Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. The Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumor necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase [PI(3)K] and its downstream target Akt (protein kinase B). Wortmannin [a PI(3)K inhibitor], dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signaling pathway that is necessary for inducing key immune and inflammatory responses (Ozes, 1999).
The mechanisms of cell proliferation and transformation are intrinsically linked to the process of apoptosis: the default of proliferating cells is to die unless specific survival signals are provided. Platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation, but the mechanisms mediating its anti-apoptotic properties are not completely understood. The transcription factor NF-kappaB is important in PDGF signaling. NF-kappaB transmits two signals: one is required for the induction of proto-oncogene c-myc and proliferation, and the second, an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. A putative pathway has been traced whereby PDGF activates NF-kappaB through Ras and phospatidylinositol-3-kinase [PI(3)K] to the PKB/Akt protein kinase and the IkappaB kinase (IKK); NF-kappaB thus appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt pathway. Upon PDGF stimulation, Akt transiently associates in vivo with IKK and induces IKK activation. These findings establish a role for NF-kappaB in growth factor signaling and define an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kappaB pathway, thus linking anti-apoptotic signaling with transcription machinery (Romashkova, 1999).
Recent work has suggested a role for the serine/threonine kinase Akt and IkappaB kinases (IKKs) in nuclear factor (NF)-kappaB activation. In this study, the involvement of these components in NF-kappaB activation through a G protein-coupled pathway was examined using transfected HeLa cells that express the B2-type bradykinin (BK) receptor. The function of IKK2, and to a lesser extent, IKK1, is suggested by BK-induced activation of their kinase activities and by the ability of their dominant negative mutants to inhibit BK-induced NF-kappaB activation. BK-induced NF-kappaB activation and IKK2 activity are markedly inhibited by RGS3T, a regulator of G protein signaling that inhibits Galpha(q), and by two Gbetagamma scavengers. Co-expression of Galpha(q) potentiates BK-induced NF-kappaB activation, whereas co-expression of either an activated Galpha(q)(Q209L) or Gbeta(1)gamma(2) induces IKK2 activity and NF-kappaB activation without BK stimulation. BK-induced NF-kappaB activation is partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that PI3K is a downstream effector of Galpha(q) and Gbeta(1)gamma(2) for NF-kappaB activation. Furthermore, BK can activate the PI3K downstream kinase Akt, whereas a catalytically inactive mutant of Akt inhibits BK-induced NF-kappaB activation. Taken together, these findings suggest that BK utilizes a signaling pathway that involves Galpha(q), Gbeta(1)gamma(2), PI3K, Akt, and IKK for NF-kappaB activation (Xie, 2000).
The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16. Akt regulates signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, since other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival (Kane, 1999).
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. Akt also regulates the activity of FKHRL1 (Drosophila homolog Foxo), a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene (Brunet, 1999).
The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX is phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB have been replaced by Ala is not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induces transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites have been replaced by Ala is detected exclusively in the cell nucleus. AFX-GFP is in the nucleus when the cells are infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stays in the cytoplasm when the cells express constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation is induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppresses this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhances DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB (Takaishi, 1999).
Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. The activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylates recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) are resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus (Biggs, 1999).
The androgen receptor (AR) controls several biological functions including prostate cell growth and apoptosis. However, the mechanism by which AR maintains its stability to function properly remains largely unknown. Akt and Mdm2 have been shown to form a complex with AR and promote phosphorylation-dependent AR ubiquitylation, resulting in AR degradation by the proteasome. The effect of Akt on AR ubiquitylation and degradation is markedly impaired in a Mdm2-null cell line compared with the wild-type cell line, suggesting that Mdm2 is involved in Akt-mediated AR ubiquitylation and degradation. Furthermore, the E3 ligase activity of Mdm2 and phosphorylation of Mdm2 by Akt are essential for Mdm2 to affect AR ubiquitylation and degradation. These results suggest that phosphorylation-dependent AR ubiquitylation and degradation by Akt require the involvement of Mdm2 E3 ligase activity, a novel mechanism that provides insight into how AR is targeted for degradation (Lin, 2002).
IGF-1 can promote AR degradation via activation of the PI3K-Akt pathway. In addition, IL-6 can also down-regulate AR protein levels via the PI3K-Akt pathway. Thus, PI3K-Akt, but not MAPK, may represent the major pathway for growth factor-induced AR protein degradation. While the mechanism responsible for this distinct effect on AR protein degradation is currently unclear, it is likely that phosphorylation of AR at distinct sites by these two pathways may result in different AR conformations, which may then contribute differentially to protein stability. In support of the role of PI3K-Akt in AR degradation, an AR mutant, which is defective in Akt-mediated AR phosphorylation, is remarkably stable compared with the wtAR. Thus, the PI3K-Akt pathway is involved in protein degradation. PI3K-Akt has been associated with p27Kip1 and insulin receptor substrate-1 (IRS-1) stability, and blockage of the PI3K-Akt pathway causes the accumulation of p27Kip1 protein and IRS-1. Therefore, the PI3K-Akt pathway may represent a central pathway for the degradation of several proteins, including AR (Lin, 2002).
The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Protein kinase B phosphorylates AFX, a human ortholog of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus (Kops, 1999).
Forkhead transcription factor FKHR (Foxo1: Drosophila homolog Foxo) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. The fate has been investigated of FKHR after cells are stimulated by insulin. Insulin treatment is shown to decrease endogenous FKHR proteins in HepG2 cells; this decrease is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation (Matsuzaki, 2003).
Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggers proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilizes the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggers the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes (Plas, 2003).
Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. Myc is shown to recruit TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. It is suggested that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes (Bouchard, 2004).
The mTOR kinase controls cell growth, proliferation, and survival through two distinct multiprotein complexes, mTORC1 and mTORC2. mTOR and mLST8 are in both complexes, while raptor and rictor are part of only mTORC1 and mTORC2, respectively. To investigate mTORC1 and mTORC2 function in vivo, mice deficient for raptor, rictor, or mLST8 were generated. Like mice null for mTOR, those lacking raptor die early in development. However, mLST8 null embryos survive until e10.5 and resemble embryos missing rictor. mLST8 is necessary to maintain the rictor-mTOR, but not the raptor-mTOR, interaction, and both mLST8 and rictor are required for the hydrophobic motif phosphorylation of Akt/PKB and PKCα, but not S6K1. Furthermore, insulin signaling to FOXO3, but not to TSC2 or GSK3β, requires mLST8 and rictor. Thus, mTORC1 function is essential in early development, mLST8 is required only for mTORC2 signaling, and mTORC2 is a necessary component of the Akt-FOXO and PKCα pathways (Guertin, 2007).
