The DAF-2 insulin receptor-like signaling pathway controls metabolism, development, longevity, and stress response in C. elegans. SGK-1, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase SGK, acts in parallel to the AKT kinases to mediate DAF-2 signaling. Loss of sgk-1 results in defective egg-laying, extended generation time, increased stress resistance, and an extension of life span. SGK-1 forms a protein complex with the AKT kinases, and is activated by and strictly depends on PI3K-like dependent kinase (PDK-1; Protein kinase 61C in Drosophila, the central mediator of cell signaling between phosphoinositide 3-kinase and various intracellular serine/threonine kinases including Akt). All three kinases of this complex are able to directly phosphorylate DAF-16/FKHRL1, yet have different functions in DAF-2 signaling. Whereas AKT-1 and AKT-2 are more important for regulating dauer formation, SGK-1 is the crucial factor for the control of development, stress response, and longevity. These data also suggest the existence of a second pathway from DAF-2 to DAF-16 that does not depend on AKT-1, AKT-2, and SGK-1 (Hertweck, 2004).
The mammalian insulin signaling pathway affects multiple downstream targets mediating a variety of cellular responses to insulin. Genetic studies have shown that a similar pathway exists in C. elegans that involves insulin-like ligands, the DAF-2 insulin/IGF-I receptor-like protein, the AAP-1 PI3K-like adaptor subunit, the AGE-1 PI3K-like catalytic subunit, the DAF-18 PTEN lipid phosphatase, and the PI3K-like dependent kinase PDK-1, which activates the Akt/PKB-like serine/threonine (S/T) protein kinases AKT-1 and AKT-2. AKT-1 and AKT-2 are candidates to antagonize the forkhead transcription factor DAF-16 to repress genes that regulate diapause, longevity, stress response, and energy storage, and to activate genes necessary for metabolism and reproductive growth. Reduction of daf-2 gene activity or other genes that are positively regulated by daf-2, like aap-1, age-1, pdk-1, or akt-1/-2, cause a constitutive developmental arrest at the dauer larval stage (Daf-c). Reduction-of-function mutations in genes that antagonize daf-2 signaling, such as daf-18 and daf-16, suppress the dauer constitutive phenotype of daf-2 and the other Daf-c mutants (Hertweck, 2004 and references therein).
According to the current model of Akt/PKB activation in mammalian cells, phosphatidylinositol-3,4-diphosphate (PIP2) and/or phosphatidylinositol-3,4,5-triphosphate (PIP3) bind to the amino-terminal pleckstrin homology (PH) domain of Akt/PKB, resulting in the recruitment of Akt to the plasma membrane. PDK1 colocalizes with Akt/PKB to the cell membrane and activates it by phosphorylation. Activated Akt/PKB is capable of promoting cell survival by phosphorylating and inactivating the mammalian DAF-16 homolog FKHRL1. However, in C. elegans, RNAi depletion of the Akt/PKB homologs akt-1 and akt-2 is not sufficient to shut down the DAF-2 insulin signaling pathway entirely. Moreover, inactivation of the Akt/PKB consensus phosphorylation sites in DAF-16 was not sufficient to induce dauer formation and life span extension. Therefore, other, yet-unidentified components have to exist downstream of pdk-1 to essentially control metabolism, development, longevity, and stress response (Hertweck, 2004 and references therein).
Serum- and glucocorticoid-inducible kinases (SGKs) are S/T protein kinases with approximately 55% sequence similarity to Akt/PKB. SGK expression is regulated by a variety of external stimuli, including growth factors like TGF-β and IGF-I, steroid or peptide hormones like insulin, cytokines, alterations in cell volume, osmotic changes, and cortical brain injury. Recent studies have revealed a role for SGK in cell survival signaling depending on PI3K. SGK is thought to be activated by PDK1 at sites that are equivalent to the Akt/PKB phosphorylation sites. PDK1 uses its hydrophobic PIF binding pocket to interact with and activate SGK. SGK may promote cell survival in parallel or complementary to Akt/PKB, since it participates in phosphorylating and inactivating FKHRL1 at sites different from those modified by Akt/PKB (Hertweck, 2004 and references therein).
