The basal flux of Akt in the mitochondria is mediated by heat shock protein 90

Akt is a known client protein of heat shock protein 90 (HSP90). We have found that HSP90 is responsible for Akt accumulation in the mitochondria in unstimulated cells. Treatment of SH-SY5Y neuroblastoma cells and human embryonic kidney cells with the HSP90 inhibitors novobiocin and geldanamycin caused substantial decreases in the level of Akt in the mitochondria without affecting the level of Akt in the cytosol. Moreover, intracerebroventricular injection of novobiocin into mice brains decreased Akt levels in cortical mitochondria. Knockdown of HSP90 expression with short interfering RNA also caused a significant decrease in Akt levels in the mitochondria without affecting total Akt levels. Using a mitochondrial import assay it was found that Akt is transported into the mitochondria. Furthermore, it was found that the mitochondrial import of Akt was independent of Akt activation as both an unmodified Akt and constitutively active mutant Akt; both readily accumulated in the mitochondria in an HSP90-dependent manner. Interestingly, incubation of isolated mitochondria with constitutively active Akt caused visible alterations in mitochondrial morphology, including pronounced remodeling of the mitochondrial matrix. This effect was blocked when Akt was mostly excluded from the mitochondria with novobiocin treatment. These results indicate that the level of Akt in the mitochondria is dependent on HSP90 chaperoning activity and that Akt import can cause dynamic changes in mitochondrial configuration.     

GM1-induced activation of phosphatidylinositol 3-kinase: involvement of Trk receptors

The ganglioside GM1 promotes neuronal growth, differentiation, survival, phenotypic expression, and function restoration, by apparently interacting with neurotrophic factors and/or their receptors. In brain, GM1 activates the Trk receptors for neurotrophins and the Raf/MEK/ERK cascade in situ and in vivo . We have expanded these studies and explored whether GM1 recruits the phosphatidylinositol 3 (PI3)-kinase pathway in brain also. Incubating striatal slices with GM1 increased the activity of PI3-kinase in phosphotyrosine immunoprecipitates in a time- and concentration-dependent manner, and the response was blocked by the PI3-kinase inhibitors wortmannin and LY294002. PI3-kinase activation following GM1 was rapid and short lasting with an EC₅₀ of 5 μmol/L. There was a temporally parallel activation of the downstream PI3-kinase target Akt, which was prevented by PI3-kinase inhibition. PI3-kinase activity was found increased in Trk and Gab1 immunoprecipitates, and co-immunoprecipitation studies demonstrated the association of Trk and Gab1 after GM1 treatment. Enhanced PI3-kinase activity associated with Trk or Gab1 immunoprecipitates was blocked by the Trk inhibitor K252a. GM1 did not appear to transactivate Trk and did not alter the efflux of neurotrophins in striatal slices. Our findings suggest that GM1 induces activation of PI3-kinase that is, in part, mediated through Trk and Gab1.     

VMP1 is a new player in the regulation of the autophagy-specific phosphatidylinositol 3-kinase complex activation

We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells.     

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.     

Angiotensin II suppresses adriamycin-induced apoptosis through activation of phosphatidylinositol 3-kinase/Akt signaling in human breast cancer cells

Angiotensin II (Ang II) stimulates tumor growth and angio-genesis in some solid cancer cells, but its anti-apoptosis role in breast cancer remains unclear. To address this issue, we investigated the effect of Ang II on adriamycin-induced apoptosis in breast cancer MCF-7 cells. Treatment of human breast cancer MCF-7 cells with adriamycin, a DNA topoisomerase IIα inhibitor, caused apoptosis. However, cells pretreated with Ang II were resistant to this apoptosis. Ang II significantly reduced the ratio of apoptotic cells and stimulation of phospho-Akt-Thr308 and phospho-Akt-Ser473 in a dose-dependent and time-dependent manner. In addition, Ang II significantly prevented apoptosis through inhibiting the cleavage of procaspase-9, a major downstream effector of Akt. TheAng II type 1 receptor (AT1R) was responsible for these effects. Among the signaling molecules downstream of AT1R, we revealed that the phosphatidylinositol 3-kinase/Akt pathway plays a predominant role in the anti-apoptotic effect of Ang II. Our data indicated that Ang n plays a critical anti-apoptotic role in breast cancer cells by a mechanism involving AT1R/phosphatidylinositol 3-kinase/Akt activation and the subsequent suppression of caspase-9 activation.     