The mammalian target of rapamycin (mTOR) kinase is the catalytic subunit of at least two distinct signaling complexes, referred to as mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Studies with cultured cell lines indicate that the complexes participate in different pathways and recognize distinct substrates, the specificity of which is determined by unique mTOR-interacting proteins. mTORC1 controls cell growth in part by phosphorylating S6 Kinase 1 (S6K1) and the eIF-4E-binding protein 1 (4E-BP1), known regulators of protein synthesis. It has been proposed that mTORC2 phosphorylates and activates Akt/PKB, which regulates cell proliferation, growth, survival, and metabolism. Full activation of Akt/PKB requires phosphorylation of S473 of the hydrophobic motif, the site proposed to depend on mTORC2, as well as phosphorylation of T308 of the activation loop by PDK1. The clinically valuable drug rapamycin specifically inhibits mTORC1 activity, although recent studies indicate that prolonged rapamycin treatment can also inhibit mTORC2 assembly and function in some cell types. Both complexes participate in signaling pathways associated with human diseases, including tuberous sclerosis complex (TSC), lymphangioleiomyomatosis (LAM), Cowden disease, Peutz-Jeghers syndrome (PJS), neurofibromatosis, familial cardiac hypertrophy, and cancers characterized by hyperactivation of PI3K/Akt (Guertin, 2007).
The roles of mTORC1 and mTORC2 during mammalian development are not well understood. In addition to mTOR, mTORC1 contains raptor (regulatory-associated protein of mTOR) and mLST8 (also called GβL). mTORC2 also contains mTOR and mLST8, but instead of raptor, this complex contains rictor (rapamycin-insensitive companion of mTOR). Germline disruption of mTOR in mice causes embryonic lethality at or around implantation. However, when these mice were engineered, it was not known that mTOR is part of two distinct complexes and pathways. With this new information, it becomes difficult to interpret the phenotypes of the mTOR null mice because these animals lack both mTORC1 and mTORC2 function. Thus, to determine the in vivo role of each branch of the mTOR signaling network, mice lacking the expression of raptor, rictor, and mLST8 were generated and characterized. It is conclude that during mammalian development, mTORC1 and mTORC2 have different, but essential, roles; that mLST8 is required only for mTORC2 function; and that mTORC2 is a crucial component of the Akt/PKB-FOXO and PKCα signaling networks (Guertin, 2007).
The serine/threonine kinase Akt is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. This study identifies an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Database searches reveal homologs in mouse, rat, and Drosophila, but no apparent matches in Caenorhabditis elegans and Dictyostelium. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 is replaced with alanine, form abnormal elongated shapes and exhibit limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility (Enomoto, 2005).
The structure of Girdin predicted by the COILS algorithm showed a tendency to assume an alpha-helical coiled-coil conformation in its middle domain, between Ala-253 and Lys-1375, with a high coiled-coil probability of 1.0. The predicted coiled-coil domain contains 135 continuous heptad repeats ([abcdefg]135) that are typical of alpha-helical coiled-coils. The 9.5 kb Girdin transcript was found to be expressed ubiquitously in various human tissues by high-stringency Northern blot analysis (Enomoto, 2005).
Four different regions can be distinguished in the Girdin molecule based on the sequences of its subunits, subcellular localization, and functions: an N-terminal region that seems to facilitate the formation of a dimer (NT), an extremely long coiled-coil region, a region that binds to the plasma membrane through the interaction with phosphoinositides (CT1), and a C-terminal region that encompasses an actin binding site (CT2). The amino acid sequence of the CT2 domain shows no homology with the calponin homology (CH) domain, a common actin binding domain that is present in most actin binding proteins such as alpha-actinin, filamins, fimbrin, spectrins, cortexillins, and dystrophin, suggesting that Girdin represents a novel class of actin binding proteins (Enomoto, 2005).
Analysis of the sequence of Girdin reveals that it includes 135 heptad repeats, (abcdefg)135, between Leu-253 and Lys-1375 that correspond to a central rod domain. Within the repeats, positions a and d are preferentially occupied by hydrophobic residues like Leu, Ile, Met, or Val; this is consistent with the signature of canonical coiled-coil structures that wind around each other in a superhelix. The oligomerization properties of coiled-coil sequences are determined by the distribution of alpha-branched residues in the a or d positions. Val and Ile in position a favor dimerization, they favor tetramerization in position d, and their presence in both a and d positions facilitate the formation of trimers. In the coiled-coil sequence of Girdin, 22 repeats have Val or Ile in the a position, whereas they are present in the d position of only 9 repeats, suggesting that the coiled-coil domain of Girdin tends to form a dimer. This is consistent with the findings suggested by gel filtration that the NT domain of Girdin forms a dimer (Enomoto, 2005).
The possession of two actin binding sites enables crosslinking or bundling proteins to link filaments and to stabilize higher-order assemblies of actin filaments. Possessing two actin binding CT2 domains in juxtaposition, the dimeric Girdin molecules seem to be designed to gather actin filaments together into bundles or a meshwork. Consistent with this possibility are the findings of immunofluorescent staining and electron microscopy that the depletion of Girdin interfers with actin networks, leading to the disruption of stress fibers, cortical actin filaments, and actin meshwork at the leading edge. During migration, the Girdin knockdown cells produce multiple protrusions, resulting in limited directional migration. These observations indicate that Girdin fulfils an essential function in determining the stability and integrity of actin bundles and meshwork. These mediate a variety of important biological processes. Eukaryotic cells have a fail-safe mechanism in the multiple actin crosslinking proteins that share overlapping functions. The phenotypic consequences of the depletion of Girdin indicate that the presence of other proteins cannot completely compensate for its loss. Because the speculated primary structure, molecular size, and putative function of Girdin are reminiscent of those of filamin, it is important to clarify the functional difference and synergism between the two (Enomoto, 2005).
