Inhibition of HMC-1 mast cell proliferation by vitamin E: involvement of the protein kinase B pathway

The effects of four natural tocopherols on the proliferation and signaling pathways were examined in the human mastocytoma cell line (HMC-1). The four tocopherols inhibited HMC-1 cell proliferation with different potency (delta > alpha = gamma > beta). Growth inhibition correlated with the reduction of PKB (protein kinase B) phosphorylation by the different tocopherols. The reduction of PKB phosphorylation led to a decrease of its activity, as judged from a parallel reduction of GSKalpha/beta phosphorylation. The translocation of PKB to the membrane, as a response to receptor stimulation by NGFbeta, is also prevented by treatment with tocopherols. In the presence of PKC or PP2A inhibitors, the reduction of PKB phosphorylation by tocopherols was still observed, thus excluding the direct involvement of these enzymes. Other pathways, such as the Ras-stimulated ERK1/2 (extracellular signal responsive kinase) pathway, were not affected by tocopherol treatment. The tocopherols did not significantly change oxidative stress in HMC-1 cells, suggesting that the observed effects are not the result of a general reduction of oxidative stress. Thus, the tocopherols interfere with PKB phosphorylation and reduce proliferation of HMC-1 cells, possibly by modulating either phosphatidylinositol 3-kinase, a kinase phosphorylating PKB (PDK1/2), or a phosphatase that dephosphorylates it. Inhibition of proliferation and PKB signaling in HMC-1 cells by vitamin E suggests a role in preventing diseases with mast cell involvement, such as allergies, atherosclerosis, and tumorigenesis.     

Modulation of gene expression by α-tocopherol and α-tocopheryl phosphate in THP-1 monocytes

The natural vitamin E analog α-tocopheryl phosphate (αTP) modulates atherosclerotic and inflammatory events more efficiently than the unphosphorylated α-tocopherol (αT). To investigate the molecular mechanisms involved, we have measured plasma levels of αTP and compared the cellular effects of αT and αTP in THP-1 monocytes. THP-1 cell proliferation is slightly increased by αT, whereas it is inhibited by αTP. CD36 surface expression is inhibited by αTP within hours without requiring transport of αTP into cells, suggesting that αTP may bind to CD36 and/or trigger its internalization. As assessed by gene expression microarrays, more genes are regulated by αTP than by αT. Among a set of confirmed genes, the expression of vascular endothelial growth factor is induced by αTP as a result of activating protein kinase B (PKB/Akt) and is associated with increased levels of reactive oxygen species (ROS). Increased Akt(Ser473) phosphorylation and induction of ROS by αTP occur in a wortmannin-sensitive manner, indicating the involvement of phosphatidylinositol kinases. The induction of Akt(Ser473) phosphorylation and ROS production by αTP can be attenuated by αT. It is concluded that αTP and αT influence cell proliferation, ROS production, and Akt(Ser473) phosphorylation in an antagonistic manner, most probably by modulating phosphatidylinositol kinases.     

Modulation of gene expression by α-tocopherol and α-tocopheryl phosphate in THP-1 monocytes

The natural vitamin E analog α-tocopheryl phosphate (αTP) modulates atherosclerotic and inflammatory events more efficiently than the unphosphorylated α-tocopherol (αT). To investigate the molecular mechanisms involved, we have measured plasma levels of αTP and compared the cellular effects of αT and αTP in THP-1 monocytes. THP-1 cell proliferation is slightly increased by αT, whereas it is inhibited by αTP. CD36 surface expression is inhibited by αTP within hours without requiring transport of αTP into cells, suggesting that αTP may bind to CD36 and/or trigger its internalization. As assessed by gene expression microarrays, more genes are regulated by αTP than by αT. Among a set of confirmed genes, the expression of vascular endothelial growth factor is induced by αTP as a result of activating protein kinase B (PKB/Akt) and is associated with increased levels of reactive oxygen species (ROS). Increased Akt(Ser473) phosphorylation and induction of ROS by αTP occur in a wortmannin-sensitive manner, indicating the involvement of phosphatidylinositol kinases. The induction of Akt(Ser473) phosphorylation and ROS production by αTP can be attenuated by αT. It is concluded that αTP and αT influence cell proliferation, ROS production, and Akt(Ser473) phosphorylation in an antagonistic manner, most probably by modulating phosphatidylinositol kinases.     

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3beta (GSK-3beta) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-alpha (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.     

Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.     

Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress.

