Putative Akt Phosphorylation Site Research Articles

DAF-2c signaling promotes taste avoidance after starvation in Caenorhabditis elegans by controlling distinct phospholipase C isozymes

Previously, we reported that DAF-2c, an axonal insulin receptor isoform in Caenorhabditis elegans, acts in the ASER gustatory neuron to regulate taste avoidance learning, a process in which worms learn to avoid salt concentrations experienced during starvation. Here, we show that secretion of INS-1, an insulin-like peptide, after starvation conditioning is sufficient to drive taste avoidance via DAF-2c signaling. Starvation conditioning enhances the salt-triggered activity of AIA neurons, the main sites of INS-1 release, which potentially promotes feedback signaling to ASER to maintain DAF-2c activity during taste avoidance. Genetic studies suggest that DAF-2c–Akt signaling promotes high-salt avoidance via a decrease in PLCβ activity. On the other hand, the DAF-2c pathway promotes low-salt avoidance via PLCε and putative Akt phosphorylation sites on PLCε are essential for taste avoidance. Our findings imply that animals disperse from the location at which they experience starvation by controlling distinct PLC isozymes via DAF-2c.

Regulation of HCN2 Current by PI3K/Akt Signaling.

It has long been known that heart rate is regulated by the autonomic nervous system. Recently, we demonstrated that the pacemaker current, If, is regulated by phosphoinositide 3-kinase (PI3K) signaling independently of the autonomic nervous system. Inhibition of PI3K in sinus node (SN) myocytes shifts the activation of If by almost 16 mV in the negative direction. If in the SN is predominantly mediated by two members of the HCN gene family, HCN4 and HCN1. Purkinje fibers also possess If and are an important secondary pacemaker in the heart. In contrast to the SN, they express HCN2 and HCN4, while ventricular myocytes, which do not normally pace, express HCN2 alone. In the current work, we investigated PI3K regulation of HCN2 expressed in HEK293 cells. Treatment with the PI3K inhibitor PI-103 caused a negative shift in the activation voltage and a dramatic reduction in the magnitude of the HCN2 current. Similar changes were also seen in cells treated with an inhibitor of the protein kinase Akt, a downstream effector of PI3K. The effects of PI-103 were reversed by perfusion of cells with phosphatidylinositol 3,4,5-trisphosphate (the second messenger produced by PI3K) or active Akt protein. We identified serine 861 in mouse HCN2 as a putative Akt phosphorylation site. Mutation of S861 to alanine mimicked the effects of Akt inhibition on voltage dependence and current magnitude. In addition, the Akt inhibitor had no effect on the mutant channel. These results suggest that Akt phosphorylation of mHCN2 S861 accounts for virtually all of the observed actions of PI3K signaling on the HCN2 current. Unexpectedly, Akt inhibition had no effect on If in SN myocytes. This result raises the possibility that diverse PI3K signaling pathways differentially regulate HCN-induced currents in different tissues, depending on the isoforms expressed.

Phosphorylation of Carma1, but not Bcl10, by Akt regulates TCR/CD28-mediated NF-κB induction and cytokine production

Previous studies from our group and others have shown that the Akt kinase can contribute to induction of NF-κB by antigen receptor signaling. However, the direct targets of Akt in this pathway are not known. Here we show that Akt-mediated NF-κB activation is mediated at least in part through direct phosphorylation of the adaptor protein Carma1, which we previously demonstrated could interact with Akt in a TCR ligation-dependent manner. The putative Akt phosphorylation sites in Carma1 are distinct from known PKC consensus sites. Mutation of S551, S637 and S645 in Carma1 to non-phosphorylatable residues decreased phosphorylation of GST-Carma1-linker construct by Akt in vitro. In addition, Carma1 S637A/S645A mutants were significantly impaired in their ability to restore TCR-mediated NF-κB activation and IL-2 expression in Carma1-deficient T cells. Thus, our data reveal Carma1 as a novel target for Akt phosphorylation and suggest that Akt-mediated phosphorylation of Carma1 is an additional regulatory mechanism tuning the NF-κB response downstream of antigen receptor and co-stimulatory signaling.

