Oleanane triterpenoid CDDO-Me inhibits Akt activity without affecting PDK1 kinase or PP2A phosphatase activity in cancer cells

Our previous studies have shown that methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane synthetic triterpenoid induces apoptosis in prostate cancer cells by inhibiting the Akt/NF-κB/mTOR signaling cascade; however, the mechanism by which CDDO-Me inhibits Akt/NF-κB/mTOR signaling has remained undetermined. Present studies show that Akt plays a critical role in the response of prostate cancer cells to CDDO-Me. Silencing of Akt sensitized PC-3 cells to CDDO-Me, whereas its overexpression rendered them resistant to CDDO-Me. Evaluation of the effect of CDDO-Me on Akt which lies upstream of NF-κB and mTOR showed that CDDO-Me directly inhibits the Akt kinase activity in cell-free kinase activity assay and in vivo without modulating the activity of PDK1, the upstream kinase that phosphorylates and activates Akt. The inhibition of Akt activity resulted in inhibition of phosphorylation/inactivation of proapoptotic procaspase-9, Bad and Foxo3a. Further, inhibition of p-Akt by CDDO-Me was not attributable to an increase in the activity of protein phosphatase 2A (PP2A) or PH domain/leucine-rich repeat protein phosphatase1 (PHLPP1) both of which dephosphorylate p-Akt. These findings show that Akt is a direct target of CDDO-Me in the Akt/NF-κB/mTOR prosurvival signaling axis.     

UBE2T-mediated Akt ubiquitination and Akt/β-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/β-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T’s effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, β-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.Subject terms: Liver cancer, Ubiquitylation     

CRKL promotes hepatocarcinoma through enhancing glucose metabolism of cancer cells via activating PI3K/Akt

Abnormal glucose metabolism may contribute to cancer progression. As a member of the CRK (v-crk sarcoma virus CT10 oncogene homologue) adapter protein family, CRKL (CRK-like) associated with the development and progression of various tumours. However, the exact role and underlying mechanism of CRKL on energy metabolism remain unknown. In this study, we investigated the effect of CRKL on glucose metabolism of hepatocarcinoma cells. CRKL and PI3K were found to be overexpressed in both hepatocarcinoma cells and tissues; meanwhile, CRKL up-regulation was positively correlated with PI3K up-regulation. Functional investigations revealed that CRKL overexpression promoted glucose uptake, lactate production and glycogen synthesis of hepatocarcinoma cells by up-regulating glucose transporters 1 (GLUT1), hexokinase II (HKII) expression and down-regulating glycogen synthase kinase 3β (GSK3β) expression. Mechanistically, CRKL promoted glucose metabolism of hepatocarcinoma cells via enhancing the CRKL-PI3K/Akt-GLUT1/HKII-glucose uptake, CRKL-PI3K/Akt-HKII-glucose-lactate production and CRKL-PI3K/Akt-Gsk3β-glycogen synthesis. We demonstrate CRKL facilitates HCC malignancy via enhancing glucose uptake, lactate production and glycogen synthesis through PI3K/Akt pathway. It provides interesting fundamental clues to CRKL-related carcinogenesis through glucose metabolism and offers novel therapeutic strategies for hepatocarcinoma.     

Negative regulation of phosphatidylinositol 3-kinase and Akt signalling pathway by PKC

