Heparanase induces endothelial cell migration via protein kinase B/Akt activation

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition of heparanase stimulated phosphatidylinositol 3-kinase-dependent endothelial cell migration and invasion. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor.     

CH-ILKBP regulates cell survival by facilitating the membrane translocation of protein kinase B/Akt

Cell survival depends on proper propagation of protective signals through intracellular signaling intermediates. We report here that calponin homology domain-containing integrin-linked kinase (ILK)-binding protein (CH-ILKBP), a widely expressed adaptor protein localized at plasma membrane-actin junctions, is essential for transmission of survival signals. Cells that are depleted of CH-ILKBP undergo extensive apoptosis despite the presence of cell-extracellular matrix contacts and soluble growth factors. The activating phosphorylation of protein kinase B (PKB/Akt), a key regulator of apoptosis, is impaired in the absence of CH-ILKBP. Importantly, loss of CH-ILKBP prevents the membrane translocation of PKB/Akt. Furthermore, forced membrane targeting of PKB/Akt bypasses the requirement of CH-ILKBP for the activating phosphorylation of PKB/Akt, suggesting that CH-ILKBP is required for the membrane translocation but not the subsequent phosphorylation of PKB/Akt. Finally, we show that loss of CH-ILKBP is also required for the full activation of extracellular signal-regulated kinase (ERK)1/2. However, restoration of the PKB/Akt activation is sufficient for protection of cells from apoptosis induced by the depletion of CH-ILKBP despite the persistent suppression of the ERK1/2 activation. Thus, CH-ILKBP is an important component of the prosurvival signaling pathway functioning primarily by facilitating the membrane translocation of PKB/Akt and consequently the activation of PKB/Akt in response to extracellular survival signals.     

S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance

Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donor-treated cells. Mutated Akt1/PKBalpha (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that S-nitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS- and/or oxidative stress-involved insulin resistance.     

Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling

Epithelial cells are major sites of malignant transformation. Atypical protein kinase C (aPKC) isoforms are overexpressed and activated in many cancer types. Using normal, highly polarized epithelial cells (MDCK and NMuMG), we report that aPKC gain of function overcomes contact inhibited growth and is sufficient for a transformed epithelial phenotype. In 2D cultures, aPKC induced cells to grow as stratified epithelia, whereas cells grew as solid spheres of nonpolarized cells in 3D culture. aPKC associated with Mst1/2, which uncoupled Mst1/2 from Lats1/2 and promoted nuclear accumulation of Yap1. Of importance, Yap1 was necessary for aPKC-mediated overgrowth but did not restore cell polarity defects, indicating that the two are separable events. In MDCK cells, Yap1 was sequestered to cell–cell junctions by Amot, and aPKC overexpression resulted in loss of Amot expression and a spindle-like cell phenotype. Reexpression of Amot was sufficient to restore an epithelial cobblestone appearance, Yap1 localization, and growth control. In contrast, the effect of aPKC on Hippo/Yap signaling and overgrowth in NMuMG cells was independent of Amot. Finally, increased expression of aPKC in human cancers strongly correlated with increased nuclear accumulation of Yap1, indicating that the effect of aPKC on transformed growth by deregulating Hippo/Yap1 signaling may be clinically relevant.     

Reelin-mediated signaling locally regulates protein kinase B/Akt and glycogen synthase kinase 3beta

Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3beta. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin-mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3beta kinase activities.     

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.     

Inactivation of host Akt/protein kinase B signaling by bacterial pore-forming toxins

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin alpha-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and alpha-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.     

The v-Crk oncogene enhances cell survival and induces activation of protein kinase B/Akt

The v-Crk oncogene encodes an adaptor protein containing an SH2 domain and an SH3 domain. v-Crk-transformed fibroblast cells display enhanced tyrosine phosphorylation levels, and the v-Crk protein localizes in focal adhesions, suggesting that transformation may be due to enhanced focal complex signaling. Here we investigated the mechanism of transformation and found that v-Crk-transformed NIH 3T3 cells display growth rates and serum requirements similar to control cells. However, v-Crk enhanced survival in conditions of serum starvation. Both an intact SH2 and SH3 domain are required; moreover, SH2 mutants displayed dominant interfering properties, enhancing cell death. Using other cell death-inducing stimuli, it appeared that v-Crk in general inhibits apoptosis and enhances cell survival. In search of the signaling pathways involved, we found that v-Crk-transformed cells show constitutively higher levels of phospho-protein kinase B (PKB)/Akt and PKB/Akt activity, especially in conditions of serum starvation. These data strongly suggest involvement of the phosphatidylinositol 3-kinase/PKB survival pathway in the v-Crk-induced protection against apoptosis. In accordance, inhibition of this pathway by wortmannin or LY924002 reduced protection against starvation-induced apoptosis. In addition to the phosphatidylinositol 3-kinase/PKB pathway, a MEK-dependent pathway and an unknown additional pathway are also implicated in resistance against apoptosis. Activation of survival pathways may be the most important function of v-Crk in its oncogenic properties.     

Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the alpha1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor beta-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.     

