The activation of Akt/PKB signaling pathway and cell survival

Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBalpha/Akt1, PKBbeta/Akt2 and PKBgamma/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.     

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.     

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.     

The role of the PI3K/AKT signalling pathway in the corneal epithelium: recent updates

Phosphatidylinositol 3 kinase (PI3K)/AKT (also called protein kinase B, PKB) signalling regulates various cellular processes, such as apoptosis, cell proliferation, the cell cycle, protein synthesis, glucose metabolism, and telomere activity. Corneal epithelial cells (CECs) are the outermost cells of the cornea; they maintain good optical performance and act as a physical and immune barrier. Various growth factors, including epidermal growth factor receptor (EGFR) ligands, insulin-like growth factor 1 (IGF1), neurokinin 1 (NK-1), and insulin activate the PI3K/AKT signalling pathway by binding their receptors and promote antiapoptotic, anti-inflammatory, proliferative, and migratory functions and wound healing in the corneal epithelium (CE). Reactive oxygen species (ROS) regulate apoptosis and inflammation in CECs in a concentration-dependent manner. Extreme environments induce excess ROS accumulation, inhibit PI3K/AKT, and cause apoptosis and inflammation in CECs. However, at low or moderate levels, ROS activate PI3K/AKT signalling, inhibiting apoptosis and stimulating proliferation of healthy CECs. Diabetes-associated hyperglycaemia directly inhibit PI3K/AKT signalling by increasing ROS and endoplasmic reticulum (ER) stress levels or suppressing the expression of growth factors receptors and cause diabetic keratopathy (DK) in CECs. Similarly, hyperosmolarity and ROS accumulation suppress PI3K/AKT signalling in dry eye disease (DED). However, significant overactivation of the PI3K/AKT signalling pathway, which mediates inflammation in CECs, is observed in both infectious and noninfectious keratitis. Overall, upon activation by growth factors and NK-1, PI3K/AKT signalling promotes the proliferation, migration, and anti-apoptosis of CECs, and these processes can be regulated by ROS in a concentration-dependent manner. Moreover, PI3K/AKT signalling pathway is inhibited in CECs from individuals with DK and DED, but is overactivated by keratitis.Subject terms: Growth factor signalling, Apoptosis, Extracellular matrix     

Structure, regulation and function of PKB/AKT--a major therapeutic target

Protein phosphorylation and dephosphorylation play a major role in intracellular signal transduction activated by extracellular stimuli. Protein kinase B (PKB/Akt) is a central player in the signal transduction pathways activated in response to growth factors or insulin and is thought to contribute to several cellular functions including nutrient metabolism, cell growth and apoptosis. Recently, several significant publications have described novel mechanisms used to regulate PKB. Since the alteration of PKB activity is associated with several human diseases, including cancer and diabetes, understanding PKB regulation is an important task if we are to develop successful therapeutics.     

Sodium vanadate inhibits apoptosis in malignant glioma cells: A role for Akt/PKB

The dual signal hypothesis of apoptosis holds that a common signal can activate both apoptotic and proliferative pathways. The fate of a cell is dependent on which of these two pathways predominates. In the MAPK family of kinases, ERK and JNK have been proposed to mediate apoptosis whereas the PI3K-stimulated kinase, Akt/PKB, has been shown to inhibit apoptosis. The object of this study was to determine the role of these kinases in a glioma model of apoptosis. We have previously shown that K252a induces apoptosis and inhibits kinase activity. In this study we confirm these results and shown that the protein tyrosine phosphatase inhibitor sodium vanadate activates ERK, JNK and Akt/PKB, but does not stimulate proliferation. Vanadate did protect T98G cells from K252a-induced apoptosis, an effect that was abolished by addition of the PI3K inhibitor wortmannin. This suggests that PI3K and Akt/PKB may be responsible for mediating vanadate's protective effect on glioma cells. We conclude that the intracellular balance between protein phosphorylation pathways is a critical determinant of both cell proliferation and cell death.     

Sphingosine-1-phosphate inhibits human keratinocyte proliferation via Akt/protein kinase B inactivation

Although sphingosine-1-phosphate (S1P) is a well-known mitogen, it has only recently been demonstrated that S1P is able to inhibit cell proliferation in human epidermal keratinocytes and hepatic myofibroblasts. In the present study, we investigated the possible signalling pathways involved in the growth inhibition of human keratinocytes. Our results show that S1P potently inhibits keratinocyte proliferation, and that this leads to the inhibition of DNA synthesis. Interestingly, the prolonged activation of extracellular signal-regulated protein kinase (ERK) and the transient inactivation of Akt/protein kinase B (PKB) were also observed in concert with the inhibition of keratinocyte proliferation by S1P. To verify further the antiproliferative action of S1P, we examined changes in cell cycle-related proteins. S1P inhibited cyclin D(2) synthesis but stimulated p21(WAF1/CIP1) (p21) and p27(KIP1) (p27) synthesis; all are inhibitors of cyclin-dependent kinase. Furthermore, we found that the growth inhibition by S1P was in part abolished by pertussis toxin (PTX) treatment, but that ERK activation and Akt/PKB inhibition were not abrogated, suggesting that S1P functions both intracellularly, as a second messenger, and extracellularly, as a ligand for cell surface receptors. Insulin-like growth factor I (IGF-I) is a well-established human keratinocyte mitogen and is known to stimulate Akt/PKB in various cell types. In the present study, S1P was found to inhibit the keratinocyte proliferation and Akt/PKB activation induced by IGF-I. Our results suggest that S1P may play an important role in the negative regulation of keratinocyte proliferation by inhibiting the Akt/PKB pathway.     