To study the role of protein kinase B (PKB) in response to cellular stress, we examined PKBalpha activity following different stress treatments. Hyperosmotic but not chemical stress resulted in inactivation of PKBalpha and prevented activation by pervanadate and mitogens. Hyperosmotic shock did not affect the MAP kinase pathway, suggesting that this inhibitory effect was specific for PKB. Our data further indicate that downregulation occurs via dephosphorylation of Thr308 and Ser473, the major regulatory phosphorylation sites of PKBalpha. Indeed, calyculin A, which inhibits protein phosphatases 1 and 2A, effectively blocked hyperosmotic stress-mediated inactivation (dephosphorylation) of PKBalpha. High osmolarity did not affect phosphatidylinositol 3-kinase activity but led to a marked increase in PI(3,4,5)P3 and a decrease in PI(3,4)P2 formation after pervanadate stimulation, suggesting that hyperosmotic stress has an inhibitory effect on a phosphatidylinositol 5-phosphatase which converts PI(3,4,5)P3 into PI(3,4)P2. Immunofluorescence studies revealed that membrane translocation, a prerequisite for PKB activation, was not affected by hyperosmotic stress. Our results indicate that hyperosmotic stress can act at two levels: (i) inhibition of phosphorylation of Thr308 and Ser473 by upstream kinases and (ii) by promoting rapid dephosphorylation of these regulatory sites.     

The expression of protein kinase B in gastric cancer cell apoptosis induced by 12-O-tetradecanoylphorbol-1, 3-acetate

Protein kinase B (PKB/Akt) is a serine-threonine kinase functioning downstream of phosphatidylinositol 3-kinase (PI-3 kinase) in response to mitogen or growth factor stimulation. In several cell types, it plays an important anti-apoptotic role. TPA is a potent regulator of the growth of many different cell types. Here, we detected that TPA could induce cell apoptosis in the gastric cancer cell line, BGC-823. We also found that TPA inhibited the expression of PKB/Akt in a TPA concentration- and time-dependent manner. Furthermore, TPA inhibited the phosphorylation of PKB at Ser473, but did not affect the phosphorylation of Thr308. It only attenuated the expression of PKB/Akt and the phosphorylation of Ser473 in the cell nucleus, whereas it did not change the PKB/Akt distribution in BGC-823 cells. These results suggest that PKB/Akt inhibition by TPA may be the important factor in the mechanism of effect of TPA on gastric cell lines.     

SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity

Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.     

Dual regulation of platelet protein kinase B

Protein kinase B (PKB) is a serine/threonine kinase that is activated by growth hormones and implicated in prevention of apoptosis, glycogen metabolism, and glucose uptake. A key enzyme in PKB activation is phosphatidylinositide 3-kinase (PI-3K), which triggers the dual phosphorylation of PKB by phosphatidylinositol-dependent kinases (PDKs). Here we report that the major PKB subtype in platelets is PKBalpha, which is activated by phosphorylation of Thr(308) and Ser(473) and has a constitutively phosphorylated Thr(450) that does not contribute to PKB activation. alpha-Thrombin and thrombopoietin activate PKBalpha via PI-3K and trigger the concurrent phosphorylation of Thr(308) (via PDK1) and Ser(473) (via a not yet identified PDK2). In addition, alpha-thrombin activates a PI-3K-independent pathway involving phospholipase Cbeta and calcium-dependent protein kinase C subtypes (PKCalpha/beta). This route is specific for phosphorylation of Ser(473) and can be initiated by direct PKC activation with phorbol ester or purified active PKC catalytic fragment in platelet lysate. Different degrees of Ser(473) and Thr(308) phosphorylation correlate with different degrees of enzyme activity. These data reveal a PI-3K-independent PKB activation in which PKCalpha/beta regulates the phosphorylation of Ser(473) in PKBalpha. The independent control of the two phosphorylation sites may contribute to fine regulation of PKBalpha activity.     

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.     

12-O-tetradecanoylphorbol-1, 3-acetate induces the negative regulation of protein kinase B by protein kinase Cα during gastric cancer cell apoptosis

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.     