Using reverse genetic approaches and biochemical analyses, the C. elegans homolog of the serum- and glucocorticoid-inducible kinase, SGK-1 is shown to function in parallel to AKT-1 and AKT-2 to mediate DAF-2 insulin receptor-like signaling. SGK-1 is activated by and strictly depends on PDK-1, and binds to AKT-1 and AKT-2 to form a multimeric protein complex. Direct evidence is provided that the AKT-1/AKT-2/SGK-1 complex transduces AGE-1/PI3K signals via PDK-1 to control the intracellular localization and activation of DAF-16/FKHRL1 by phosphorylation. In this respect, AKT-1/AKT-2/SGK-1 compete with a parallel branch within the DAF-2 pathway. Moreover, inactivation of sgk-1, but not of akt-1 or akt-2, results in a retarded postembryonic development, defective egg-laying, extended life span, and increased stress tolerance. It is concluded that SGK-1 is the critical kinase of the AKT-1/AKT-2/SGK-1 regulatory complex that controls aspects of development, longevity, and stress response (Hertweck, 2004).
The cellular response to genotoxic stress involves the integration of multiple prosurvival and proapoptotic signals that dictate whether a cell lives or dies. In mammals, AKT/PKB regulates cell survival by modulating the activity of several apoptotic proteins, including p53. In Caenorhabditis elegans, akt-1 and akt-2 regulate development in response to environmental cues by controlling the FOXO transcription factor daf-16, but the role of these genes in regulating p53-dependent apoptosis is not known. In this study, it was shown that akt-1 and akt-2 negatively regulate DNA-damage-induced apoptosis in the C. elegans germline. The antiapoptotic activity of akt-1 is independent of its target gene daf-16 but dependent on cep-1/p53. Although only akt-1 regulates the apoptotic activity of cep-1, both akt-1 and akt-2 modulate the intensity of the apoptotic response independently of the transcriptional activity of CEP-1. Finally, it was shown that AKT-1 regulates apoptosis but not cell-cycle progression downstream of the HUS-1/MRT-2 branch of the DNA damage checkpoint (Quevedo, 2007).
In C. elegans, mrt-2, hus-1, and clk-2 encode checkpoint proteins that transmit DNA-damage signals to the core apoptotic pathway through CEP-1/p53. HUS-1 and MRT-2 form part of the 9:1:1 complex, whereas CLK-2 functions in parallel to the 9:1:1 complex. In addition to activating apoptosis, these checkpoint genes also promote cell-cycle arrest in the mitotic region of the germline in response to DNA damage independently of cep-1. Checkpoint mutants also produce inviable embryos after treatment with IR because they are unable to repair damaged DNA. Because AKT-1 appears to act upstream of CEP-1/p53, it was asked whether akt-1 also has a role in the checkpoint response. It was found that germline cell-cycle arrest was not altered in either akt-1 gain-of-function or loss-of-function mutants, and the survival of progeny from akt-1(mg144) and akt-1(ok525) worms were no more sensitive to IR than wild-type worms. Therefore, these results indicate that AKT-1 does not act as a checkpoint protein but likely lies downstream of the DNA damage checkpoint to regulate the apoptotic activity of CEP-1/p53. To test this, double mutants were generated between akt-1(ok525) and loss-of-function alleles in the clk-2, mrt-2, and hus-1 checkpoint genes. It was found that clk-2(qm37);akt-1(ok525) double mutants were as resistant to damage-induced apoptosis as clk-2(qm37) single mutants, indicating that akt-1 does not act downstream of clk-2. However, irradiated mrt-2(e2663);akt-1(ok525) or hus-1(op244);akt-1(ok525) double mutants exhibited similar levels of apoptosis as irradiated wild-type controls, indicating that AKT-1 acts downstream of, or in parallel to, the 9:1:1 checkpoint. This suggests that inhibition of AKT-1 