RIP1, a kinase on the crossroads of a cell's decision to live or die

Binding of inflammatory cytokines to their receptors, stimulation of pathogen recognition receptors by pathogen-associated molecular patterns, and DNA damage induce specific signalling events. A cell that is exposed to these signals can respond by activation of NF-kappaB, mitogen-activated protein kinases and interferon regulatory factors, resulting in the upregulation of antiapoptotic proteins and of several cytokines. The consequent survival may or may not be accompanied by an inflammatory response. Alternatively, a cell can also activate death-signalling pathways, resulting in apoptosis or alternative cell death such as necrosis or autophagic cell death. Interplay between survival and death-promoting complexes continues as they compete with each other until one eventually dominates and determines the cell's fate. RIP1 is a crucial adaptor kinase on the crossroad of these stress-induced signalling pathways and a cell's decision to live or die. Following different upstream signals, particular RIP1-containing complexes are formed; these initiate only a limited number of cellular responses. In this review, we describe how RIP1 acts as a key integrator of signalling pathways initiated by stimulation of death receptors, bacterial or viral infection, genotoxic stress and T-cell homeostasis.     

Mechanical stretch stimulates protein kinase B/Akt phosphorylation in epidermal cells via angiotensin II type 1 receptor and epidermal growth factor receptor

Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.     

Angiostatin and endostatin inhibit endothelial cell migration in response to FGF and VEGF without interfering with specific intracellular signal transduction pathways

The anti-angiogenic agents angiostatin and endostatin have been shown to affect endothelial cell migration in a number of studies. We have examined the effect of these agents on intracellular signalling pathways known to regulate endothelial cell migration and proliferation/survival. Both agents inhibited fibroblast growth factor (FGF)-, and vascular endothelial growth factor (VEGF)-mediated migration of primary human microvascular endothelial cells and affected vascular formation in the embryoid body model. However, using phosphospecific antibodies we could not detect any effect of angiostatin or endostatin on phospholipase C-gamma (PLC-gamma), Akt/PKB, p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and p21-activated kinase (PAK) activity. Furthermore, using a glutathione S-transferase (GST)-PAK pull-down assay, we could not detect any effect on Rac activity. We conclude that angiostatin and endostatin inhibit chemotaxis, without affecting intracellular signalling pathways known to regulate endothelial migration and proliferation/survival.     

The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB

The serine/threonine kinase Akt, or protein kinase B (PKB), has recently been a focus of intense research. It appears that Akt/PKB lies in the crossroads of multiple cellular signaling pathways and acts as a transducer of many functions initiated by growth factor receptors that activate phosphatidylinositol 3-kinase (PI 3-kinase). Akt/PKB is particularly important in mediating several metabolic actions of insulin. Another major activity of Akt/PKB is to mediate cell survival. In addition, the recent discovery of the tumor suppressor PTEN as an antagonist of PI 3-kinase and Akt/PKB kinase activity suggests that Akt/PKB is a critical factor in the genesis of cancer. Thus, elucidation of the mechanisms of Akt/PKB regulation and its physiological functions should be important for the understanding of cellular metabolism, apoptosis, and cancer.     

Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt

The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth, glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein, called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and apoptosis in many cell types.     

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.     

Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death

The phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK-3 in response to insulin and growth factors, which inhibits GSK-3 activity and leads to the modulation of multiple GSK-3 regulated cellular processes. We show that the novel potent and selective small-molecule inhibitors of GSK-3; SB-415286 and SB-216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3-kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK-3 activity and modulation of GSK-3 substrates tau and beta-catenin. Thus, in addition to the previously assigned roles of GSK-3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK-3 activity in primary neurones is sufficient to prevent death, implicating GSK-3 as a physiologically relevant principal regulatory target of the PI 3-kinase/PKB neuronal survival pathway.     

Apoptosis: live or die--hard work either way!

This review presents a brief overview of the cell's apoptotic machinery, including specific and indirect death signals. Specific death signals are transferred via death ligands, death receptors, and their intracellular signalling pathways. Indirect death signals cumulate a wide range of stimuli that potentially harm survival of cells. These include intercalating drugs, irradiation or altered intracellular signalling. Herein, a focal point is the mitochondrial control of specific death enzymes--so called caspases--by members of the pro-apoptotic Bax and BH3 subfamily or the anti-apoptotic Bcl-2 subfamily. While the initiation of cell death happens through a variety of signalling systems, the activation of caspases plays a pivotal role in the progression towards the final morphologic findings in cells undergoing apoptosis. Caspases appear to directly cleave and inactivate substrates that are clinical for the maintenance of cell structure and function but also regulate the activity of other enzymes that induce the apoptotic phenotype within the cell. The insulin-like growth factors (IGFs) are potent proliferation factors and potently inhibit apoptosis acting via the ubiquitously expressed IGF-I receptor. Within IGF-I receptor signalling, key to the inhibition of apoptosis are the RAS/RAF/mitogen-activated protein (MAP)-kinase pathway and the PI 3'-kinase pathway. To give an example of high clinical relevance of apoptosis within endocrine disorders, apoptotic death of pancreatic beta cells in type 1 diabetes disease and the involvement of IGF-II in beta cell survival and beta cell function is discussed in detail. Finally, further understanding of signalling systems that are involved in proliferation or in apoptosis might provide novel tools to treat or even heal disorders like type I diabetes.     

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.