Although substantial studies have begun to explore the regulation of phosphatidylinositol 3-kinase/Akt cascade by different signalling pathways, whether protein kinase C (PKC) activity plays a crucial role remains as yet unclear. In this study, we found that in A549 and HEK293 cells non-selective PKC inhibitors Ro 31-8220 and bisindolylmaleimide VIII, and PKCbeta inhibitor LY 379196, caused Akt/PKB phosphorylation at Ser 473 and increased the upstream activator, integrin-linked kinase (ILK) activity. The increased Akt phosphorylation was blocked by phosphatidylinositol 3-kinase inhibitor wortmannin and the newly identified PIP(3)-dependent kinases (PDK) inhibitor SB 203580. In contrast to the Akt stimulation caused by PKC inhibitors, PMA attenuated Akt/PKB phosphorylation. We also found that this stimulating effect on Akt phosphorylation by PKC inhibitors was not the result of phosphatase inhibition, since treatment with PP2A, PP2B and tyrosine phosphatase inhibitors (okadaic acid, FK506 and sodium orthovanadate, respectively) had no effect. We conclude that phosphatidylinositol 3-kinase/Akt signalling pathway is regulated by PKC in a negative manner.     

Protein phosphatase 1-dependent dephosphorylation of Akt is the prime signaling event in sphingosine-induced apoptosis in Jurkat cells

Sphingosine (SPH) is an important bioactive lipid involved in mediating a variety of cell functions including apoptosis. However, the signaling mechanism of SPH-induced apoptosis remains unclear. We have investigated whether SPH inhibits survival signaling in cells by inhibiting Akt kinase activity. This study demonstrates that treatment of Jurkat cells with SPH leads to Akt dephosphorylation as early as 15 min, and the cells undergo apoptosis after 6 h. This Akt dephosphorylation is not mediated through deactivation of upstream kinases, since SPH does not inhibit the upstream phosphoinositide-dependent kinase 1 (PDK1) phosphorylation. Rather, sensitivity to the Ser/Thr protein phosphatase inhibitors (calyculin A, phosphatidic acid, tautomycin, and okadaic acid) indicates an important role for protein phosphatase 1 (PP1) in this process. In vitro phosphatase assay, using Akt immunoprecipitate following treatment with SPH, reveals an increase in Akt-PP1 association as determined by immunoprecipitation analysis. Moreover, SPH-induced dephosphorylation of Akt at Ser(473) subsequently leads to the activation of GSK-3β, caspase 3, PARP cleavage, and ultimately apoptosis. Pre-treatment with caspase 3 inhibitor z-VAD-fmk and Ser/Thr phosphatase inhibitor abrogates the effect of SPH on facilitating apoptosis. Altogether, these results demonstrate that PP1-mediated inhibition of the key anti-apoptotic protein, Akt, plays an important role in SPH-mediated apoptosis in Jurkat cells.     

Protein phosphatase 2A negatively regulates insulin's metabolic signaling pathway by inhibiting Akt (protein kinase B) activity in 3T3-L1 adipocytes

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C lambda (PKC lambda) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC lambda activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC lambda expression or PKC lambda siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC lambda and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.     

Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt

A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B', B' gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aalpha scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aalpha paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aalpha knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aalpha with a mutant with selective B' subunit binding deficiency implicated PP2A/B' heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Balpha and Bdelta led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Balpha and Bdelta in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties.     

DMH1 Increases Glucose Metabolism through Activating Akt in L6 Rat Skeletal Muscle Cells

DMH1(4-[6-(4-Isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline) is a compound C analogue with the structural modifications at the 3- and 6-positions in pyrazolo[1,5-a]pyrimidine backbone. Compound C was reported to inhibit both AMPK and Akt. Our preliminary work found that DMH1 activated Akt. Since Akt was involved in glucose metabolism, we aimed to identify the effects of DMH1 on glucose metabolism in L6 rat muscle cells and the potential mechanism. Results showed that DMH1 increased lactic acid release and glucose consumption in L6 rat muscle cells in a dose-dependent manner. DMH1 activated Akt in L6 cells. Akt inhibitor inhibited DMH1-induced Akt activation and DMH1-induced increases of glucose uptake and consumption. DMH1 had no cytotoxicity in L6 cells, but inhibited mitochondrial function and reduced ATP production. DMH1 showed no effect on AMPK, but in the presence of Akt inhibitor, DMH1 significantly activated AMPK. Compound C inhibited DMH1-induced Akt activation in L6 cells. Compound C inhibited DMH1-induced increase of glucose uptake, consumption and lactic acid release in L6 cells. DMH1 inhibited PP2A activity, and PP2A activator forskolin reversed DMH1-induced Akt activation. We concluded that DMH1 increased glucose metabolism through activating Akt and DMH1 activated Akt through inhibiting PP2A activity in L6 rat muscle cells. In view of the analogue structure of DMH1 and compound C and the contrasting effects of DMH1 and compound C on Akt, the present study provides a novel leading chemical structure targeting Akt with potential use for regulating glucose metabolism.     