Phosphorylation of CLK2 at Serine 34 and Threonine 127 by AKT Controls Cell Survival after Ionizing Radiation

AKT phosphorylates components of the intrinsic cell survival machinery and promotes survival to various stimuli. In the present study, we identified CDC-like kinase 2 (CLK2) as a new substrate of AKT activation and elucidated its role in cell survival to ionizing radiation. AKT directly binds to and phosphorylates CLK2 on serine 34 and threonine 127, in vitro and in vivo. CLK2 phosphorylation was detected in HeLa cells overexpressing active AKT. In addition, we demonstrated that ionizing radiation induces CLK2 phosphorylation via AKT activation. In contrast, the suppression of endogenous AKT expression by siRNA inhibited CLK2 phosphorylation in response to 2 gray of γ-ray or insulin. Furthermore, we examined the effect of CLK2 on the survival of irradiated CCD-18Lu cells overexpressing Myc-CLK2. CLK2 overexpression significantly increased cell growth and inhibited cell death induced by 2 gray. The role of CLK2 in cell survival to ionizing radiation was dependent on the phosphorylation of serine 34 and threonine 127. Our results suggest that AKT activation controls cell survival to ionizing radiation by phosphorylating CLK2, revealing an important regulatory mechanism required for promoting cell survival.

The E3 Ligase TTC3 Facilitates Ubiquitination and Degradation of Phosphorylated Akt

The serine threonine kinase Akt is a core survival factor that underlies a variety of human diseases. Although regulatory phosphorylation and dephosphorylation have been well documented, the other posttranslational mechanisms that modulate Akt activity remain unclear. We show here that tetratricopeptide repeat domain 3 (TTC3) is an E3 ligase that interacts with Akt. TTC3 contains a canonical RING finger motif, a pair of tetratricopeptide motifs, a putative Akt phosphorylation site, and nuclear localization signals, and is encoded by a gene within the Down syndrome (DS) critical region on chromosome 21. TTC3 is an Akt-specific E3 ligase that binds to phosphorylated Akt and facilitates its ubiquitination and degradation within the nucleus. Moreover, DS cells exhibit elevated TTC3 expression, reduced phosphorylated Akt, and accumulation in the G(2)M phase, which can be reversed by TTC3 siRNA or Myr-Akt. Thus, interaction between TTC3 and Akt may contribute to the clinical symptoms of DS.

Activation-induced Degradation of FLIPL Is Mediated via the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway in Macrophages

Cellular FLIP (Flice-like inhibitory protein) is critical for the protection against death receptor-mediated cell apoptosis. In macrophages, FLIP long (FLIP(L)) and FLIP short (FLIP(S)) mRNA was induced by tumor necrosis factor (TNF) alpha, mediated through NF-kappaB. However, we observed TNFalpha reduced the protein level of FLIP(L), but not FLIP(S), at 1 and 2 h. Similar results were observed with lipopolysaccharide. The reduction of FLIP(L) by TNFalpha was not mediated by caspase 8, or through JNK or Itch, but was suppressed by inhibition of the phosphatidylinositol 3-kinase/Akt pathway employing chemical inhibitors, a dominant negative Akt-1, or Akt-1 small interfering RNA. The reduction of FLIP(L) resulted in the short term induction of caspase 8-like activity, which augmented NF-kappaB activation. A co-immunoprecipitation assay demonstrated that Akt-1 physically interacts with FLIP(L). Moreover, TNFalpha enhanced FLIP(L) serine phosphorylation, which was increased by activated Akt-1. Serine 273, a putative Akt-1 phosphorylation site in FLIP(L), was critical for the activation-induced reduction of FLIP(L). Thus, these observations document a novel mechanism where by TNFalpha facilitates the reduction of FLIP(L) protein, which is dependent on the phosphatidylinositol 3-kinase/Akt signaling.