is part of the mechanism by which the HUS-1/MRT-2 complex signals to activate CEP-1/p53-dependent apoptosis in response to DNA damage. To assess this, CEP-1/p53 transcriptional activity was measured in hus-1(op244);akt-1(ok525) and mrt-2(e2663);akt-1(ok525) double-mutant animals. Although CEP-1/p53 is modestly activated in hus-1(op244) and mrt-2(e2663) single mutants treated with IR, presumably because the CLK-2 checkpoint is active, this activation was not enhanced by the akt-1(ok525) allele. Therefore, the increased germ-cell apoptosis observed in mrt-2(e2663);akt-1(ok525) and hus-1(op244);akt-1(ok525) double mutants treated with IR was not due to an increase in CEP-1/p53 transcriptional activity. Because AKT-2 is able to regulate apoptosis without affecting CEP-1/p53 transcriptional activity, hus-1(op244);akt-2(ok393) double mutants were created and similar levels of apoptosis were observed in these mutants as with the hus-1(op244);akt-1(ok525) strain. Because the increased IR-induced apoptosis observed in akt-1(ok525) mutants requires functional cep-1, these results suggest that CEP-1 may also regulate apoptosis independently of its transcriptional activity, as described in mammalian cells. The possibility that CEP-1 regulates the transcription of genes, other than egl-1, that also regulate germline apoptosis cannot be ruled out. A third possibility is that AKT-1/2 can modulate the magnitude of the apoptotic response independently of CEP-1, perhaps by regulating components of the core apoptotic pathway (Quevedo, 2007).
TRANCE, a TNF family member, and its receptor, TRANCE-R, are critical regulators of dendritic cell and osteoclast function. TRANCE activates the antiapoptotic serine/threonine kinase Akt/PKB through a signaling complex involving c-Src and TRAF6. A deficiency in c-Src or the addition of Src family kinase inhibitors blocks TRANCE-mediated PKB activation in osteoclasts. c-Src and TRAF6 interact with each other and with TRANCE-R upon receptor engagement. TRAF6, in turn, enhances the kinase activity of c-Src leading to tyrosine phosphorylation of downstream signaling molecules such as c-Cbl. These results define a mechanism by which TRANCE activates Src family kinases and PKB and provide evidence of cross-talk between TRAF proteins and Src family kinases (Wong, 1999).
The serine/threonine kinase Akt (also known as protein kinase B) (Akt/PKB) is activated upon T-cell antigen receptor (TCR) engagement or upon expression of an active form of phosphatidylinositide (PI) 3-kinase in T lymphocytes. The small GTPase Rac1 is implicated in this pathway, connecting the receptor with the lipid kinase. In Jurkat cells, activated forms of Rac1 or Cdc42, but not Rho, stimulate an increase in Akt/PKB activity. TCR-induced Akt/PKB activation is inhibited either by PI 3-kinase inhibitors (LY294002 and wortmannin) or by overexpression of a dominant negative mutant of Rac1 but not Cdc42. Accordingly, triggering of the TCR rapidly stimulates a transient increase in GTP-Rac content in these cells. Similar to TCR stimulation, L61Rac-induced Akt/PKB kinase activity is also LY294002 and wortmannin sensitive. However, induction of Akt/PKB activity by constitutively active PI 3-kinase is unaffected when dominant negative Rac1 is coexpressed, placing Rac1 upstream of PI 3-kinase in the signaling pathway. When analyzing the signaling hierarchy in the pathway leading to cytoskeleton rearrangements, it was found that Rac1 acts downstream of PI 3-kinase, a finding that is in accordance with numerous studies in fibroblasts. These results reveal a previously unrecognized role of the GTPase Rac1, acting upstream of PI 3-kinase in linking the TCR to Akt/PKB. This is the first report of a membrane receptor employing Rac1 as a downstream transducer for Akt/PKB activation (Genot, 2000).