A novel Epac-Rap-PP2A signaling module controls cAMP-dependent Akt regulation

Rap1b has been implicated in the transduction of the cAMP mitogenic signal. It is phosphorylated and activated by cAMP, and its expression in models where cAMP is mitogenic leads to proliferation and tumorigenesis. Akt is a likely downstream effector of cAMP-Rap1 action. cAMP elevation induced a rapid and transient Akt inhibition that required activated and phosphorylated Rap1b. However, the mechanism(s) by which cAMP-Rap regulates Akt remains unclear. Here we show that (i) upstream regulators, PIK and PDK1, are not the target(s) of the cAMP inhibitory action; (ii) constitutively active Akt and calyculin A-stimulated Akt are resistant to cAMP inhibition, suggesting the action of a phosphatase; (iii) cAMP increases the rate of Akt dephosphorylation, directly implicating an Akt-phosphatase; (iv) Epac- and protein kinase A (PKA)-specific analogs synergistically inhibit Akt, indicating the involvement of both cAMP-dependent effector pathways; (v) H89 and dominant negative Epac 279E block cAMP-inhibitory action; (vi) Epac associates in a complex with Akt and PP2A, and the associated-phosphatase activity is positively modulated by cAMP in a PKA- and Rap1-dependent manner; (vii) like its action on Akt inhibition, PKA- and Epac-specific analogs synergistically activate Epac-associated PP2A; and (viii) dominant negative PP2A blocks cAMP-inhibitory action. Thus, we uncovered a novel cAMP-Epac/PKA-Rap1b-PP2A signaling module involved in Akt regulation that may represent a physiological event in the process of cAMP stimulation of thyroid mitogenesis.     

Integrin alpha 2 beta 1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3 beta

Serine/threonine kinase Akt is a downstream effector protein of phosphatidylinositol-3-kinase (PI-3K). Many integrins can function as positive modulators of the PI-3K/Akt pathway. Integrin alpha 2 beta 1 is a collagen receptor that has been shown to induce specific signals distinct from those activated by other integrins. Here, we found that, in contrast what was found for cells adherent to fibronectin, alpha 2 beta 1-mediated cell adhesion to collagen leads to dephosphorylation of Akt and glycogen synthase kinase 3 beta (GSK3 beta) and concomitantly to the induction of protein serine/threonine phosphatase 2A (PP2A) activity. PP2A activation can be inhibited by mutation in the alpha 2 cytoplasmic domain and by a function-blocking anti-alpha 2 antibody. Akt can be coprecipitated with PP2A, and coexpression of Akt with PP2Ac (catalytic subunit) inhibits Akt kinase activity. Integrin alpha 2 beta 1-related activation of PP2A is dependent on Cdc42. These results indicate that cell adhesion to collagen modulates Akt activity via the alpha 2 beta 1-induced activation of PP2A.     