The CT1 domain of Girdin associates with the plasma membrane through the cluster of basic amino acid residues Arg-1389 to Lys-1407. This positively charged sequence is related to a consensus sequence for PI(4,5)P2 binding, which has been found in gelsolin, villin, profilin, vinculin, and other various cytoskeletal proteins. Unexpectedly, the basic amino acid cluster in Girdin does not bind to PI(4,5)P2, but binds to PI(4)P and binds weakly to PI(3)P. Considering that PI(4)P, but not PI(3)P, is abundant in mammalian cells, it is plausible to conclude that Girdin binds to PI(4)P, which resides in the membranes of mammalian cells in an amount equal to that of PI(4,5)P2. It is speculated that it stabilizes the cortical actin filaments by anchoring them at the plasma membrane (Enomoto, 2005).
Akt phosphorylates Girdin in vitro and in intact cells. The phosphorylation of Girdin is induced by EGF and during cell migration, suggesting a significance for phosphorylation in physiological cellular events. In migrating Vero fibroblasts, the phosphorylated Girdin preferentially localizes to lamellipodia at the leading edge, which is in line with observations that activated Akt is also localized at the leading edge during migration in mammalian cells. It is plausible that Akt, activated downstream of PI3K, translocates from the cytosol to the leading edge through its PH domain, and subsequently phosphorylates Girdin on the actin filaments at the front of the cells (Enomoto, 2005). How does Akt regulate the function of Girdin by phosphorylation? Insight into this issue comes from the observation that the phosphorylation of the CT domain of Girdin affects in vitro binding to PI(4)P. Because the phosphorylation site is present in the neighborhood of the phosphoinositide binding site, it is speculated that phosphorylation induces a conformational change around these sites, and this change in turn alters affinity for the phosphoinositide. It was further found that the phosphorylated CT domain retains the property of actin binding, and its affinity for F-actin is comparable to that of the nonphosphorylated form. Based on these observations, it is speculated that phosphorylation by Akt releases Girdin from PI(4)P and allows it to localize at the leading edge in order to crosslink the newly generated actin filaments in the lamellipodium network (Enomoto, 2005).
BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) is inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, is activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt are found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway (del Paso, 1997).
Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).
In eukaryotes, entry into M-phase of the cell cycle is induced by activation of cyclin B-Cdc2 kinase. At G2-phase, the activity of its inactivator, a member of the Wee1 family of protein kinases (see Drosophila Wee1), exceeds that of its activator, Cdc25C phosphatase. However, at M-phase entry the situation is reversed, such that the activity of Cdc25C exceeds that of the Wee1 family. The mechanism of this reversal is unclear. In oocytes from the starfish Asterina pectinifera, the kinase Akt (or protein kinase B) phosphorylates and downregulates Myt1, a member of the Wee1 family. This switches the balance of regulator activities and causes the initial activation of cyclin B-Cdc2 at the meiotic G2/M-phase transition. These findings identify Myt1 as a new target of Akt, and demonstrate that Akt functions as an M-phase initiator (Okumura, 2002).
Growth factors and hormones activate protein translation by phosphorylation and inactivation of the translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive signaling pathway. The mechanism by which signals emanating from extracellular signals lead to phosphorylation of 4E-BPs is not well understood. The activity of the serine/threonine kinase Akt/PKB is shown to be required in a signaling cascade that leads to phosphorylation and inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1. Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1 by Akt requires the activity of FRAP/mTOR. FRAP/mTOR may lie downstream of Akt in this signaling cascade. These results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1 (Gingras, 1998).
Investigated were the roles of Akt (protein kinase B) and the atypical lambda isoform of protein kinase C (PKClambda), both of which act downstream of phosphoinositide 3-kinase, in the activation of glycogen synthase and phosphorylation of 4E-BP1 (PHAS-1) in response to insulin. A mutant Akt (Akt-AA) in which the phosphorylation sites targeted by growth factors are replaced by alanine was shown to inhibit insulin-induced activation of both Akt and glycogen synthase in L6 myotubes. Expression of a mutant Akt in which Lys179 in the kinase domain was replaced by aspartate also inhibits insulin-induced activation of glycogen synthase but has no effect on insulin activation of endogenous Akt. A kinase-defective mutant of PKClambda (lambdaDeltaNKD), which prevents insulin-induced activation of PKClambda, does not affect the activation of glycogen synthase by insulin. Insulin-induced phosphorylation of 4E-BP1 is inhibited by Akt-AA in Chinese hamster ovary cells. However, lambdaDeltaNKD has no effect on 4E-BP1 phosphorylation induced by insulin. These data suggest that Akt, but not PKClambda, is required for insulin activation of glycogen synthase and for insulin-induced phosphorylation of 4E-BP1 (Takata, 1999).
Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1: see Drosophila RPS6-p70-protein kinase) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. The importance of PKB signaling in S6K1 activation was examined. In parallel, both the inactivation of glycogen synthase kinase 3beta (GSK-3beta) and the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) were monitored as respective markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independent of its intracellular location (Dufner, 1999).
The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast-based two-hybrid system was employed that identified inosine-5' monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associate in vitro and this association moderately activates IMPDH. Purified PKB/Akt also associates with IMPDH in vitro. PKB/Akt or IMPDH can be pulled-down from mammalian cell lysates using glutathione-S-transferase (GST)-IMPDH or GST-PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH can be co-immunoprecipitated from COS cell lysates and active PKB/Akt can phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins (Ingleyab, 2000).
The role of the protein kinase Akt in cell migration is incompletely understood. Sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T236 residue. Transactivation of EDG-1 by Akt is not required for Gi-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequesters Akt and acts as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration. How GPCRs regulate Rac is poorly understood. EDG-1 activates Rac activity in endothelial cells and transfected CHO cells. Akt activity is required for EDG-1 to activate Rac. Akt and Rac are involved in a complex regulatory network to modulate actin dynamics and cell migration. Since EDG-1 phosphorylation by Akt is needed for Rac activation, it is likely that phosphorylated EDG-1 interacts with upstream mediators of Rac -- for example, the exchange factors such as Tiam. Indeed, S1P treatment of endothelial cells results in translocation of Tiam I and Rac to cell membrane (Lee, 2001).