Thyroid hormone is a MAPK-dependent growth factor for thyroid cancer cells and is anti-apoptotic

Thyroid hormone (l-thyroxine, T(4), or 3,5,3'-triiodo-l-thyronine, T(3)) treatment of human papillary and follicular thyroid cancer cell lines resulted in enhanced cell proliferation, measured by proliferating cell nuclear antigen (PCNA). Thyroid hormone also induced activation of the Ras/MAPK (ERK1/2) signal transduction pathway. ERK1/2 activation and cell proliferation caused by thyroid hormone were blocked by an iodothyronine analogue, tetraiodothyroacetic acid (tetrac), that inhibits binding of iodothyronines to the cell surface receptor for thyroid hormone on integrin alphaVbeta3. A MAPK cascade inhibitor at MEK, PD 98059, also blocked hormone-induced cell proliferation. We then assessed the possibility that thyroid hormone is anti-apoptotic. We first established that resveratrol (10 microM), a pro-apoptotic agent in other cancer cells, induced p53-dependent apoptosis and c-fos, c-jun and p21 gene expression in both papillary and follicular thyroid cancer cells. Induction of apoptosis by the stilbene required Ser-15 phosphorylation of p53. Resveratrol-induced gene expression and apoptosis were inhibited more than 50% by physiological concentrations of T(4). T(4) activated MAPK in the absence of resveratrol, caused minimal Ser-15 phosphorylation of p53 and did not affect c-fos, c-jun and p21 mRNA abundance. Thus, plasma membrane-initiated activation of the MAPK cascade by thyroid hormone promotes papillary and follicular thyroid cancer cell proliferation in vitro.     

Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells

The objective of this project was to identify some possible mechanisms by which two common phytochemicals, resveratrol and beta-sitosterol, inhibit the growth of human prostate cancer PC-3 cells. These mechanisms include the effect of the phytochemicals on apoptosis, cell cycle progression, prostaglandin synthesis and the production of reactive oxygen species (ROS). Prostaglandins have been known to play a role in regulating cell growth and apoptosis. PC-3 cells were supplemented with 50 microM resveratrol or 16 microM beta-sitosterol alone or in combination for up to 5 days. Phytochemical supplementation resulted in inhibition in cell growth. beta-Sitosterol was more potent than resveratrol and the combination of the two resulted in greater inhibition than supplementation with either alone. Long-term supplementation with resveratrol or beta-sitosterol elevated basal prostaglandin release but beta-sitosterol was much more potent than resveratrol in this regard. beta-Sitosterol was more effective than resveratrol in inducing apoptosis and the combination had an intermediate effect after 1 day of supplementation. Cells supplemented with resveratrol were arrested at the G1 phase and at the G2/M phase in the case of beta-sitosterol while the combination resulted in cell arrest at the two phases of the cell cycle. beta-Sitosterol increased ROS production while resveratrol decreased ROS production. The combination of the two phytochemicals resulted in an intermediate level of ROS. The observed changes in prostaglandin levels and ROS production by these two phytochemicals may suggest their mediation in the growth inhibition. The reduction in ROS level and increase by resveratrol supplementation in PC-3 cells reflects the antioxidant properties of resveratrol. It was concluded that these phytochemicals may induce the inhibition of tumor growth by stimulating apoptosis and arresting cells at different locations in the cell cycle and the mechanism may involve alterations in ROS and prostaglandin production.     

Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI

Phosphoinositide 3-kinase (PI3K) is a critical component of the signaling pathways that control the activation of platelets. Here we have examined the regulation of protein kinase B (PKB), a downstream effector of PI3K, by the platelet collagen receptor glycoprotein (GP) VI and thrombin receptors. Stimulation of platelets with collagen or convulxin (a selective GPVI agonist) resulted in PI3K-dependent, and aggregation independent, Ser(473) and Thr(308) phosphorylation of PKBalpha, which results in PKB activation. This was accompanied by translocation of PKB to cell membranes. The phosphoinositide-dependent kinase PDK1 is known to phosphorylate PKBalpha on Thr(308), although the identity of the kinase responsible for Ser(473) phosphorylation is less clear. One candidate that has been implicated as being responsible for Ser(473) phosphorylation, either directly or indirectly, is the integrin-linked kinase (ILK). In this study we have examined the interactions of PKB, PDK1, and ILK in resting and stimulated platelets. We demonstrate that in platelets PKB is physically associated with PDK1 and ILK. Furthermore, the association of PDK1 and ILK increases upon platelet stimulation. It would therefore appear that formation of a tertiary complex between PDK1, ILK, and PKB may be necessary for phosphorylation of PKB. These observations indicate that PKB participates in cell signaling downstream of the platelet collagen receptor GPVI. The role of PKB in collagen- and thrombin-stimulated platelets remains to be determined.     

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.     

Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1

Protein kinase B (PKB/Akt) has been well established as an important signaling intermediate, and its deregulation has been implicated in the development of human cancer and diabetes (reviewed in). Full activation of PKB requires phosphorylation on residues Thr308 and Ser473. While the Thr308 kinase, named 3-phosphoinositide-dependent kinase-1 (PDK1), has been extensively characterized (reviewed in ), the identity of the Ser473 kinase remains unclear. We have focused our study on the plasma membrane (PM) fraction because membrane localization is sufficient to activate PKB, and this suggests that PKB upstream kinases are constitutively active at the membrane. Here, we report the identification of a constitutively active PKB Ser473 kinase activity enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity was released from the membrane by high salt, and gel filtration analysis showed that the kinase responsible is present in a large complex of >500 kDa. Two major phosphoproteins and integrin-linked kinase (ILK) were detected in partially purified PKB Ser473 kinase preparations. In contrast to previous observations, however, ILK immunoprecipitates did not retain Ser473 kinase activity. Thus, we have identified a novel raft-associated PKB Ser473 kinase, implicating a role for lipid rafts in PKB signaling.     

Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS-1 knockdown HepG2 cells

Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS-1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB-alpha (HepG2-CA-Akt/PKB). IRS-1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS-1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2-CA-Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS-1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2-CA-Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS-1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2-CA-Akt/PKB cells. These results suggest that knockdown of IRS-1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS-1 knockdown in cells overexpressing constitutively active Akt1/PKB-alpha either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells.     

Conditional knock-out of integrin-linked kinase demonstrates an essential role in protein kinase B/Akt activation

Protein kinase B (PKB/Akt) plays a pivotal role in signaling pathways downstream of phosphatidylinositol 3-kinase, regulating fundamental processes such as cell survival, cell proliferation, differentiation, and metabolism. PKB/Akt activation is regulated by phosphoinositide phospholipid-mediated plasma membrane anchoring and by phosphorylation on Thr-308 and Ser-473. Whereas the Thr-308 site is phosphorylated by PDK-1, the identity of the Ser-473 kinase has remained unclear and controversial. The integrin-linked kinase (ILK) is a potential regulator of phosphorylation of PKB/Akt on Ser-473. Utilizing double-stranded RNA interference (siRNA) as well as conditional knock-out of ILK using the Cre-Lox system, we now demonstrate that ILK is essential for the regulation of PKB/Akt activity. ILK knock-out had no effect on phosphorylation of PKB/Akt on Thr-308 but resulted in almost complete inhibition of phosphorylation on Ser-473 and significant inhibition of PKB/Akt activity, accompanied by significant stimulation of apoptosis. The inhibition of PKB/Akt Ser-473 phosphorylation was rescued by kinase-active ILK but not by a kinase-deficient mutant of ILK, suggesting a role for the kinase activity of ILK in the stimulation of PKB/Akt phosphorylation. ILK knock-out also resulted in the suppression of phosphorylation of GSK-3beta on Ser-9 and cyclin D1 expression. These data establish ILK as an essential upstream regulator of PKB/Akt activation.     

In rat hepatocytes glucagon increases mammalian target of rapamycin phosphorylation on serine 2448 but antagonizes the phosphorylation of its downstream targets induced by insulin and amino acids

The major function of mammalian target of rapamycin (mTOR) is the control of cell growth. Insulin and amino acids regulate the mTOR pathway, and both are needed to promote its maximal activation. To further understand mTOR regulation by insulin and amino acids, we have studied the enzyme in primary cultures of hepatocytes. We show that insulin increases mTOR phosphorylation on Ser2448, a consensus phosphorylation site for protein kinase B (PKB). Ser2448 phosphorylation is also increased by amino acids, although they do not activate PKB. Furthermore, insulin and amino acids have an additive effect, indicating that they act through distinct pathways. We also show that phosphorylation of Ser2448 does not seem to modulate in vitro phosphorylation of eukaryotic initiation factor 4E-binding protein 1 by mTOR. However, stimulation of hepatocytes with insulin and amino acids leads to an increase in mTOR kinase activity. Rapamycin has no effect on insulin-, glucagon-, and 8-(4-chlorophenylthio)adenosine-cAMP-induced amino acid transport. Surprisingly, glucagon and 8-(4-chlorophenylthio)adenosine-cAMP, which do not activate PKB, stimulate the phosphorylation on Ser2448 of mTOR. However, glucagon inhibits amino acid- and insulin-induced activation of ribosomal S6 protein kinase 1 and phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1. Our results demonstrate that glucagon, which is not able to activate but rather inhibits the mTOR pathways, stimulates the phosphorylation of mTOR on Ser2448. This finding suggests that phosphorylation of this site might not be sufficient for mTOR kinase activity but is likely to be involved in other functions.