Activation of the Akt/protein kinase B signaling pathway is associated with granulosa cell survival

Follicles from the hen ovary that have been selected into the preovulatory hierarchy are committed to ovulation and rarely become atretic under normal physiological conditions. In part, this is attributed to the resistance of the granulosa layer to apoptosis. The present studies were conducted to evaluate the role of the phosphatidylinositol (PI) 3-kinase/Akt signaling pathway in hen granulosa cell survival and, by implication, follicle viability. Cloning of the chicken akt2 homologue revealed a high degree of amino acid homology to its mammalian counterparts within the catalytic domain, plus complete conservation of the putative Thr(308) and Ser(474) phosphorylation sites. Treatment of granulosa cells from the three largest preovulatory follicles with insulin-like growth factor (IGF)-I and, to a lesser extent, transforming growth factor (TGF)-alpha induces rapid phosphorylation of Akt, and such phosphorylation is effectively blocked by the PI 3-kinase-inhibitor LY294006. Serum withdrawal from cultured cells for 33-44 h initiates oligonucleosome formation, an indicator of apoptotic cell death, whereas cotreatment with IGF-I prevents this effect. Moreover, treatment of cultured cells for 20 h with LY294006 induces apoptosis. The potential for nonspecific cell toxicity following LY294006 treatment is considered unlikely because of the ability of either LH or 8-bromo cAMP cotreatment to block LY294006-induced cell death. Finally, both IGF-I and TGF-alpha also activate mitogen-activated protein (MAP) kinase signaling, at least in part, through the phosphorylation of ERK: However, treatment with neither U0126 nor PD98059 (inhibitors of MAP kinase kinase) induced cell death in cultured granulosa cells, despite the ability of each inhibitor to effectively block Erk phosphorylation. Taken together, these results provide evidence for a role of the Akt signaling pathway in promoting cell survival within the preovulatory follicle granulosa layer. In addition, the data indicate the importance of an alternative survival pathway mediated via gonadotropins and protein kinase A independent of Akt signaling.     

Inhibition of integrin-linked kinase/protein kinase B/Akt signaling: mechanism for ganglioside-induced apoptosis

Ganglioside GT1b inhibits keratinocyte attachment to and migration on a fibronectin matrix by binding to alpha(5)beta(1) and preventing alpha(5)beta(1) interaction with fibronectin. The role of gangliosides in triggering keratinocyte apoptosis, however, is unknown. Addition of GT1b to keratinocyte-derived SCC12 cells, grown in serum-free medium but exposed to fibronectin, suppressed Bad phosphorylation, activated caspase-9, and inhibited cyclin D and E expression, resulting in cell cycle arrest at G(1) phase and initiation of apoptosis. The mechanism of GT1b activation of caspase-9 involved inhibition of beta(1) integrin serine/threonine phosphorylation and decreased phosphorylation of both integrin-linked kinase and protein kinase B/Akt at its Ser-473 site, leading to cytochrome c release from mitochondria. Consistently, blockade of GT1b function with anti-GT1b antibody specifically activated the Ser-473 site of Akt, markedly suppressing apoptosis. The ganglioside-induced inhibition of Akt phosphorylation was GT1b-specific and was not observed when cells were treated with other keratinocyte gangliosides, including GD3. These studies suggest that the modulation of keratinocyte cell cycle and survival by GT1b is mediated by its direct interaction with alpha(5)beta(1) and resultant inhibition of the integrin/integrin-linked kinase/protein kinase B/Akt signaling pathway.     

Microtubule network is required for insulin signaling through activation of Akt/protein kinase B: evidence that insulin stimulates vesicle docking/fusion but not intracellular mobility

The microtubule network has been shown to be required for insulin-dependent GLUT4 redistribution; however, the precise molecular function has not been elucidated. In this article, we used fluorescence recovery after photobleaching (FRAP) to evaluate the role of microtubules in intracellular GLUT4 vesicle mobility. A comparison of the rate of fluorescence recovery (t((1/2))), and the maximum fluorescence recovered (F(max)) was made between basal and insulin-treated cells with or without nocodazole treatment to disrupt microtubules. We found that intracellular mobility of fluorescently tagged GLUT4 (HA-GLUT4-GFP) was high in basal cells. Mobility was not increased by insulin treatment. Basal mobility was dependent upon an intact microtubule network. Using a constitutively active Akt to signal GLUT4 redistribution, we found that microtubule-based GLUT4 vesicle mobility was not obligatory for GLUT4 plasma membrane insertion. Our findings suggest that microtubules organize the insulin-signaling complex and provide a surface for basal mobility of GLUT4 vesicles. Our data do not support an obligatory requirement for long range microtubule-based movement of GLUT4 vesicles for insulin-mediated GLUT4 redistribution to the cell surface. Taken together, these findings suggest a model in which insulin signaling targets membrane docking and/or fusion rather than GLUT4 trafficking to the cell surface.     

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.     

Chronic hypoxia impairs the differentiation of 3T3-L1 fibroblast in culture: Role of sustained protein kinase C activation

The effect of hypoxia on 3T3-L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 ± 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H₇ exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3-L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process. © 1994 Wiley-Liss, Inc.     

Mechanism of activation of protein kinase B by insulin and IGF-1.

Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).     

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.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.