Basement membranes (BM) are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Analysis of a fibroblast growth factor (FGF) receptor mutation has raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. This hypothesis was tested; constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, was shown to induce the synthesis of laminin-1 and collagen IV isotypes and cause their translocation to the BM. By using promoter-reporter constructs, constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB was shown to activate, whereas dominant negative Akt/PKB was shown to inhibit, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of BM formation. Thus, Akt/PKB activates laminin and collagen IV at the level of transcription. Akt/PKB controls numerous transcription factors. Whether the forkhead family, the NFkappa B system, or other mechanisms connect Akt/PKB activation with the transcription of laminin and collagen IV chains remains to be determined. It is tempting, nevertheless, to speculate that this regulation represents the positive side of BM remodeling, whereas metalloproteinases represent its negative side. Such positive regulation of BM formation could result in the local amplification of cell signaling mediated by the various signaling molecules associated with the BM (Li, 2001).
Heregulin (HRG)-induced tyrosine phosphorylation of the Gab2 docking protein is enhanced by pretreatment with wortmannin, indicating negative regulation via a PI3-kinase-dependent pathway. This represents phosphorylation by the serine/threonine kinase protein kinase B (PKB), since PKB constitutively associates with Gab2 (Drosophila homolog: Daughter of sevenless), phosphorylates Gab2 on a consensus phosphorylation site (Ser159) in vitro and inhibits Gab2 tyrosine phosphorylation. However, expression of Gab2 mutated at this site (S159A Gab2) not only enhances HRG-induced Gab2 tyrosine phosphorylation and association with Shc and ErbB2, but also markedly increased tyrosine phosphorylation of ErbB2 and other cellular proteins and amplifies activation of the ERK and PKB pathways. The impact of this negative regulation is further emphasized by a potent transforming activity for S159A Gab2, but not wild-type Gab2, in fibroblasts. These studies establish Gab2 as a proto-oncogene, and a model is presented in which receptor recruitment of Gab2 is tightly regulated via an intimate association with PKB. Release of this negative constraint enhances growth factor receptor signaling, possibly since Gab2 binding limits dephosphorylation and disassembly of receptor-associated signaling complexes (Lynch, 2002).
Overactivation of ionotropic glutamate receptors can induce neuronal death, a phenomenon known as excitotoxicity. Cell survival during this response is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. A novel interaction is described between Akt1 and JNK interacting protein 1 (JIP1), a JNK pathway scaffold. Direct association between Akt1 and JIP1 is observed in primary neurons. Neuronal exposure to an excitotoxic stimulus decreases the Akt1-JIP1 interaction and concomitantly increases association between JIP1 and JNK. Akt1 interaction with JIP1 inhibits JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Consistent with this view, neurons from Akt1-deficient mice exhibited higher susceptibility to kainate excitotoxicity than wild-type littermates. Overexpression of Akt1 mutants that bind JIP1 reduced excitotoxic apoptosis. These results suggest that Akt1 binding to JIP1 acts as a regulatory gate preventing JNK activation, which is released under conditions of excitotoxic injury (Yano, 2002).
In several systems, the JNK pathway plays a positive role in apoptosis. Glutamate or kainate exposure activates the JNK pathway in primary neurons; this activation is responsible for subsequent apoptotic death. How is an excitotoxicity-specific JNK response generated? The JIP family of JNK scaffolds (also islet-brain [IB] or JNK/stress-activated kinase-associated protein [JSAP]) has been suggested to play a critical role in assembling specific JNK signaling pathway components. Each member of this scaffold family can bind JNK, MKK7, and a mixed-lineage kinase (MLK, a MAPKKK family) on different regions of JIP. Experiments using transiently transfected cell lines have suggested that JIP1 can amplify MLK-induced JNK activation. Recent evidence from mice genetically deficient in JIP1 has demonstrated that this scaffold is a stimulus-specific, positive regulator of JNK activity in vivo. Significantly, JIP1 gene deletion confers higher resistance to kainate-induced neuronal death in mice and neuronal culture, indicating that JIP1 plays an positive role in AMPA/kainate receptor-mediated apoptotic signaling (Yano, 2002).
JNK activity can be antagonized by Akt kinase activity in numerous cell systems, and this crosstalk may underlie many of the prosurvival effects of Akt. The Akt family of Ser/Thr-directed protein kinases (Akt1-3 or protein kinase Balpha-gamma) are important mediators of cell survival in response to growth factors and stimuli that elicit calcium influx. Akt kinases have been suggested to phosphorylate a number of proapoptotic proteins directly, thereby leading to suppression of death signals. This study describes a novel mechanism of Akt-JNK crosstalk in neurons undergoing excitotoxic apoptosis. Evidence that Akt1 binding to JIP1 decreases JIP1's ability to enhance JNK activity by interfering with JIP1-mediated assembly of an active JNK signaling complex. Excitotoxic kainate exposure decreases the neuronal interaction between Akt1 and JIP1 and increases formation of JNK-JIP1 complexes, suggesting that Akt1 interaction with JIP1 acts as a negative switch for JNK activity. Consistent with this model, Akt1 gene deletion renders neurons more susceptible to kainate-induced neuronal death, and ectopic expression of Akt1 binding mutants decreases kainate toxicity (Yano, 2002).
In the search for neuroprotective factors in Huntington's disease, it was found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. huntingtin is a substrate of Akt and phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Akt is altered in Huntington's disease patients. Taken together, these results support a potential role of the Akt pathway in Huntington's disease (Humbert, 2002).
Although the complete neuroprotective effect mediated by IGF-1 requires phosphorylation of mutant huntingtin at S421, in the absence of phosphorylation of huntingtin, IGF-1 is still able to induce some neuroprotection. This suggests that in the context of mutant huntingtin-induced cell death, IGF-1 mediates its neuroprotective effect not only via a direct action of Akt on huntingtin protein but also via the phosphorylation of other substrates that increase neuronal survival. Several substrates of Akt such as Bad, FOXOs, and caspase-9 that promote neuronal survival when phosphorylated by Akt have been described. Those substrates, as well as others that remain to be identified, could also participate in mediating the neuroprotective effect elicited by IGF-1 and Akt on mutant huntingtin-induced cell death (Humbert, 2002).
The role of the PI 3-kinase cascade in regulation of cell growth is well established. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects and the role it plays in initiation of S phase. PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas constitutively active PKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery (Shtivelman, 2002).
An affinity purification method has been used to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown to be the Yes-associated protein (YAP: Drosophila homolog Yorkie), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage (Basu, 2003).
