Heregulin regulation of Akt/protein kinase B in breast cancer cells.

In the present studies, we demonstrate that heregulin is a potent and rapid activator of the serine/threonine kinase called Akt in the MCF-7 breast cancer cell line but not in 3 other breast cancer cell lines (T47D, HBL-100, and MDA-231). The extent of activation of Akt in the 4 cell lines correlated with the ability of heregulin to activate phosphatidylinositol 3-kinase and inhibition of the kinase blocked Akt activation. A monoclonal antibody to HER2 inhibited the ability of heregulin to activate Akt in the MCF-7 cells. BT474, a breast cancer cell line which overexpresses HER2, had high basal Akt enzymatic activity. This high basal activity was lowered when cells were pre-incubated with an anti-HER2 monoclonal antibody which is used to treat breast cancer patients. Our results indicate that heregulin is a potent activator of Akt and that overexpression of HER2 in breast cancers could also lead to activation of Akt.     

蛋白激酶B/Akt抑制剂

磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路在细胞生长与存活中起着关键作用,PI3K/Akt通路的过度激活在多种肿瘤中常见。Akt激酶本身以及Akt激酶上游调节分子,例如PTEN和PI3K,在超过50%的人类肿瘤中均有异常变化。因此Akt成为肿瘤预防和肿瘤靶向治疗的热点之一。许多小分子化合物通过不同机制抑制Akt活性,根据小分子抑制剂与激酶的结合部位和化学结构不同,主要分为ATP竞争性抑制剂、Akt变构抑制剂和磷脂酰肌醇类似物抑制剂。本文综述了PI3K/Akt通路与肿瘤的关系和Akt抑制剂的研究现状,为新型抗癌药物的设计研究提供参考。     

Activation of protein kinase B (Akt/RAC-protein kinase) by cellular stress and its association with heat shock protein Hsp27

Protein kinase B (PKB, also named as Akt or RAC-protein kinase), that is activated by cellular stress such as heat shock and hyperosmotic treatment, was revealed to be activated by oxidative stress and by chemical stressors of CdCl₂ and NaAsO₂ by measuring the activity of the enzyme immunoprecipitated from the transfected COS-7 cells. Upon stress treatment, a 30-kDa phosphoprotein was co-immunoprecipitated with PKB from the cells metabolic labeled with [³²P]orthophosphate. The phosphoprotein was identified as Hsp27, a small heat shock protein, by immunoblot analysis and co-immunoprecipitation. The association of Hsp27 was specific to PKB as the heat shock protein was not co-immunoprecipitated with other protein kinases such as protein kinase C and PKN. When the cells were treated with H₂O₂, PKB was activated gradually and the association of Hsp27 with PKB increased concurrently with the enhancement of PKB activity. In heat-shocked cells, activation of PKB and the association of Hsp27 were detected immediately after the treatment, and the association of the heat shock protein decreased while PKB kept stimulated activity when the cells were further incubated at 37°C. These results suggest that Hsp27 is involved in the activation process of PKB in the signal transduction pathway of various forms of stress.     

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.     

Protein kinase Cepsilon activates protein kinase B/Akt via DNA-PK to protect against tumor necrosis factor-alpha-induced cell death

We have previously shown that protein kinase Cepsilon (PKCepsilon) protects breast cancer cells from tumor necrosis factor-alpha (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCepsilon is mediated via Akt and the mechanism by which PKCepsilon regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser473 in MCF-7 cells. Overexpression of PKCepsilon in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCepsilon by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCepsilon-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCepsilon on TNF-mediated cell death. Akt was constitutively associated with PKCepsilon and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCepsilon enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCepsilon. These results suggest that PKCepsilon activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.     

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.     

Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits

While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells. In HEK-293 and COS-7 cells transiently transfected with beta(2)-adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type Galpha(i1), and was not duplicated by co-transfection of the constitutively active Galpha(s)-Q227L or Galpha(i)-Q204L mutant. Co-transfection of Gbeta(1), Gbeta(2) but not Gbeta(5) together with Ggamma(2) activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four Galpha subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080-19085) that described activation of Akt in response to mutationally activated Galpha(q) and Galpha(i) transfection in COS cells. To the contrary, in our experiments Galpha(q)-Q209L inhibited Akt activation resulting from betagamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells Galpha(q)-Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser(473) of endogenous Akt in an atropine-reversible fashion. We conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by Gbetagamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by Galpha(q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.     

