of 5 NBM samples (range % kill of AML and normal CFC after treatment with 3μM; 0 – 86% and 0 – 23%, respectively). The IC90 for this inhibitor was ≤ 10μM for 9 of 27 AML and 0 of 5 NBMs (range % kill 6 – 99 and 30 – 68, respectively, for AML and normal CFC treated with 10μM QLT0267). Interestingly, AML-CFC from 4 AML samples in which ILK protein could not be detected were resistant to killing with QLT0267. Thus, ILK is expressed in a large proportion of AML samples. Inhibition of ILK kinase is cytotoxic to leukemic progenitors suggesting that this molecule is important for the survival of these cells. Approximately one third of AML samples tested were more susceptible to killing by ILK inhibition than NBM cells suggesting that selective targeting of malignant rather than normal hematopoietic cells may be achieved in some cases. Furthermore, it may be possible to predict which AML samples will be sensitive to inhibitors such as QLT0267 by measuring expression of the target protein.

scholarly journals CTLA-4 and PD-1 Receptors Inhibit T-Cell Activation by Distinct Mechanisms

2005 ◽  
Vol 25 (21) ◽  
pp. 9543-9553 ◽  
Author(s):  
Richard V. Parry ◽  
Jens M. Chemnitz ◽  
Kenneth A. Frauwirth ◽  
Anthony R. Lanfranco ◽  
Inbal Braunstein ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Direct Evidence ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Pi3k Activation ◽  
Cellular Phenotypes ◽  
Induced Changes

ABSTRACT CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.

scholarly journals Phosphatidylinositol 3-kinase activity is important for progesterone-induced Xenopus oocyte maturation.

1993 ◽  
Vol 13 (11) ◽  
pp. 6661-6666 ◽  
Author(s):  
A J Muslin ◽  
A Klippel ◽  
L T Williams
Keyword(s):  
Oocyte Maturation ◽  
Kinase Activity ◽  
Sh2 Domain ◽  
Pi3 Kinase ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Phosphatidylinositol 3 Kinase ◽  
Kinase Activation ◽  
Phosphatidylinositol 3

In somatic cells, phosphatidylinositol 3-kinase (PI3 kinase) is a critical intermediary in growth factor-induced mitogenesis. We have examined the role of this enzyme in meiotic maturation of Xenopus laevis oocytes. PI3 kinase activity was present in immunoprecipitates of the p85 subunit of PI3 kinase from immature oocytes and markedly increased following progesterone stimulation. Injection of bacterially expressed protein corresponding to the C-terminal SH2 domain of p85 (SH2-C) inhibited progesterone-induced PI3 kinase activation and meiotic maturation. Injection of protein corresponding to the N-terminal SH2 domain or the SH3 domain of p85 did not inhibit PI3 kinase activation or maturation. SH2-C did not inhibit oocyte maturation induced by c-mos RNA injection. In addition, radiolabelled SH2-C was used to probe oocyte lysates, revealing that a novel 200-kDa protein bound to SH2-C. This protein may be an important mediator of progesterone-induced lipid metabolism in oocytes.

Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct-ligated rats

2003 ◽  
Vol 284 (5) ◽  
pp. G837-G852 ◽  
Author(s):  
Marco Marzioni ◽  
Gene D. LeSage ◽  
Shannon Glaser ◽  
Tushar Patel ◽  
Carla Marienfeld ◽  
...  
Keyword(s):  
Bile Duct ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Caspase 8 ◽  
Phosphatidylinositol 3 Kinase ◽  
Intrahepatic Bile Ducts ◽  
Duct Ligation ◽  
Phosphorylated Akt ◽  
Bile Acid Transporter ◽  
Expression And Activity

The aim of this study was to determine whether taurocholate prevents vagotomy-induced cholangiocyte apoptosis. After bile duct ligation (BDL) + vagotomy, rats were fed taurocholate for 1 wk in the absence or presence of wortmannin. Caspase involvement was evaluated by measurement of caspase 8, 9, and 3 activities. Proliferation was determined by morphometry and PCNA immunoblots. Changes in phosphatidylinositol 3-kinase (PI3-kinase) activity were estimated by the expression of the phosphorylated Akt protein. Apically located Na+-dependent bile acid transporter (ABAT) expression and activity were evaluated by immunoblots and [3H]taurocholate uptake, respectively. Cholangiocyte apoptosis increased, whereas proliferation decreased in BDL + vagotomy rats. Taurocholate feeding prevented vagotomy effects on cholangiocyte functions, which were abolished by wortmannin. ABAT expression and activity as well as phosphorylated Akt protein expression were reduced by vagotomy but restored by taurocholate. The activities of caspase 8, 9, and 3 increased in BDL + vagotomy rats but were restored by taurocholate. The protective effect of taurocholate was associated with maintenance of ABAT activity, downregulation of caspase 8, 9, and 3, and activation of PI3-kinase. Bile acids are important in modulating cholangiocyte proliferation in denervated livers.

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