PKB Alpha Research Articles

Synthesis, Biological Evaluation and Molecular Docking Study of Novel Benzhydryl Piperazine‐1,2,3‐Triazoline Hybrids

AbstractHerein, a novel series of 1,5‐disubstituted‐1,2,3‐triazolines containing 1‐(4‐chlorobenzhydryl) piperazine moiety (8–18) were synthesised and evaluated for their anticancer activity across eight human tumor cell lines. Remarkably, compound 11 substituted with 3‐acetylphenyl group was the most potent anticancer agent against three selected human cancer cell lines (HL‐60, Z138, and DND‐41) with IC50 values of 16.80, 18.50, and 19.20 μM, respectively. In contrast, analogue 10 demonstrated activity against HL‐60, Z138, and DND‐41 cell lines, with IC50 values of 19.90, 18.00, and 18.50 μM, respectively. Moreover, derivative 13 substituted with 4‐bromophenyl moiety displayed activity with IC50=19.90 μM against the DND‐41 cell line. However, all analogues showed IC50 values ranging from 22.95 to 58.45 μM when tested against other investigated cancer cell lines. These findings suggest that derivative 11 holds promise as a potential candidate for synthesizing novel anticancer agents. Furthermore, compounds 8–18 were screened for their antioxidant activity. Molecular docking studies of compound 11 on crystal structures of two proteins, CDK2/cyclin A2 (PDB: 7B7S) and kinase Akt1 PKB alpha (PDB: 4GV1) have been studied.

Abstract 4613: SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer

Abstract v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), a serine/threonine-protein kinase also known as protein kinase B Alpha (PKB Alpha), is a key mediator of a signaling pathway that governs various cellular processes regulating cell growth, survival, glucose metabolism, genome stability and neovascularization. It is known that dysregulation of AKT1 activity is one of the critical factors for the development and/or progression of a variety of human cancers. Posttranslational modifications of AKT1, including phosphorylation, ubiquitination and glycosylation, are well-analyzed, and it is known that the phosphorylation status of AKT1 is strongly correlated with its oncogenic activity. However, biological functions of methylation on AKT1 have never been analyzed so far. In the present study, we demonstrated that the protein lysine methyltrasnferase SMYD3 methylates lysine 14 in the PH domain of AKT1, which is known to play a critical role in the regulation of AKT1 activity through binding to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308, which is the major indicator of AKT1 activity, than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 plays a central role in the oncogenic activity of AKT1. Although Phosphatidylinositide 3-kinase (PI3K)-dependent AKT1 phosphorylation by Pyruvate Dehydrogenase Kinase, Isozyme 1 (PDK1) and mTOR Complex 2 (mTORC2) has long been considered to be the primary mechanism accounting for AKT1 activation, these members are not sufficient enough to explain how AKT1 hyperactivation can occur in tumors with normal levels of PI3K/Phosphatase And Tensin Homolog (PTEN) activity. Our findings unveiled the novel mechanism of constitutive hyperphosphorylation on AKT1 in cancer cells, mediated by SMYD3-mediated methylation. The inhibition of this methylation pathway appears to be a promising strategy to develop anti-cancer drugs. Citation Format: Yuichiro Yoshioka, Yusuke Nakamura, Ryuji Hamamoto. SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4613.

Effects of Bushen Tongmai Recipe (补肾通脉方) on protein kinase Bα expression in polycystic ovary rats with insulin resistance

To observe the effects of Bushen Tongmai Recipe (, BSTMR) on mRNA and protein expressions of protein kinase B alpha (PKB alpha) in hepatic, adipose, muscular, and ovarian tissues of polycystic ovary (PCO) rats with insulin resistance (IR) and to explore the possible molecular mechanism of BSTMR in treating IR and ovulation dysfunction. Female 22-day-old SD rats were injected subcutaneously with sodium prasterone sulfate (9 mg.100g(-1).d(-1)) for 20 days and fed with high-fat diet for 80 days to induce PCO rats with IR. Then, the PCO rats were randomly divided into the model group (n=23) and the treated group (n=21). The treated group was administered with BSTMR for 2 weeks. Meanwhile, a group with 15 rats of the same age was used as the control group. The histological changes in the ovaries were examined. Fasting blood glucose (FBG) was determined by the glucose oxidase method. Serum fasting insulin (Fins) was determined by radioimmunoassay (RIA). The mRNA level of PKBalpha was measured by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry staining and Western blot analysis were employed to detect the protein expression in target tissues. Compared with the control group, the ovaries in the model group showed multiple follicular cysts, levels of FBG and Fins in the model group increased markedly (P<0.05 or P<0.01, respectively), and the insulin sensitive index (ISI) decreased obviously (P<0.01). The mRNA and protein expressions of PKBalpha in target tissues in the model group were dramatically lower than those in the control group (P<0.05 or P<0.01). Compared with the model group, the stratum granulosum of the ovarian follicle in the treated group increased markedly, the level of Fins in the treated group decreased obviously (P<0.01), ISI in the treated group improved markedly (P<0.01), and the mRNA and protein expressions of PKBalpha in target tissues of the treated rats were elevated significantly (P<0.05 or P<0.01). BSTMR could improve IR and ovulation dysfunction in PCO rats with IR, and its molecular mechanisms might be closely related with the elevation of mRNA and protein expressions of PKBalpha in target tissues of PCO rats with IR.