The protein kinase B/Akt signalling pathway in human malignancy

Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which in mammals comprises three highly homologous members known as PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). PKB/Akt is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. The activation mechanism remains to be fully characterised but occurs downstream of phosphoinositide 3-kinase (PI-3K). PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a lipid second messenger essential for the translocation of PKB/Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. PKB/Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer. Constitutive PKB/Akt activation can occur due to amplification of PKB/Akt genes or as a result of mutations in components of the signalling pathway that activates PKB/Akt. Although the mechanisms have not yet been fully characterised, constitutive PKB/Akt signalling is believed to promote proliferation and increased cell survival and thereby contributing to cancer progression. This review surveys recent developments in understanding the mechanisms and consequences of PKB/Akt activation in human malignancy.     

Akt/protein kinase B in skeletal muscle physiology and pathology

The Akt/protein kinase B is critical regulator of cellular homeostasis with diminished Akt activity being associated with dysregulation of cellular metabolism and cell death while Akt over‐activation has been linked to inappropriate cell growth and proliferation. Although the regulation of Akt function has been well characterized in vitro, much less is known regarding the function of Akt in vivo. Here we examine how skeletal muscle Akt expression and enzymatic activity are controlled, the role of Akt in the regulation of skeletal muscle contraction, stress response glucose utilization, and protein metabolism, and the potential participation of this important molecule in skeletal muscle atrophy, aging, and cancer. J. Cell. Physiol. 226: 29–36, 2010. © 2010 Wiley‐Liss, Inc.     

Akt/protein kinase B promotes organ growth in transgenic mice

One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.     

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.     

Design and synthesis of pyridine-pyrazolopyridine-based inhibitors of protein kinase B/Akt

Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our indazole-pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The structure-activity relationships of the new pyridine-pyrazolopyridine series of Akt inhibitors and their structural features when bound to PKA are also discussed.     

Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B

Recent data have implicated the serine/threonine protein kinase Akt/protein kinase B (PKB) in a diverse array of physiological pathways, raising the question of how biological specificity is maintained. Partial clarification derived from the observation that mice deficient in either of the two isoforms, Akt1/PKBalpha or Akt2/PKBbeta, demonstrate distinct abnormalities, i.e. reduced organismal size or insulin resistance, respectively. However, the question still persists as to whether these divergent phenotypes are due exclusively to tissue-specific differences in isoform expression or distinct capacities for signaling intrinsic to the two proteins. Here we show that Akt2/PKBbeta-/- adipocytes derived from immortalized mouse embryo fibroblasts display significantly reduced insulin-stimulated hexose uptake, clearly establishing that the partial defect in glucose disposal in these mice derives from lack of a cell autonomous function of Akt2/PKBbeta. Moreover, in adipocytes differentiated from primary fibroblasts or immortalized mouse embryo fibroblasts, and brown preadipocytes the absence of Akt2/PKBbeta resulted in reduction of insulin-induced hexose uptake and glucose transporter 4 (GLUT4) translocation, whereas Akt1/PKBalpha was dispensable for this effect. Most importantly, hexose uptake and GLUT4 translocation were completely restored after re-expression of Akt2/PKBbeta in Akt2/PKBbeta-/- adipocytes, but overexpression of Akt1/PKBalpha at comparable levels was ineffective at rescuing insulin action to normal. These results show that the Akt1/PKBalpha and Akt2/PKBbeta isoforms are uniquely adapted to preferentially transmit distinct biological signals, and this property is likely to contribute significantly to the ability of Akt/PKB to play a role in diverse processes.     

Synthesis and SAR of indazole-pyridine based protein kinase B/Akt inhibitors

A series of heteroaryl-pyridine containing inhibitors of Akt are reported. The synthesis and structure-activity relationships are discussed, leading to the discovery of a indazole-pyridine analogue (K(i)=0.16 nM). These compounds bind in the ATP binding site, are potent, ATP competitive, and reversible inhibitors of Akt activity. No selectivity amongst the Akt isoforms is observed for this analogue, but there is good selectivity against an panel of other kinases. It is least selective for other members of the AGC family of kinases but is nonetheless 40-fold selective for Akt over PKA. The compound shows cellular activity and significantly slows tumor growth in vivo.     

Functional role of protein kinase B/Akt in gastric acid secretion

Epidermal growth factor (EGF) stimulates gastric acid secretion and H(+)/K(+)-ATPase alpha-subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H(+)/K(+)-ATPase gene expression by Northern blotting using a canine H(+)/K(+)-ATPase alpha-subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV-beta-gal, which expresses beta-galactosidase. Ad.Myr-Akt induced H(+)/K(+)-ATPase alpha-subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H(+)/K(+)-ATPase production, measured by Western blots with an anti-H(+)/K(+)-ATPase alpha-subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H(+)/K(+)-ATPase expression.     

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.     

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.