The protein kinase Akt plays a central role in a number of key biological functions including protein synthesis, glucose homeostasis, and the regulation of cell survival or death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has been recently defined. In contrast, the mechanism of activation of Akt by other cell surface receptors is much less well understood. For G protein-coupled receptors (GPCRs), conflicting data suggest that these receptors stimulate Akt in a cell type-specific manner by a yet to be fully elucidated mechanism. Here, advantage was taken of the availability of cells, where Akt activity could not be enhanced by agonists acting on this large family of cell surface receptors, such as NIH 3T3 cells, to investigate the pathway linking GPCRs to Akt. Evidence is presented that expression of phosphatidylinositol 3-kinase (PI3K) beta is necessary and sufficient to transmit signals from G proteins to Akt in these murine fibroblasts and the activation of PI3Kbeta may represent the most likely mechanism whereby GPCRs stimulate Akt, since the vast majority of cells do not express PI3Kgamma, a known G protein-sensitive PI3K isoform. Furthermore, available evidence indicates that GPCRs activate Akt by a pathway distinct from that utilized by growth factor receptors, since it involves the tyrosine phosphorylation-independent activation of PI3Kbeta by G protein betagamma dimers (Murga, 2000).
Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all G(i) and G(q) alpha subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT(1B) receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT(1B) receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation is attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. Since RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways (Lione, 2000).
The protein-tyrosine phosphatase Shp-2 is a positive modulator of the Ras/mitogen-activated protein kinase pathway and a putative substrate of the transforming non-receptor tyrosine kinase v-Src. To characterize the role of Shp-2 in cellular transformation and signaling by v-Src, v-Src was expressed in normal and Shp-2-deficient mouse embryo fibroblasts. Expression of Shp-2 is necessary for morphological transformation by v-Src: Shp-2+/+ cells become rounded or spindly upon v-Src expression, whereas Shp-2-deficient cells remain relatively flat. v-Src-induces reorganization of the actin cytoskeleton and the formation of podosomes are compromised in Shp-2-deficient cells. Shp-2 deficiency also reduces v-Src-induced activation of the anti-apoptotic protein kinase Akt. The reduced activation of Akt in Shp-2-deficient cells correlates with a reduction in the association of the p85 regulatory subunit of PI3-kinase with the adapter protein Cbl. Activation of PI3-kinase by v-Src may be mediated by the association of the adapter protein Cbl with the p85 subunit. Since activation of Akt is dependent on PI3-kinase, this suggests that the effect of Shp-2 on Akt activation may be mediated, at least in part, by its effects on the interaction between PI3-kinase and Cbl. The defect in activation of the Akt survival pathway also correlates with enhanced sensitivity of Shp-2-deficient cells to an apoptosis-inducing agent. These results implicate Shp-2 in v-Src-induced cytoskeletal reorganization and activation of the Akt cell survival pathway (Hakak, 2000).
The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools [Galpha(i3) > Galpha(i2) >> Galpha(0)], were both inhibited following betagamma sequestration. The sst[2(a)] receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) are dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst[2(b)] receptors are blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone has no effect on the activity of either p38 or Akt but markedly enhances p38 phosphorylation mediated by sst[2(a)] receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediate a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) is detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst[2(a)] receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enables a proliferative response to be detected in sst[2(a)] receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades (Sellers, 2000).
The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis. Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various tissues. The relationship between ceramide signaling and the activation of an important signaling pathway, phosphatidylinositol (PI) 3-kinase and its downstream target, protein kinase B (PKB), has been examined. PKB activation is observed following stimulation of cells with the cytokine granulocyte-macrophage colony-stimulating factor. Addition of cell-permeable ceramide analogs, C(2)- or C(6)-ceramide, causes a partial loss (50%-60%) of PKB activation. This reduction is not a result of decreased PI(3,4,5)P(3) or PI(3,4)P(2) generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation is consistently reduced by treatment with ceramide, whereas threonine 308 phosphorylation remains unaffected. In further experiments, ceramide appears to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in serine 473 phosphorylation is inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation is abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of threonine 308 phosphorylation. These results demonstrate that PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 is the principal target of a ceramide-activated phosphatase (Schubert, 2000).