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility

BackgroundAkt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells.ResultsWe confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1α. EF1α contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1α expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1α siRNAs with specific pAkt inhibitors whereas EF1α downregulation slightly attenuated the decreased invasion induced by Akt inhibitors.ConclusionWe show here that EF1α is a pAkt-interacting protein which regulates pAkt levels. Since EF1α is often overexpressed in breast cancer, the consequences of EF1α increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

Phosphorylation of Telomeric Repeat Binding Factor 1 (TRF1) by Akt Causes Telomere Shortening

Telomeric repeat binding factor 1 (TRF1) belongs to the shelterin complex, which modulates the telomere structures. Akt/protein kinase B activation caused genomic instability and contributes to tumorigenesis, although the molecular mechanism remained little known. Here, we show the direct interaction between Akt and TRF1. In vitro kinase assays showed the phosphorylation of a putative Akt phosphorylation site (Threonine 273) in wild type TRF1, but not the mutant TRF1 (T273A), by Akt. Overexpression of Akt decreased telomere length in a HTC cell line. These results indicate that Akt plays an important role in telomere length regulation, contributing to genomic instability and tumorigenesis.

A Combination of HNF-4 and Foxo1 Is Required for Reciprocal Transcriptional Regulation of Glucokinase and Glucose-6-phosphatase Genes in Response to Fasting and Feeding

Glucokinase (GK) and glucose-6-phosphatase (G6Pase) regulate rate-limiting reactions in the physiologically opposed metabolic cascades, glycolysis and gluconeogenesis, respectively. Expression of these genes is conversely regulated in the liver in response to fasting and feeding. We explored the mechanism of transcriptional regulation of these genes by nutritional condition and found that reciprocal function of HNF-4 and Foxo1 plays an important role in this process. In the GK gene regulation, Foxo1 represses HNF-4-potentiated transcription of the gene, whereas it synergizes with HNF-4 in activating the G6Pase gene transcription. These opposite actions of Foxo1 concomitantly take place in the cells under no insulin stimulus, and such gene-specific action was promoter context-dependent. Interestingly, HNF-4-binding elements (HBEs) in the GK and G6Pase promoters were required both for the insulin-stimulated GK gene activation and insulin-mediated G6Pase gene repression. Indeed, mouse in vivo imaging showed that mutating the HBEs in the GK and G6Pase promoters significantly impaired their reactivity to the nutritional states, even in the presence of intact Foxo1-binding sites (insulin response sequences). Thus, in the physiological response of the GK and G6Pase genes to fasting/feeding conditions, Foxo1 distinctly decodes the promoter context of these genes and differently modulates the function of HBE, which then leads to opposite outcomes of gene transcription.

Hsp70/Hsc70 regulates the effect phosphorylation has on stabilizing ataxin‐1

Spinocerebellar ataxia type 1 (SCA1) is an inherited neurodegenerative disorder. The mutation causing SCA1 is an expansion in the polyglutamine tract of the ATXN1 protein. Previous work demonstrated that phosphorylation of mutant ATXN1 at serine 776 (S776), a putative Akt phosphorylation site, is critical for pathogenesis. To examine this pathway further, we utilized a cell-transfection system that allowed the targeting of Akt to either the cytoplasm or the nucleus. In contrast to HeLa cells, we found that Akt targeted to the cytoplasm increased the degradation of ATXN1 in Chinese hamster ovary cells. However, Akt targeted to the cytoplasm failed to destabilize ATXN1 if Hsp70/Hsc70 was present. Thus, Hsp70/Hsc70 can regulate ATXN1 levels in concert with phosphorylation of ATXN1 at S776.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

AILIM/ICOS-mediated elongation of activated T cells is regulated by both the PI3-kinase/Akt and Rho family cascade