A New Strategy for Studying Protein Kinase B and Its Three Isoforms. Role of Protein Kinase B in Phosphorylating Glycogen Synthase Kinase-3, Tuberin, WNK1, and ATP Citrate Lyase

Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, beta, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described. These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta, or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets. Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was <6% blocked insulin-stimulated phosphorylation of endogenous GSK-3alpha and GSK-3beta isoforms by 67% and 45%, respectively, showing that GSK-3alpha and GSK-3beta are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3alpha and GSK-3beta phosphorylation, with PKBbeta having the predominant role. Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3alpha and GSK-3beta, and a pathway involving atypical PKCs, zeta/lambda, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.

Human Glycated Albumin Affects Glucose Metabolism in L6 Skeletal Muscle Cells by Impairing Insulin-induced Insulin Receptor Substrate (IRS) Signaling through a Protein Kinase Cα-mediated Mechanism

Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.

Protein Kinase Bα/Akt1 Regulates Placental Development and Fetal Growth

Protein kinase B alpha (PKB alpha/Akt1) is implicated in the regulation of metabolism, transcription, cell survival, angiogenesis, cell migration, growth, and tumorigenesis. Previously, it was reported that PKB alpha-deficient mice are small with increased neonatal mortality (Cho, H., Thorvaldsen, J. L., Chu, Q., Feng, F., and Birnbaum, M. J. (2001) J. Biol. Chem. 276, 38349-38352 and Chen, W. S., Xu, P. Z., Gottlob, K., Chen, M. L., Sokol, K., Shiyanova, T., Roninson, I., Wenig, W., Suzuki, R., Tobe, K., Kadowaki, T., and Hay, N. (2001) Genes Dev. 15, 2203-2208). Here we show that PKB alpha is widely expressed in placenta including all types of trophoblast and vascular endothelial cells. Pkb alpha-/- placentae display significant hypotrophy, with marked reduction of the decidual basalis and nearly complete loss of glycogen-containing cells in the spongiotrophoblast, and exhibit decreased vascularization. Pkb alpha-/- placentae also show significant reduction of phosphorylation of PKB and endothelial nitric-oxide synthase. These defects may cause placental insufficiency, fetal growth impairment, and neonatal mortality. These data represent the first evidence for the role of PKB alpha and endothelial nitricoxide synthase in regulating placental development and provide an animal model for intrauterine growth retardation.

Insulin Increases the Association of Akt-2 with Glut4-containing Vesicles

Expression of a constitutively active, membrane-associated Akt-1 (PKB alpha) construct in 3T3L1 adipocytes was shown to induce glucose uptake in the absence of insulin by stimulating Glut4 translocation to the plasma membrane (Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996) J. Biol. Chem. 271, 31372-31378). However, in rat fat cell the vast majority of Akt-1 is cytosolic and shows no re-distribution to the plasma membrane in response to insulin. On the other hand, little work has been done with other Akt family members such as Akt-2 (PKB beta) or Akt-3 (PKB gamma). In this report, an analysis of the subcellular distribution of Akt-2 in rat adipocytes shows that Akt-2 is present in significant amounts in various membrane compartments, as well as in the cytosol, and the former include the light microsomes where Glut4 is present in the basal state. The distribution of Akt-2 in resting adipocytes was found to substantially overlap with that of Glut4 when light microsomes were subfractionated by a sucrose velocity gradient indicating possible co-localization. We confirmed co-localization of Akt-2 and Glut4 in the basal state by immunopurification of Glut4 vesicles, which exhibited a 5.5-fold increase in Akt-2 in response to insulin relative to the amount of Glut4. These results are consistent with the possibility that Akt-2 may be involved in Glut4 vesicle translocation.

Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells.

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.