YAP is a 65 kDa protein (sometimes termed YAP65 or YAP1) that was originally identified due to its interaction with the Src family tyrosine kinase Yes. YAP contains either one or two WW domains depending on alternative splicing and also a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. YAP has been reported to interact with p53 binding protein-2, an important regulator of the apoptotic activity of p53. Through its carboxyl terminus, YAP binds to the PDZ-containing protein EBP50, a submembranous scaffolding protein. YAP is a transcriptional coactivator that binds and activates Runx transcription factors and the four TEAD/TEF transcription factors. YAP is homologous to TAZ (45% identity), a transcriptional coactivator that is regulated by interaction with 14-3-3 and PDZ domain-containing proteins. YAP also interacts with the p53 family member p73, resulting in an enhancement of p73's transcriptional activity. YAP phosphorylation by Akt suppresses its ability to promote p73-mediated transcription of proapoptotic genes in response to DNA damaging agents and the resulting cell death. This extends the range of mechanisms whereby Akt can promote cellular survival in the face of apoptotic stimuli (Basu, 2003).
The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity is clearly observed when immunoprecipitations are conducted with the mTOR antibody, mTAb2. Insulin also increases by severalfold the 32P content of mTOR, determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affects neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreases the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity are abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimics insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. These findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB (Scott, 1999).
Components of intracellular signaling that mediate the stimulation-dependent recycling of integrins are being identified, but key transport effectors that are the ultimate downstream targets remain unknown. ACAP1, a GAP for ARF6, has been shown to function as a transport effector in the cargo sorting of transferrin receptor (TfR) that undergoes constitutive recycling. This study shows that ACAP1 also participates in the regulated recycling of integrin β1 to control cell migration. However, in contrast to TfR recycling, the role of ACAP1 in β1 recycling requires its phosphorylation by Akt, which is, in turn, regulated by a canonical signaling pathway. Disrupting the activities of either ACAP1 or Akt, or their assembly with endosomal β1, inhibits β1 recycling and cell migration. These findings advance an understanding of how integrin recycling is achieved during cell migration, and also address a basic issue of how intracellular signaling can interface with transport to achieve regulated recycling (Li, 2005).
The ARF family of small GTPases initiates intracellular transport by regulating the recruitment of coat proteins and other cargo-sorting adaptors from the cytosol to membrane. The GAPs for these small GTPases in the better-characterized transport pathways have been shown to function not only as negative upstream regulators of ARFs, but also as their effectors, by being components of coat complexes. An important implication of the cumulative findings on ACAP1 as a cargo-sorting device is that this role will be relevant for a broad range of cellular activities that are known to involve endocytic recycling. Besides cell migration, which itself underlies a wide range of physiologic and pathologic events, other important examples that require endocytic recycling include insulin-stimulated recycling of glucose transporters, cell polarity, cytokinesis, and phagocytosis. Thus, the future investigation of a potential role for ACAP1 in these examples will likely contribute to a better mechanistic understanding of how these events are achieved (Li, 2005),
In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. Activation of PKB and CDK1 was monitored during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation (Kalous, 2005).
Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. The kinase activity of Akt/PKB kinase is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a pathway involving a heterotrimeric G protein and PI3-kinase. Dictyostelium contains several kinases structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement and cellular morphogenesis during development. PKBR-1 has a kinase and a carboxy-terminal domain related to those of Akt/PKB, but no PH domain. Instead, it has an amino-terminal myristoylation site, which is required for its constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G-protein-dependent pathway, but does not require PI3-kinase, probably because of the constitutive membrane localization of PKBR-1. This is supported by experiments demonstrating the requirement for membrane association for activation and in vivo function of PKBR-1. PKBR-1 protein is found in all cells throughout early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented by Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell-polarity defects than Akt/PKB null cells. These results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during Dictyostelium multicellular development. The results suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB functions predominantly during aggregation to control cell polarity and chemotaxis, whereas PKBR-1 is required for morphogenesis during multicellular development (Meili, 2000).
Studies in Dictyostelium have shown that the p110-related phosphatidylinositol-3-kinases PI3K1 and PI3K2 are required for proper development, pinocytosis chemotaxis, and chemoattractant-mediated activation of PKB. Insights into the mechanism by which PI3K regulates chemotaxis derive from studies on PKB in mammalian leukocytes and Dictyostelium cells. PKB activation requires its translocation to the plasma membrane by binding of its PH domain to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 produced upon activation of PI3K, leading to PKB activation. In leukocytes and Dictyostelium cells, chemoattractants mediate PKB activation through a G-protein-coupled pathway that requires the activity of the respective PI3Ks. Chemoattractant stimulation of neutrophils and Dictyostelium cells results in a transient localization of a GFP fusion of the PH domains from the Dictyostelium and mammalian PKBs to the plasma membrane. When these cells are placed in a chemoattractant gradient, membrane localization of the PKB-PH-GFP fusion is restricted to the leading edge, as is the case for other PH-domain-containing proteins in Dictyostelium. In Dictyostelium, translocation of the PKB-PH domain GFP fusion is PI3K-dependent. PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, a structural homolog of mammalian PAKs (p21-activated kinase) that is required for myosin II assembly. PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis (Chung, 2001).
The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Disruption is reported of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt-/- mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt-/- mice, upon exposure to genotoxic stress, is shorter. However, Akt-/- mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt-/- male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt-/- mice, and Akt-/- thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt-/- mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal (Chen, 2001).
The relatively subtle phenotype of Akt1-/- mice suggests that Akt2 and Akt3 may substitute to some extent for Akt1, as was shown for the Akt1 and Akt2 in Caenorhabditis elegans. Since Akt2 and Akt3 are expressed in both testes and thymus, it is not clear why only these particular organs are affected by the ablation of Akt1. One possibility is that germ cells and thymus cells are exclusively dependent on Akt for their survival and therefore even a reduced threshold level of Akt activity is sufficient to affect their survival. Alternatively, despite a similar level of expression of the other Akt isoforms in these organs, Akt1 is more profoundly activated in the cells of these organs and/or may have exclusive protein substrates in these cells. Further studies including deletions of akt2 and akt3 genes are required to verify these possibilities (Chen, 2001).