T-cell migration and movement is a critical component of a fully functional immune system. Activation-inducible lymphocyte immunomediatory molecule/inducible co-stimulator (AILIM/ICOS), which is a member of CD28 co-stimulatory receptor family, induces both activated T-cell migration underneath tumor necrosis factor alpha-treated human umbilical vein endothelial cell layers and also the morphological polarization of activated T cells. In our current study, we have investigated the signaling mechanisms underlying the morphological polarization of activated T cells, initiated by AILIM/ICOS signaling. AILIM/ICOS signaling induces the activation of phosphoinositide-3 (PI3)-kinase, the product of which, phosphatidylinositol 3,4,5-trisphosphate (PIP3), was found to be localized in the lamellipodia at the front part of the cells. Phosphorylated Akt is also co-localized with PIP3 and filamentous actin in lamellipodia and the PI3-kinase/Akt signaling cascade has critical roles in T-cell polarization and lamellipodia formation via the re-organization of the actin cytoskeleton. Rho family members and their downstream effectors, Rho-associated kinase and p21-activated kinase (PAK), are also involved in AILIM/ICOS-mediated elongation. The PAK family members are serine/threonine kinase downstream effectors of both Rac and Cdc42. PAK3 is induced by the activation of T cells, whereas PAK1 is constitutively expressed in both naive and activated T cells. During the elongation, not only PAK1 but also PAK3 play an essential role through the phosphorylation of their conservative autophosphorylation sites and catalytic domain. Ser-244 phosphorylation, which is a putative Akt phosphorylation site, on PAK3 but not on PAK1 also regulates the morphological polarization of activated T cells by AILIM/ICOS signaling. Both the PI3-kinase/Akt and Rho family cascades operate coordinately to induce the forward migration of activated T cells by AILIM/ICOS signaling.

Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

The Valosin-containing Protein (VCP) Is a Target of Akt Signaling Required for Cell Survival

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-kappaB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.

Akt Phosphorylates and Regulates Pdcd4 Tumor Suppressor Protein

Programmed cell death 4 (Pdcd4) is a tumor suppressor protein that interacts with eukaryotic initiation factor 4A and inhibits protein synthesis. Pdcd4 also suppresses the transactivation of activator protein-1 (AP-1)-responsive promoters by c-Jun. The Akt (protein kinase B) serine/threonine kinase is a key mediator of phosphoinositide 3-kinase pathway involved in the regulation of cell proliferation, survival, and growth. Because Pdcd4 has two putative Akt phosphorylation sites at Ser(67) and Ser(457), we investigated whether Akt phosphorylates and regulates Pdcd4. Our results show that Akt specifically phosphorylates Ser(67) and Ser(457) residues of Pdcd4 in vitro and in vivo. We further show that phosphorylation of Pdcd4 by Akt causes nuclear translocation of Pdcd4. Using luciferase assay, we show that phosphorylation of Pdcd4 by Akt also causes a significant decrease of the ability of Pdcd4 to interfere with the transactivation of AP-1-responsive promoter by c-Jun.

Telomerase down‐regulation does not mediate PC12 pheochromocytoma cell differentiation induced by NGF, but requires MAP kinase signalling

Telomerase is a ribonucleoprotein complex that maintains chromosomal telomere homeostasis and underlies continuous renewal of stem cells and immortalization of neoplastic cells. Telomerase is down-regulated during cell differentiation, but the mechanisms of down-regulation are largely unknown. Here, we examined roles of mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3) kinase signalling pathways in telomerase down-regulation triggered by nerve growth factor (NGF), and the role of telomerase down-regulation in NGF-induced neural differentiation in PC12 cells. We report that NGF-induced telomerase down-regulation requires MAP kinase signalling. While mutations of all putative Akt phosphorylation sites in telomerase reverse transcriptase (TERT) has no effect on telomerase activity, inhibition of MAP kinase signalling by PD98059 or U0126 abolishes NGF-induced telomerase down-regulation in a concentration-dependent manner. Reversal of NGF-induced telomerase down-regulation by TERT overexpression does not prevent NGF-induced neural differentiation. Down-regulation of telomerase by silencing TERT gene expression does not trigger cell differentiation in the absence of NGF, nor enhances NGF-induced differentiation. Thus, telomerase, withdraws by a mechanism at TERT gene transcription level involving MAP kinase signalling while cells cease proliferation and undergo differentiation. The withdrawal of telomerase is not required to mediate NGF-induced PC12 cell differentiation and re-establishment of telomerase activity at significant levels does not inhibit differentiation.