NT3 inhibits FGF2-induced neural progenitor cell proliferation via the PI3K/GSK3 pathway

Neurotrophin 3 (NT3), a member of the neurotrophin family, antagonizes the proliferative effect of fibroblast growth factor 2 (FGF2) on cortical precursors. However, the mechanism by which NT3 inhibits FGF2-induced neural progenitor (NP) cell proliferation is unclear. Here, using an FGF2-dependent rat neurosphere culture system, we found that NT3 inhibits both FGF2-induced neurosphere growth and bromodeoxyuridine (BrdU) incorporation in a dose-dependent manner. U0126, a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, both inhibited FGF2-induced BrdU incorporation, suggesting that the extracellular signal-regulated kinase1/2 (ERK1/2) and PI3K pathways are required for FGF2-induced NP cell proliferation. NT3 significantly inhibited FGF2-induced phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta), a downstream kinase of Akt, whereas phosphorylation of ERK1/2 was unaffected. The inhibitory effect of NT3 on FGF2-induced NP cell proliferation was abolished by LY294002, and treatment with SB216763, a specific GSK3 inhibitor, antagonized the NT3 effect, rescuing both neurosphere growth and BrdU incorporation. Moreover, experiments with anti-NT3 antibody revealed that endogenous NT3 also plays a role in inhibiting FGF2-induced NP cell proliferation, and that anti-NT3 antibody enhanced phospho-Akt and phospho-GSK3beta levels in the presence of FGF2. These findings indicate that FGF2-induced NP cell proliferation is inhibited by NT3 via the PI3K/GSK3 pathway.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium

The regulatory influences of glycogen synthase kinase-3 beta (GSK3 beta) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH-SY5Y cells. Activation of Akt (protein kinase B) with serum-increased phospho-serine-9-GSK3 beta (the inactive form of the enzyme), inhibited GSK3 beta activity, and increased CREB DNA binding activity. Inhibition of GSK3 beta by another paradigm, treatment with the selective inhibitor lithium, also increased CREB DNA binding activity. The inhibitory regulation of CREB DNA binding activity by GSK3 beta also was evident in differentiated SH-SY5Y cells, indicating that this regulatory interaction is maintained in non-proliferating cells. These results demonstrate that inhibition of GSK3 beta by serine-9 phosphorylation or directly by lithium increases CREB activation. Conversely, overexpression of active GSK3 beta to 3.5-fold the normal levels completely blocked increases in CREB DNA binding activity induced by epidermal growth factor, insulin-like growth factor-1, forskolin, and cyclic AMP. The inhibitory effects due to overexpressed GSK3 beta were reversed by treatment with lithium and with another GSK 3beta inhibitor, sodium valproate. Overall, these results demonstrate that GSK3 beta inhibits, and lithium enhances, CREB activation.     

Naturally occurring ether-linked phosphatidylcholine activates phosphatidylinositol 3-kinase and stimulates cell growth