In Drosophila, the PTEN/PI 3-kinase/Akt signaling pathway is associated with cell survival, organism size and metabolism. Disruption of the Akt gene in Drosophila impairs normal cell survival during embryogenesis, and results in a decreased cell size. The disruption of the akt-1 gene in the mouse, which by itself does not impair embryogenesis, has been shown here to affect cell survival and organism size, as well as cause growth retardation in adult mice. It remains to be seen if the combined disruption of the three akt genes in the mouse will result in embryonic lethality as a result of impaired cell survival during embryogenesis (Chen, 2001).
Surprisingly, despite multiple downstream effectors of Akt and the ubiquitous expression of Akt1, ablation of Akt1 by itself does not have a gross phenotypic impact. This observation implies that reduced threshold level of Akt activity can be tolerated and therefore suggests that small molecules aimed at reducing Akt activity could be excellent therapeutic regimens for the treatment of cancers in which the PI 3-kinase/Akt pathway is constitutively activated (Chen, 2001).
A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. Two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation (Paradis, 1998).
Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways exert opposing effects on muscle cell hypertrophy. Manipulation of these pathways during muscle differentiation indicates that inhibition of the Ras-Raf-MEK-ERK pathway promotes differentiation, whereas inhibition of PI3K blocks differentiation. However, the roles of these two pathways in the process of skeletal muscle hypertrophy has not previously been evaluated. C2C12 myoblasts normally proliferate and are mononucleated. When deprived of serum at confluence, they fuse and differentiate into postmitotic, elongated, and multinucleated myotubes. The hypertrophic action of insulin-like growth factor-1 (IGF-1) on muscle cells in vivo is mimicked by the addition of IGF-1 during the differentiation of C2C12 myotubes in vitro, resulting in the generation of thicker myotubes. In addition to inducing hypertrophy of myotubes in vivo, IGF-1 has been shown to activate both the Raf-MEK-ERK pathway and the PI3K-Akt pathway. The roles of these two pathways in the differentiation and hypertrophy of C2C12 myotubes were examined by genetic manipulation. Expression of a constitutively active form of Raf (c.a.-Raf) results in the generation of smaller and thinner myotubes, whereas expression of a dominant negative form of Raf (d.n.-Raf) results in markedly thicker myotubes. Thus, inhibition of the Raf-MEK-ERK pathway induces a hypertrophic phenotype similar to that elicited by IGF-1 treatment. In contrast, activation of the Akt pathway by expression of a constitutively active form of Akt (c.a.-Akt) results in a hypertrophic phenotype more pronounced than that observed with d.n.-Raf and characterized by multinucleated myotubes that are both thickened and shortened. Thus, genetic manipulation of the Raf-MEK-ERK and PI3K-Akt pathways reveals opposing phenotypic effects of these pathways during muscle differentiation, with the Raf-MEK-ERK pathway inhibiting development of the hypertrophic phenotype and the PI3K-Akt pathway promoting it. The PI3K-Akt pathway inhibits the Raf-MEK-ERK pathway; this cross-regulation depends on the differentiation state of the cell: Akt activation inhibits the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlates with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex (Rommel, 1999).
The Akt/protein kinase B serine/threonine kinase is a downstream effector of phosphoinositide 3-kinase (PI3K). Akt is an important component of mitogenic and antiapoptotic signaling pathways and is implicated in neoplastic transformation. Thyroid cells in culture retain a differentiated phenotype consisting of epithelial cell morphology and the expression of several tissue-specific genes. The survival and proliferation of these cells depend on thyrotropin and a mixture of five additional hormones that includes insulin. The regulation of proliferation and the expression of the thyroid differentiation program are intimately connected processes. As a result, oncogenes that induce hormone-independent proliferation invariably impair the expression of the thyroid-specific differentiation markers. Given that thyrotropin and insulin stimulate Akt activation in thyroid cells, the effects of Akt on thyroid cell proliferation, survival, and differentiation were determined. To this end, constitutively active myristylated Akt (myrAkt) was expressed in PC Cl 3 thyroid cells. The myrAkt-expressing cells continue to proliferate, even in the absence of hormones, and they are resistant to programmed cell death induced by starvation. These effects are paralleled by the induction of the G1 cyclins D3 and E and by the inhibition of induction of the proapoptotic Fas, Fas ligand, and BAD genes in starved cells. However, in marked contrast with several other oncogenes, myrAkt does not interfere with the expression of thyroid differentiation functions. These results unveil the existence of an Akt-triggered thyroid cell pathway that modulates proliferation and survival without affecting the expression of the thyroid cell differentiated phenotype (De Vita, 2000).
The serine/threonine kinase protein kinase B (PKB)/Akt mediates cell survival in a variety of systems. Transgenic mice expressing a constitutively active form of PKB (gag-PKB) have been generated to examine the effects of PKB activity on T lymphocyte survival. Thymocytes and mature T cells overexpressing gag-PKB display increased active PKB, enhanced viability in culture, and resistance to a variety of apoptotic stimuli. PKB activity prolongs the survival of CD4(+)CD8(+) double positive (DP) thymocytes in fetal thymic organ culture, but is unable to prevent antigen-induced clonal deletion of thymocytes expressing the major histocompatibility complex class I-restricted P14 T cell receptor (TCR). In mature T lymphocytes, PKB can be activated in response to TCR stimulation, and peptide-antigen-specific proliferation is enhanced in T cells expressing the gag-PKB transgene. Both thymocytes and T cells overexpressing gag-PKB display elevated levels of the antiapoptotic molecule Bcl-X(L). In addition, the activation of peripheral T cells leads to enhanced NF-kappaB activation via accelerated degradation of the NF-kappaB inhibitory protein IkappaBalpha. These data highlight a physiological role for PKB in promoting survival of DP thymocytes and mature T cells, and provide evidence for the direct association of three major survival molecules [PKB, Bcl-X(L), and NF-kappaB] in vivo in T lymphocytes (Jones, 2000).
Phosphoinositide 3-kinase (PI3K) has been shown to regulate cell and organ size in Drosophila, but the role of PI3K in vertebrates in vivo is not well understood. To examine the role of PI3K in intact mammalian tissue, transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart have been created and characterized. Cardiac-specific expression of constitutively active PI3K results in mice with larger hearts, while dominant-negative PI3K results in mice with smaller hearts. The increase or decrease in heart size is associated with comparable increase or decrease in myocyte size. Cardiomyopathic changes, such as myocyte necrosis, apoptosis, interstitial fibrosis or contractile dysfunction, are not observed in either of the transgenic mice. Thus, the PI3K pathway is necessary and sufficient to promote organ growth in mammals (Shioi, 2000).