Akt Phosphorylates Tal1 Oncoprotein and Inhibits Its Repressor Activity

The helix-loop-helix transcription factor Tal1 is required for blood cell development and its activation is a frequent event in T-cell acute lymphoblastic leukemia. The Akt (protein kinase B) kinase is a key player in transduction of antiapoptotic and proliferative signals in T cells. Because Tal1 has a putative Akt phosphorylation site at Thr90, we investigated whether Akt regulates Tal1. Our results show that Akt specifically phosphorylates Thr90 of the Tal1 protein within its transactivation domain in vitro and in vivo. Coimmunoprecipitation experiments showed the presence of Tal1 in Akt immune complexes, suggesting that Tal1 and Akt physically interact. We further showed that phosphorylation of Tal1 by Akt causes redistribution of Tal1 within the nucleus. Using luciferase assay, we showed that phosphorylation of Tal1 by Akt decreased repressor activity of Tal1 on EpB42 (P4.2) promoter. Thus, these data indicate that Akt interacts with Tal1 and regulates Tal1 by phosphorylation at Thr90 in a phosphatidylinositol 3-kinase-dependent manner.

ArgBP2γ Interacts with Akt and p21-activated Kinase-1 and Promotes Cell Survival

Akt/protein kinase B is a major cell survival pathway through phosphorylation of proapoptotic proteins Bad and Bax and of additional apoptotic pathways linked to Forkhead proteins glycogen synthase kinase-3beta and ASK1. To further explore the mechanism by which Akt regulates cell survival, we identified an Akt interaction protein by yeast two-hybrid screening. It is highly homologous to ARG-binding protein 2 (ArgBP2) with splicing exon 8 of the coding region of the ArgBP2. As two splicing isoforms (ArgBP2alpha and -beta) of ArgBP2 have been identified (Wang, B., Golemis, E. A., and Kruh, G. D. (1997) J. Biol. Chem. 272, 17542-17550), it was named ArgBP2gamma. ArgBP2gamma contains four Akt phosphorylation consensus sites, a SoHo motif, and three Src homology (SH) 3 domains and binds to C-terminal proline-rich motifs of Akt through its first and second SH3 domains. It also interacts with p21-activated protein kinase (PAK1) via its first and third SH3 domains, indicating the SH3 domains of ArgBP2gamma as docking sites for Akt and PAK1. Akt phosphorylates ArgBP2gamma in vitro and in vivo. Expression of ArgBP2gamma induces PAK1 activity and overrides apoptosis induced by ectopic expression of Bad or DNA damage. Nonphosphorylatable ArgBP2gamma-4A and SH3 domain-truncated mutant ArgBP2gamma inhibit Akt-induced PAK1 activation and reduce Akt and PAK1 phosphorylation of Bad and antiapoptotic function. These data indicate that ArgBP2gamma is a physiological substrate of Akt, functions as an adaptor for Akt and PAK1, and plays a role in Akt/PAK1 cell survival pathway.

Both estrogen and raloxifene protect against β-amyloid-induced neurotoxicity in estrogen receptor α-transfected PC12 cells by activation of telomerase activity via Akt cascade

Although estrogen is known to protect against beta-amyloid (Abeta)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Abeta-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Abeta-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) alpha gene (PCER). Raloxifene, like 17beta-estradiol (E2), significantly inhibited Abeta-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Abeta, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2- and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor kappaB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer's disease.

Pseudosubstrate peptides inhibit Akt and induce cell growth inhibition.

We have designed peptide inhibitors that potently inhibit Akt both in vitro and inside cells. These peptide inhibitors are selective for Akt versus other closely related kinases. The peptides inhibit the in vitro phosphorylation of a biotinylated Bad peptide by Akt with potency up to 100 nM. We have shown that the binding between Akt1 and these peptide inhibitors requires MgATP. Mutating the two putative Akt phosphorylation sites to Ala (nonsubstrate) in these peptides increases the inhibitory potency while mutating the sites to aspartic acid (phosphorylation mimetic) reduces the potency. When delivered into cells, these peptide inhibitors can inhibit cellular Akt activity and cell growth. Thus, these Akt-specific peptide inhibitors provide prototypes for peptide mimetic drugs as well as very useful tools to dissect cellular functions of Akt.