Renewal of the gastrointestinal epithelium involves a coordinated process of terminal differentiation and programmed cell death. Integrins have been implicated in the control of apoptotic processes in various cell types. The role of integrins in the regulation of apoptosis in gastrointestinal epithelial cells has been examined with the use of a rat small intestinal epithelial cell line (RIE1) as a model. Overexpression of the integrin alpha5 subunit in RIE1 cells confers protection against several proapoptotic stimuli. In contrast, overexpression of the integrin alpha2 subunit has no effect on cell survival. The antiapoptotic effect of the alpha5 subunit is partially retained by a mutated version that has a truncation of the cytoplasmic domain. The antiapoptotic effects of the full-length or truncated alpha5 subunit are reversed upon treatment with inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase), suggesting that the alpha5beta1 integrin might interact with the PI-3-kinase/Akt survival pathway. When cells overexpressing alpha5 are allowed to adhere to fibronectin, there is a moderate activation of protein kinase B (PKB)/Akt, whereas no such effect is seen in alpha2-overexpressing cells adhering to collagen. Furthermore, in cells overexpressing alpha5 and adhering to fibronectin, there is a dramatic enhancement of the ability of growth factors to stimulate PKB/Akt; again, this is not seen in cells overexpressing alpha2 subunit and adhering to collagen or fibronectin. Expression of a dominant negative version of PKB/Akt in RIE cells blocks to ability of alpha5 to enhance cell survival. Thus, the alpha5beta1 integrin seems to protect intestinal epithelial cells against proapoptotic stimuli by selectively enhancing the activity of the PI-3-kinase/Akt survival pathway (Lee, 2000).
The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation: beta(1C) inhibits proliferation, whereas beta(1A) promotes it. The ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival was investigated in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) do not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) show significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity are not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 leads to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induces activation of both proteins. Ras activation is strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and expression of constitutively active Ras [Ras 61 (L)] which rescues beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines is attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescues beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals (Fornaro, 2000).
The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP [beta(3)-LNCaP] cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. Alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. It is concluded that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that alpha(v)beta(3) can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner (Zheng, 2000).
The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. The role of PI3-K/Akt during the cellular response to oxidant injury has been examined. H2O2 treatment elevates Akt activity in multiple cell types in a time- and dose-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevent H2O2-induced Akt activation. Akt activation by H2O2 also depends on epidermal growth factor receptor (EGFR) signaling; H2O2 treatment leads to EGFR phosphorylation, and inhibition of EGFR activation prevents Akt activation by H2O2. Since H2O2 causes apoptosis of HeLa cells, whether or not alterations of PI3-K/Akt signaling would affect this response was investigated. Wortmannin and LY294002 treatment significantly enhances H2O2-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibits cell death. Constitutive expression of v-Akt likewise enhances survival of H2O2-treated NIH3T3 cells. These results suggest that H2O2 activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis (Wang, 2000).
NGF is a target-derived growth factor for developing sympathetic neurons. Application of NGF exclusively to distal axons of sympathetic neurons leads to an increase in PI3-K signaling in both distal axons and cell bodies. In addition, there is a more critical dependence on PI3-K for survival of neurons supported by NGF acting exclusively on distal axons as compared to neurons supported by NGF acting directly on cell bodies. Interestingly, PI3-K signaling within both cell bodies and distal axons contributes to survival of neurons. The requirement of PI3-K signaling in distal axons for survival may be explained by the finding that inhibition of PI3-K in the distal axons attenuates retrograde signaling. Therefore, a single TrkA effector, PI3-K, has multiple roles within spatially distinct cellular locales during retrograde NGF signaling (Kuruvilla, 2000).