What are the downstream targets of PI3K that are involved in the regulation of organ size? The available information does not provide a definitive answer to this question. However, Akt, a well characterized downstream target of PI3K, is likely to be one of the major mediators of this process. Akt is necessary and sufficient for phosphorylation and subsequent inactivation of 4E-BP1, a repressor of mRNA translation. Akt can also activate p70S6K in some contexts, although activation of p70S6K might not be solely dependent on Akt. p70S6K is upregulated in caPI3K hearts and downregulated in dnPI3K hearts. The amount of phosphorylated S6 protein correlates with the activation of p70S6K. The inhibition of the p70S6K pathway by rapamycin at nanomolar concentrations selectively suppresses an increase in protein synthesis of cultured neonatal myocytes in response to growth factors. Interestingly, rapamycin does not inhibit other phenotypic changes associated with myocyte hypertrophy, such as re-activation of fetal genes and sarcomere organization. This raises the possibility that p70S6K may selectively regulate cell size via controlling the rate of protein synthesis. Gene disruption of p70S6K is known to result in smaller body size in mice. In Drosophila, deficiency of the S6K gene is associated with a reduction in body size associated with smaller cells (Shioi, 2000 and references therein).
Nerve growth factor (NGF) induces dramatic axon growth from responsive embryonic peripheral neurons. However, the roles of the various NGF-triggered signaling cascades in determining specific axon morphological features remain unknown. Activated and inhibitory mutants of Trk effectors were transfected into sensory neurons lacking the proapoptotic protein Bax. This allowed axon growth to be studied in the absence of NGF, enabling the contributions of individual signaling mediators to be observed. While Ras is both necessary and sufficient for NGF-stimulated axon growth, the Ras effectors Raf and Akt induce distinct morphologies. Activated Raf-1 causes axon lengthening comparable to NGF, while active Akt increases axon caliber and branching. These results suggest that the different Trk effector pathways mediate distinct morphological aspects of developing neurons (Markus, 2002).
The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Akt activity has been shown to increase along with cellular senescence; inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology (Miyauchi, 2004).
Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. The present study assessed the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells (Lee, 2007).
Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induce phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 is defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylates recombinant Casp9 in vitro on serine-196 and inhibits its protease activity. Mutant pro-Casp9(Ser196Ala) is resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation (Cardone, 1998).
Caspase-9 is one caspase upstream of caspase-3 and its activation is stimulated by Apaf-1/cytochrome c and inhibited by Akt signals. BAD phosphorylation by Akt is an essential step for growth factor-mediated inhibition of caspase activation. Recently, it has been shown that human caspase-9 is phosphorylated by Akt and that its protease activity is reduced. To clarify the molecular mechanism of regulation of caspase-9 activation in neuronal apoptosis, two alternative splicing products of mouse caspase-9, caspase-9L and caspase-9S, were isolated from a P19 embryonal carcinoma cell cDNA library. Curiously, the Akt phosphorylation sites and motifs found in human caspase-9 are absent in both mouse caspase-9L and -9S. Mouse caspase-9 is not phosphorylated by activated Akt in vitro. Reverse transcription polymerase chain reaction analysis shows that the absent Akt motif is not limited to caspase-9 expressed in P19 embryonal carcinoma cells but also occurs in caspase-9 expressed in mouse, rat, and monkey. These results suggest that inhibition of caspase-9 activation by Akt-dependent phosphorylation is not generalized across species (Fujita, 1999).
Phosphoinositide 3 kinase/Akt pathway plays an essential role in neuronal survival. However, the cellular mechanisms by which Akt suppresses cell death and protects neurons from apoptosis remain unclear. Transient expression of constitutively active Akt inhibits ceramide-induced death of hybrid motor neuron 1 cells. Stable expression of either constitutively active Akt or Bcl-2 inhibits apoptosis, but only Bcl-2 prevents the release of cytochrome c from mitochondria, suggesting that Akt regulates apoptosis at a postmitochondrial level. Consistent with this, overexpressing active Akt rescues cells from apoptosis without altering expression levels of endogenous Bcl-2, Bcl-x, or Bax. Akt inhibits apoptosis induced by microinjection of cytochrome c and lysates from cells expressing active Akt inhibit cytochrome c-induced caspase activation in a cell-free assay, while lysates from Bcl-2-expressing cells have no effect. Addition of cytochrome c and dATP to lysates from cells expressing active Akt do not activate caspase-9 or -3 and immunoprecipitated Akt added to control lysates blocks cytochrome c-induced activation of the caspase cascade. Taken together, these data suggest that Akt inhibits activation of caspase-9 and -3 by posttranslational modification of a cytosolic factor downstream of cytochrome c and before activation of caspase-9 (Zhou, 2000).
A growing number of downstream targets of Akt have been identified, including glycogen synthase kinase-3, BAD, human caspase-9, and transcription factors such as CREB, Forkhead, and NFkappaB. While each of these has been implicated as an important target for Akt to inhibit apoptosis, in the present case, caspase-9 appears to be the most likely candidate since Akt phosphorylates human caspase-9 and inhibits its activity, which would provide a potential mechanism for Akt to inhibit caspase activation at a postmitochondrial level. However, [32P]orthophosphate labeling of cells expressing active Akt indicates that mouse caspase-9 is not phosphorylated by Akt. This may be explained by recent reports that the consensus Akt phosphorylation sites on human caspase-9 are not conserved in caspase-9 from other species such as mouse caspase-9 in HMN1 cells. However, the data also suggest that there exists an additional, more general mechanism by which Akt can suppress activation of caspases by cytochrome c at a postmitochondrial stage. When added into vector-cell extracts, immunoisolated active Akt is sufficient to inhibit caspase activation induced by cytochrome c, suggesting that active Akt has a direct effect on inhibiting cytochrome c-induced caspase activation. One such direct target for Akt may be Apaf-1 (Drosophila homolog: Apaf-1-related-killer), which forms a holoenzyme with caspase-9 and regulates caspase-9 activity. Akt phosphorylates Apaf-1 in vitro; however, further studies are needed to determine whether cellular Apaf-1 is a direct target of Akt, and how the phosphorylation status of Apaf-1 may regulate caspase-9 activation (Zhou, 2000).