Akt1/Akt2 and mammalian target of rapamycin/Bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors

Akt, also known as protein kinase B, is a serine/threonine protein kinase with antiapoptotic activities; also, it is a downstream target of phosphatidylinositol 3-kinase. Here we show that Akt1/Akt2 play a critical role in osteoclast differentiation but not cell survival and that mammalian target of rapamycin (mTOR) and Bim, a pro-apoptotic Bcl-2 family member, are required for cell survival in isolated osteoclast precursors. To investigate the function of Akt1, Akt2, mTOR, and Bim, we employed a retroviral system for delivery of small interfering RNA into cells. Loss of Akt1 and/or Akt2 protein inhibited osteoclast differentiation due to down-regulation of IkappaB-kinase (IKK) alpha/beta activity, phosphorylation of IkappaB-alpha, nuclear translocation of nuclear factor-kappaB (NFkappaB) p50, and NFkappaB p50 DNA-binding activity. Surprisingly, deletion of Akt1 and/or Akt2 protein did not stimulate cleaved caspase-3 activity and failed to promote apoptosis. Conversely, loss of mTOR protein induced apoptosis due to up-regulation of cleaved caspase-3 activity. In addition, we found that mTOR is downstream of phosphatidylinositol 3-kinase (but not Akt) and that macrophage colony-stimulating factor regulates Bim expression through mTOR activation for cell survival. These results demonstrate that Akt1/Akt2 are key elements in osteoclast differentiation and that the macrophage colony-stimulating factor stimulation of mTOR leading to Bim inhibition is essential for cell survival in isolated osteoclast precursors.     

Protein phosphatase 2A-linked and -unlinked caspase-dependent pathways for downregulation of Akt kinase triggered by 4-hydroxynonenal

We studied the signal pathways for regulation of serine/threonine protein kinase Akt in Jurkat cells that had been treated with 4-hydroxynonenal (HNE) for caspase-dependent apoptosis induction. Treatment of cells with HNE led to a decrease in the level of Akt activity due to the dephosphorylation at Ser473, a major regulatory phosphorylation site. HNE-mediated dephosphorylation of Akt was prevented by a protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and by a caspase-3 inhibitor, DEVD-CHO. HNE treatment resulted in an increase in the total level of PP2A activity, release of active tyrosine-dephosphorylated PP2A from the cytoskeleton and PP2A-Akt association, which were all dependent on caspase-3 activation. These results suggest that the level of PP2A activity is at least in part determined by its tyrosine phosphorylation, which is dually controlled by okadaic acid-sensitive phosphatases and protein-tyrosine kinases. Possibly underlying the mechanism of caspase-mediated activation of PP2A, HNE treatment resulted in downregulation of the activity of Src kinase, as a representative caspase-sensitive kinase to phosphorylate PP2A at tyrosine. In addition, activated caspase-3 partially cleaved Akt at a late stage of the apoptosis. These results indicate the existence of two distinct caspase-dependent signal pathways for downregulation of Akt that works as a mechanism of positive feedback regulation for HNE-triggered apoptotic signals.     

A triangular connection between Cyclin G,PP2A and Akt1 in the regulation of growth and metabolism in Drosophila

Size and weight control is a tightly regulated process, involving the highly conserved Insulin receptor/target of rapamycin (InR/TOR) signaling cascade. We recently identified Cyclin G (CycG) as an important modulator of InR/TOR signaling activity in Drosophila. cycG mutant flies are underweight and show a disturbed fat metabolism resembling TOR mutants. In fact, InR/TOR signaling activity is disturbed in cycG mutants at the level of Akt1, the central kinase linking InR and TORC1. Akt1 is negatively regulated by protein phosphatase PP2A. Notably the binding of the PP2A B′-regulatory subunit Widerborst (Wdb) to Akt1 is differentially regulated in cycG mutants, presumably by a direct interaction of CycG and Wdb. Since the metabolic defects of cycG mutant animals are abrogated by a concomitant loss of Wdb, CycG presumably influences Akt1 activity at the PP2A nexus. Here we show that Well rounded (Wrd), another B' subunit of PP2A in Drosophila, binds CycG similar to Wdb, and that its loss ameliorates some, but not all, of the metabolic defects of cycG mutants. We propose a model, whereby the binding of CycG to a particular B′-regulatory subunit influences the tissue specific activity of PP2A, required for the fine tuning of the InR/TOR signaling cascade in Drosophila.     