Dissociated sympathetic neurons obtained from newborn rat superior cervical ganglia and grown in compartmentalized cultures were to assess the subcellular distribution and state of activation of PI3-K and its downstream effector Akt (protein kinase B). Neurons were maintained under conditions in which cell bodies and proximal axons (hereafter referred to as the cell body compartment) were exposed to medium containing a neutralizing antibody directed against NGF (alpha-NGF), while distal axons, which are >1 mm away from cell bodies, were exposed to medium containing NGF. These conditions resemble in vivo conditions in which neurons are maintained by NGF acting exclusively on distal axons (Kuruvilla, 2000).
It was asked whether binding of NGF to receptors exclusively on distal axons regulates the activities of PI3-K and Akt in distal axons and/or cell bodies. For these experiments, NGF was removed from medium bathing distal axons for 24 hr. Then, distal axons were exposed to the same medium (control) or medium containing NGF for various times. The activation states of TrkA and Akt were assessed in extracts prepared from cell body and distal axon compartments by immunoblotting using antibodies that recognize the activated, phosphorylated forms of these proteins. P-Trk (Y490) antibodies recognize TrkA when phosphorylated on Tyr-490, which is the Shc recognition site. P-Akt antibodies recognize Akt when phosphorylated on Ser-473, which is necessary for its catalytic activity. Application of NGF to distal axons results in increased levels of P-TrkA (Y490) and P-Akt within distal axons, which are maximal after 20 min. Increases in both P-TrkA (Y490) and P-Akt are also detected in cell bodies but with slower kinetics. A small but reproducible increase in both P-TrkA (Y490) and P-Akt is detected in extracts of cell bodies within 20 min, and a more robust increase is seen at 8 hr. The appearance of P-TrkA (Y490) and P-Akt in both distal axons and cell bodies is coincident with the appearance of PI3-K activity associated with phosphotyrosine immunoprecipitates. Additionally, withdrawal of NGF from distal axons of neurons, which had been grown with medium containing a high concentration of NGF (100 ng/ml) on distal axons and alpha-NGF on cell bodies, leads to a decrease in the levels of both P-TrkA (Y490) and P-Akt in distal axons and in cell bodies. Thus, NGF acting on TrkA receptors on distal axons regulates the phosphorylation/activation of TrkA, PI3-K, and Akt both locally within distal axons and retrogradely to proximal axons and cell bodies of sympathetic neurons (Kuruvilla, 2000).
These results support the idea that PI3-K signaling within both cell bodies and distal axons is necessary for survival of neurons supported by NGF acting on distal axons. Moreover, the requirement of PI3-K signaling in distal axons is more apparent when a submaximal concentration of NGF is used to support survival. How does PI3-K signaling within distal axons contribute to survival? It was found that PI3-K activity in distal axons controls retrograde NGF transport and retrograde signaling, which may be critical for survival. Complete inhibition of PI3-K in distal axons, as assessed by levels of P-Akt, attenuates retrograde transport of NGF by ~80% in two compartment chambers and 65% in three compartment chambers. Thus, there is a small but significant amount of retrograde transport that occurs in a PI3-K-independent manner. These observations may account for the finding that inhibition of PI3-K in distal axons has more dire consequences for neurons supported by 0.5 ng/ml NGF acting on distal axons than for those supported by 50 ng/ml NGF acting on distal axons. Neurons grown in a low, submaximal concentration of NGF are more vulnerable than neurons supported by a high concentration of NGF to a 65%-80% reduction in retrograde signaling (Kuruvilla, 2000). The precise role of PI3-K signaling in distal axons for ligand-dependent internalization, retrograde transport, and retrograde signaling is not clear. It is possible that products of the PI3-K catalyzed reaction are critical for the ligand-dependent production of clathrin-coated pits, into which NGF and TrkA are initially internalized. In support of this idea, there is an essential role for the pleckstrin homology (PH) domain of the GTPase dynamin for receptor-mediated endocytosis. Further, dynamin is required for retrograde transport of NGF in sympathetic neurons. Since the dynamin PH domain binds to phosphoinositide products of the PI3-K-catalyzed reaction, PI3-K activity associated with TrkA may be critical for recruitment of dynamin to regions of the plasma membrane destined to invaginate to form NGF/TrkA-containing clathrin-coated signaling organelles. Similarly, AP-2, which is involved in clathrin coat formation and vesicle sorting at the plasma membrane, contains an amino-terminal phosphoinositide binding domain that is required for its targeting to the plasma membrane. Thus, it is tempting to speculate that PI3-K signaling in distal axons is needed for survival because this TrkA effector controls membrane recruitment of key regulators of NGF/TrkA endocytosis and retrograde TrkA signaling (Kuruvilla, 2000).