The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a mitochondrial permeability transition pore (PT pore) component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to glucose metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. Evidence is provided that such coupling serves as a downstream effector function for Akt: (1) Akt increases mitochondria-associated hexokinase activity; (2) the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase and (3) ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. It is therefore proposed that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane (Gottlob, 2001).
Spermatogonial stem cells have unique properties to self-renew and support spermatogenesis throughout their lifespan. Although glial cell line-derived neurotrophic factor (GDNF) has recently been identified as a self-renewal factor for spermatogonial stem cells, the molecular mechanism of spermatogonial stem cell self-renewal remains unclear. In the present study, the role of the phosphoinositide-3 kinase (PI3K)-Akt pathway was assessed using a germline stem (GS) cell culture system that allows in vitro expansion of spermatogonial stem cells. Akt was rapidly phosphorylated when GDNF was added to the GS cell culture, and the addition of a chemical inhibitor of PI3K prevented GS cell self-renewal. Furthermore, conditional activation of the myristoylated form of Akt-Mer (myr-Akt-Mer) by 4-hydroxy-tamoxifen induced logarithmic proliferation of GS cells in the absence of GDNF for at least 5 months. The myr-Akt-Mer GS cells expressed spermatogonial markers and retained androgenetic imprinting patterns. In addition, they supported spermatogenesis and generated offspring following spermatogonial transplantation into the testes of infertile recipient mice, indicating that they are functionally normal. These results demonstrate that activation of the PI3K-Akt pathway plays a central role in the self-renewal division of spermatogonial stem cells (Lee, 2007).
Three members have been identified in the protein kinase B (PKB) family: Akt/PKB alpha, AKT2/PKB beta, and AKT3/PKB gamma. Previous studies have demonstrated that only AKT2 is predominantly involved in human malignancies and has oncogenic activity. However, the mechanism of transforming activity of AKT2 is still not well understood. The activation of AKT2 has been demonstrated in human ovarian epithelial cancer cells with several growth factors, including epidermal growth factor, insulin-like growth factor I, insulin-like growth factor II, basic fibroblast growth factor, platelet-derived growth factor, and insulin. The kinase activity and the phosphorylation of AKT2 are induced by the growth factors and blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, and dominant-negative Ras (N17Ras). Moreover, the activated Ras and v-Src, two proteins that transduce growth factor-generated signals, also activate AKT2, and this activation is not significantly enhanced by growth factor stimulation but is abrogated by wortmannin. These results indicate that AKT2 is a downstream target of PI 3-kinase and that Ras and Src function upstream of PI 3-kinase and mediate the activation of AKT2 by growth factors. The findings also provide further evidence that AKT2, in cooperation with Ras and Src, is important in the development of some human malignancies (Liu, 1998).
The Akt/PKB protein kinase is implicated in the control of cell cycle progression and the suppression of apoptosis in cancer cells. A conditionally active form of Akt/PKB (M+ Akt:ER*) was used to study the ability of this protein to influence biological processes that are central to the process of oncogenic transformation of mammalian cells. Activation of M+ Akt:ER* in Rat1 cells elicits alterations in cell morphology and promotes anchorage-independent growth in agarose with high efficiency. Consistent with these observations, activation of M+ Akt:ER* suppresses the apoptosis of Rat1 cells that occurs after the detachment of these cells from extracellular matrix. Furthermore, activation of M+ Akt:ER* is sufficient to promote the progression of quiescent Rat1 cells into the S and G2-M phases of the cell cycle. In accord with this is the observation that activation of M+ Akt:ER* leads to decreased expression of the cyclin-dependent kinase inhibitor p27Kip1 with a concomitant increase in cyclin-dependent kinase-2 activity. Perhaps surprisingly, activation of M+ Akt:ER* or expression of a constitutively active form of Akt leads to rapid activation of MAP/ERK kinase (MEK) and the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases in Rat1 cells. However, pharmacological inhibition of MEK by PD098059 does not inhibit the morphological alterations of Rat1 cells that occur after M+ Akt:ER* activation. These data suggest that M+ Akt:ER* can activate a number of pathways in Rat1 cells, leading to significant alterations in a number of biological processes. The conditional transformation system described here will allow further elucidation of the ability of Akt to contribute to both the normal response of cells to mitogenic stimulation and the aberrant proliferation observed in cancer cells (Mirza, 2000).
v-Crk induces cellular tyrosine phosphorylation and transformation of chicken embryo fibroblasts (CEF). The molecular mechanism of the v-Crk-induced transformation was studied. Experiments with Src homology (SH)2 and SH3 domain mutants revealed that the induction of tyrosine phosphorylation of cellular proteins requires only the SH2 domain, but both the SH2 and SH3 domains are required for complete transformation. Analysis of three well defined signaling pathways, the mitogen-activated protein kinase (MAPK) pathway, the Jun N-terminal kinase (JNK) pathway, and the phosphoinositide 3-kinase (PI3K)/AKT pathway, demonstrate that only the PI3K/AKT pathway is constitutively activated in v-Crk-transformed CEF. Both the SH2 and SH3 domains are required for this activation of the PI3K/AKT pathway in CEF. The colony formation of CEF is strongly induced by a constitutively active PI3K mutant, and a PI3K inhibitor, LY294002, suppresses the v-Crk-induced transformation. These results strongly suggest that constitutive activation of the PI3K/AKT pathway plays an essential role in v-Crk-induced transformation of CEF (Akagi, 2000).
Gliomas are the most common primary malignant brain tumors and are classified into four clinical grades, with the most aggressive tumors being grade 4 astrocytomas (also known as glioblastoma multiforme; GBM). Frequent genetic alterations in GBMs result in stimulation of common signal transduction pathways involving Ras, Akt and other proteins. It is not known which of these pathways, if any, are sufficient to induce GBM formation. In a tissue-specific manner, genes encoding activated forms of Ras and Akt have been transferred to astrocytes and neural progenitors in mice. Although neither activated Ras nor Akt alone is sufficient to induce GBM formation, the combination of activated Ras and Akt induces high-grade gliomas with the histological features of human GBMs. These tumors appear to arise after gene transfer to neural progenitors, but not after transfer to differentiated astrocytes. Increased activity of RAS is found in many human GBMs, and Akt activity is increased in most of these tumors, implying that combined activation of these two pathways accurately models the biology of this disease (Holland, 2000).
PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. The integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. The activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy (Persad, 2000).
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