Phosphatidylcholine (PC) from marine invertebrates is enriched in ether-linked forms. PCs from ray fish, Dasyatis sp., and bivalve, Macoma birmanica, used in the present study, contain 65% and 75% (w/w of total PC) of ether-linked forms, respectively. Ether-linked PCs also occur in mammalian membranes. Agonist-mediated hydrolysis of PC generates second messengers which participate in cellular responses. In this study, we tested whether PCs from marine invertebrates directly affect mammalian cell growth and activity of phosphatidylinositol (PI-3-kinase). PI-3-kinase participates in mitogenesis initiated by a variety of growth factors. PI-3-kinase converts polyphosphoinositides to 3′ phosphorylated isomers and these products accumulate in response to mitogenic stimuli. Whether cell membrane lipids regulate PI-3-kinase activity is not known. The marine animal–derived PCs and dioleoyl DAG (dioleoylglycerol) stimulated growth of murine pre-B lymphocytes, whereas chicken PC (egg lecithin) inhibited growth of these cells. Egg lecithin is also a potent inhibitor of PI-3-kinase activity in vitro. We studied the effect of PCs and DAG on PI-3-kinase activity. Unlike egg lecithin, marine animal PCs enhanced PI-3-kinase activity. We investigated the effect of lipids on PI-3-kinase substrate utilization. PCs enriched in ether-linked species increased utilization of substrates by PI-3-kinase. PCs purified from marine organisms also contain a substantially higher percentage of the cis-unsaturated fatty acids, especially of the ? ω3 series (25% and 30% of total fatty acids for Dasyatis sp. and Macoma birmanica, respectively), as compared to vertebrate sources. In spite of differences in fatty acid composition, marine PCs and dioleoyl DAG showed similar effects on cell growth and PI-3-kinase activity. These findings indicate that ether-linked phospholipids activate PI-3-kinase and may participate in mitogenic responses. © 1994 Wiley-Liss, Inc.     

Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3-kinase

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor-mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) on neurotransmitter release were studied in rat PC12 cells. Brief treatment with EGF and IGF-1 enhanced Ca2+-dependent dopamine release in a concentration-dependent manner. EGF activated both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase) pathways, and the EGF-dependent enhancement of DA release was suppressed by a MAPK kinase inhibitor as well as by PI3-kinase inhibitors. In striking contrast, IGF-1 activated the PI3-kinase pathway but not the MAPK pathway, and IGF-1-dependent enhancement was suppressed by a PI3-kinase inhibitor but not by a MAPK kinase inhibitor. The enhanced green fluorescent protein-tagged pleckstrin homology (PH) domain of protein kinase B, which selectively binds to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, was translocated to the plasma membrane after treatment with either EGF or NGF. By contrast, no significant redistribution was induced by IGF-1. These results indicate that PI3-kinase participates in the enhancement of neurotransmitter release by two distinct mechanisms: EGF and NGF activate PI3-kinase in the plasma membrane, whereas IGF-1 activates PI3-kinase possibly in the intracellular membrane, leading to enhancement of neurotransmitter release in a MAPK-dependent and -independent manner respectively.     

Activation of Akt/PKB, increased phosphorylation of Akt substrates and loss and altered distribution of Akt and PTEN are features of Alzheimer's disease pathology

Studies suggest that activation of phosphoinositide 3-kinase-Akt may protect against neuronal cell death in Alzheimer's disease (AD). Here, however, we provide evidence of increased Akt activation, and hyperphosphorylation of critical Akt substrates in AD brain, which link to AD pathogenesis, suggesting that treatments aiming to activate the pathway in AD need to be considered carefully. A different distribution of Akt and phospho-Akt was detected in AD temporal cortex neurons compared with control neurons, with increased levels of active phosphorylated-Akt in particulate fractions, and significant decreases in Akt levels in AD cytosolic fractions, causing increased activation of Akt (phosphorylated-Akt/total Akt ratio) in AD. In concordance, significant increases in the levels of phosphorylation of total Akt substrates, including: GSK3beta(Ser9), tau(Ser214), mTOR(Ser2448), and decreased levels of the Akt target, p27(kip1), were found in AD temporal cortex compared with controls. A significant loss and altered distribution of the major negative regulator of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10), was also detected in AD neurons. Loss of phosphorylated-Akt and PTEN-containing neurons were found in hippocampal CA1 at end stages of AD. Taken together, these results support a potential role for aberrant control of Akt and PTEN signalling in AD.     