If PI3-K in distal axons is required for retrograde signaling, what is the role of PI3-K in cell bodies in neurons supported by NGF acting exclusively on distal axons? Inhibition of PI3-K in cell bodies leads to near complete apoptosis of neurons within 48 hr, but inhibition of PI3-K exclusively in cell bodies does not affect retrograde transport of NGF. Under these conditions, P-Akt in cell bodies is completely blocked, but levels of P-Akt in distal axons are unaffected. These observations indicate that PI3-K and Akt signaling in distal axons alone cannot support neuronal survival. Since constitutively active PI3-K and Akt can support survival of sympathetic neurons, it is speculated that PI3-K signaling in cell bodies is necessary for survival because it supports Akt signaling and phosphorylation of Akt substrates that mediate the prosurvival effects of PI3-K. Indeed, it seems likely that many of the substrates of Akt function, at least in part, within cell bodies. Substrates of Akt include BAD, caspase-9, IKK, the transcription factor forkhead, and, possibly, CREB. By extension, these data support the idea that phosphorylation of Akt substrates within distal axons cannot support neuronal survival. This may be because critical substrates of Akt are either not present in distal axons or that they are present in distal axons but cannot move in the phosphorylated forms from distal axons to cell bodies to affect the apoptotic machinery. P-Akt itself does not move from distal axons to cell bodies to an appreciable extent so the same is likely to be true for products of Akt-catalyzed phosphorylation reactions (Kuruvilla, 2000).
Growth factor signal transduction mechanisms in neurons are arguably more complex than in most other cell types due to the striking morphological specializations of neurons. Most neurons have long axons that can extend centimeters or even one meter from their cell bodies, and target-derived growth factor signals must be propagated over long distances to influence survival and gene expression within cell bodies. These retrograde signals must be integrated with signals coming from dendrites and those emanating from the membrane of the cell body itself. The present study shows that the same NGF effector pathway, the PI3-K pathway, can have different functions in distinct parts of the same neuron during long-range retrograde signaling. Interestingly, the activity of the PI3-K signaling in distal axons indirectly regulates TrkA signaling pathways in cell bodies, including the PI3-K effector pathway. Thus, there exists interdependence of TrkA effector pathways in distinct cellular locales whereby ligand-dependent TrkA effector signaling in one compartment, the distal axon, controls effector signaling in another, the cell body (Kuruvilla, 2000).
The role of integrins in leukocyte apoptosis is unclear: some studies suggest enhancement, others inhibition. ß2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of alphaMß2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNFalpha, or UV irradiation, ligation of active alphaMß2 results in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins does not show this proapoptotic effect and continues to inhibit apoptosis. This discrepancy can be attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive alphaMß2 is capable of stimulating the kinases ERK and Akt. Activated alphaMß2 stimulates Akt, but not ERK. When proapoptotic stimuli are combined with either alphaMß2 clustering or activation, Akt activity is blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive alphaMß2 continues to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, ß2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes (Whitlock, 2000).