The Molecular Mechanism Underlying Morphine-Induced Akt Activation: Roles of Protein Phosphatases and Reactive Oxygen Species

Although Akt is reported to play a role in morphine’s cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser⁴⁷³, indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K_ATP channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia–reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine’s protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.     

Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B

The sphingolipid ceramide negatively regulates insulin action by inhibiting Akt/protein kinase B (PKB), a serine/threonine kinase that is a central regulator of glucose uptake and anabolic metabolism. Despite considerable attention, the molecular mechanism accounting for this action of ceramide has remained both elusive and controversial. Herein we utilized deletion constructs encoding two different functional domains of Akt/PKB to identify which region of the enzyme conferred responsiveness to ceramide. Surprisingly the findings obtained with these separate domains reveal that ceramide blocks insulin stimulation of Akt/PKB by two independent mechanisms. First, using the isolated pleckstrin homology domain, we found that ceramide specifically blocks the translocation of Akt/PKB, but not its upstream activator phosphoinositide-dependent kinase-1, to the plasma membrane. Second, using a construct lacking this pleckstrin homology domain, which does not require translocation for activation, we found that ceramide stimulates the dephosphorylation of Akt/PKB by protein phosphatase 2A. Collectively these findings identify at least two independent mechanisms by which excessive ceramide accumulation in peripheral tissues could contribute to the development of insulin resistance. Moreover the results obtained provide a unifying theory to account for the numerous dissenting reports investigating the actions of ceramide toward Akt/PKB.     

Proteasome-dependent inactivation of Akt is essential for 12-O-tetradecanoylphorbol 13-acetate-induced apoptosis in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. Here we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on SMC apoptosis. We found that TPA treatment induced SMC apoptosis through the rapid downregulation of Akt phosphorylation. The inhibition of Akt activation by TPA was markedly reduced by inhibitors of protein phosphatase 2A and proteasome. Moreover, TPA promoted the ubiquitination of p-Akt, whereas inhibition of TPA-induced PKC activation suppressed the downregulation and ubiquitination of p-Akt. Taken together, these results demonstrate that TPA triggers inactivation of Akt, at least in part, through PKC and Ubiquitin–proteasome degradation, thereby contributing to SMC apoptosis.     

14-3-3β Promotes Migration and Invasion of Human Hepatocellular Carcinoma Cells by Modulating Expression of MMP2 and MMP9 through PI3K/Akt/NF-κB Pathway

14-3-3β has been demonstrated to possess the oncogenic potential, and its increased expression has been detected in multiple types of carcinomas. However, majority of previous studies focused on the role of 14-3-3β in tumor cell proliferation and apoptosis, leaving much to be elucidated about its function in tumor cell invasion and metastasis. Hence, the present study aimed to investigate the role of 14-3-3β in the invasion of hepatocellular carcinoma (HCC) cells and the implications in the prognosis of HCC patients. We first examined the expression of 14-3-3β in the primary tumors of HCC patients with or without portal vein tumor thrombus (PVTT), and found that 14-3-3β expression was higher in the primary tumors with PVTT, and the level was even higher in the PVTTs. Kaplan-Meier curves and multivariate analysis revealed that high expression of 14-3-3β was associated with overall survival (OS) and time to recurrence (TTR) of HCC patients. In addition, ectopic expression of 14-3-3β in HCC cell lines led to enhanced migration ability and invasiveness, as well as up-regulation of matrix metalloproteinase 2 and 9, which could be suppressed by inhibiting the activation of Akt and nuclear factor-κB (NF-κB) signaling. Furthermore, we identified a correlated elevation of 14-3-3β and p-Akt in the primary tumors of HCC patients, and showed that a combinatory detection of 14-3-3β and p-Akt could better predict post-surgical outcome of HCC patients.     

3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.