Unravelling the activation mechanisms of protein kinase B/Akt

Over the past decade, protein kinase B (PKB, also termed Akt) has emerged as an important signaling mediator between extracellular cues and modulation of gene expression, metabolism, and cell survival. The enzyme is tightly controlled and consequences of its deregulation include loss of growth control and oncogenesis. Recent work has better characterized the mechanism of PKB activation, including upstream regulators and secondary binding partners. This minireview refreshes some old concepts with new twists and highlights current outstanding questions.     

Pathophysiological concentrations of amyloid beta proteins directly inhibit rat brain and recombinant human type II phosphatidylinositol 4-kinase activity

We previously found that pathophysiological concentrations (< or = 10 nm) of an amyloid beta protein (Abeta25-35) reduced the plasma membrane phosphatidylinositol monophosphate level in cultured rat hippocampal neurons with a decrease in phosphatidylinositol 4-monophosphate-dependent Cl- -ATPase activity. As this suggested an inhibitory effect of Abeta25-35 on plasma membrane phosphatidylinositol 4-kinase (PI4K) activity, in vitro effects of Abetas on PI4K activity was examined using rat brain subcellular fractions and recombinant human type II PI4K (PI4KII). Abeta25-35 (10 nm) inhibited PI4KII activity, but neither PI 3-kinase (PI3K) nor type III PI4K (PI4KIII) activity, in microsomal fractions, while 100 nm Abeta25-35 inhibited PI3K activity in mitochondrial fractions. In plasma membrane-rich fractions, Abetas (> 0.5 nm) dose-dependently inhibited PI4KII activity, the maximal inhibition to 77-87% of control being reached around 10 nm of Abetas without significant changes in apparent Km values for ATP and PI, suggesting non-competitive inhibition by Abetas. The inhibition by 10 nm Abeta25-35 was reversible. In recombinant human PI4KIIalpha, inhibition profiles of Abetas were similar to those in rat brain plasma membranes. Therefore, pathophysiological concentrations of Abetas directly and reversibly inhibited plasma membrane PI4KII activity, suggesting that plasma membrane PI4KII is a target of Abetas in the pathogenesis of Alzheimer's disease.     

Calcineurin dephosphorylates glycogen synthase kinase-3 beta at serine-9 in neuroblast-derived cells

This study examined the role of calcineurin, a major calcium-dependent protein phosphatase, in dephosphorylating Ser-9 and activating glycogen synthase kinase-3β (GSK-3β). Treatment with calcineurin inhibitors increased phosphorylation of GSK-3β at Ser-9 in SH-SY5Y human neuroblastoma cells. The over-expression of a constitutively active calcineurin mutant, calcineurin A beta (1–401), led to a significant decrease in phosphorylation at Ser-9, an increase in the activity of GSK-3β, and an increase in the phosphorylation of tau. K_m of calcineurin for a GSK-3β phosphopeptide was 469.3 μM, and specific activity of calcineurin was 15.2 nmol/min/mg. In addition, calcineurin and GSK-3β were co-immunoprecipitated in neuron-derived cells and brain tissues, and calcineurin formed a complex only with dephosphorylated GSK-3β. We conclude that in vitro, calcineurin can dephosphorylate GSK-3β at Ser-9 and form a stable complex with GSK-3β, suggesting the possibility that calcineurin regulates the dephosphorylation and activation of GSK-3β in vivo .     

Akt activation prevents Apop-1-induced death of cells

Apop-1 is a novel protein identified in cultured atherosclerotic smooth muscle cells of ApoE-deficient mice, and the expression of the Apop-1 protein induces the death of cultured cells. Insulin-like growth factor-1 (IGF-1) is a well-characterized survival factor for VSMC; however, the interaction between Apop-1 and survival factor IGF-1 in the mediation of cell death is poorly understood. In this report, we show that the IGF-1 signaling cascade protects VSMC against Apop-1-induced death. Furthermore, our data indicate that the inhibition of Apop-1-induced death by IGF-1 is mediated by the activation of the PI3K/Akt signaling pathway.