Protein kinase B (PKB/Akt) is a regulator of cell survival and apoptosis. To become fully activated, PKB/Akt requires phosphorylation at two sites, threonine 308 and serine 473, in a phosphatidylinositol (PI) 3-kinase-dependent manner. The kinase responsible for phosphorylation of threonine 308 is the PI 3-kinase-dependent kinase-1 (PDK-1), whereas phosphorylation of serine 473 has been suggested to be regulated by PKB/Akt autophosphorylation in a PDK-1-dependent manner. However, the integrin-linked kinase (ILK: see Drosophila Integrin linked kinase) has also been shown to regulate phosphorylation of serine 473 in a PI 3-kinase-dependent manner. Whether ILK phosphorylates this site directly or functions as an adapter molecule has been debated. In-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry show that biochemically purified ILK can phosphorylate PKB/Akt directly. Co-immunoprecipitation analysis of cell extracts demonstrates that ILK can complex with PKB/Akt as well as PDK-1 and that ILK can disrupt PDK-1/PKB association. The amino acid residue serine 343 of ILK within the activation loop is required for kinase activity as well as for its interaction with PKB/Akt. Mutational analysis of ILK further shows a crucial role for arginine 211 of ILK within the phosphoinositide phospholipid binding domain in the regulation of PKB-serine 473 phosphorylation. A highly selective small molecule inhibitor of ILK activity also inhibits the ability of ILK to phosphorylate PKB/Akt in vitro and in intact cells. These data demonstrate that ILK is an important upstream kinase for the regulation of PKB/Akt (Persad, 2001).
PDK1 functions as a master kinase, phosphorylating and activating PKB/Akt, S6K and RSK (See Drosophila RSK ). To learn more about the roles of PDK1, mice were generated that either lacked PDK1 or possessed PDK1 hypomorphic alleles, expressing only ~10% of the normal level of PDK1. PDK1-/- embryos die at embryonic day 9.5, displaying multiple abnormalities including lack of somites, forebrain and neural crest derived tissues; however, development of hind- and mid-brain proceed relatively normally. In contrast, hypomorphic PDK1 mice are viable and fertile, and insulin injection induces the normal activation of PKB, S6K and RSK. Nevertheless, these mice are 40%-50% smaller than control animals. The organ volumes from the PDK1 hypomorphic mice are reduced proportionately. The volume of a number of PDK1-deficient cells is reduced by 35%-60%; PDK1 deficiency does not affect cell number, nuclear size or proliferation. Genetic evidence is provided that PDK1 is essential for mouse embryonic development, and regulates cell size independently of cell number or proliferation, as well as insulin's ability to activate PKB, S6K and RSK (Lawlor, 2002).
Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian cells. However, this study demonstrates that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. Akt is activated by expression of Rac and Cdc42; colocalization of endogenous phosphorylated Akt with Rac and Cdc42 is observed at the leading edge of fibroblasts. Importantly, expression of active Akt but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells (Higuchi, 2002).
Blood vessel formation is a complex morphological process that is only beginning to be understood at the molecular level. A novel and critical role is demonstrated for the small GTPase, RhoB, in vascular development. RhoB null mice have retarded vascular development in the retina characterized by altered sprout morphology. Moreover, pharmaceutical means to deplete RhoB in neonatal rats is associated with apoptosis in the sprouting endothelial cells of newly forming vessels. Similarly, acute depletion of RhoB by antisense or dominant-negative strategies in primary endothelial cell culture models leads to apoptosis and failures in tube formation. A novel link has been identified between RhoB and the Akt survival signaling pathway to explain these changes. Confocal microscopy reveals that RhoB is highly localized to the nuclear margin with a small percentage found inside the nucleus. Similarly, total Akt is found throughout the cell but has increased accumulation at the nuclear margin, and active phosphorylated Akt is found primarily inside the nucleoplasm, where it partially colocalizes with the RhoB therein. This colocalization is functionally relevant, because when RhoB is depleted, Akt is excluded from the nucleus and total cellular Akt protein is decreased in a proteosome-dependent manner. Because the function of RhoB in vivo appears to be rate limiting only for endothelial cell sprouting, it is proposed that RhoB has a novel stage-specific function to regulate endothelial cell survival during vascular development. RhoB may offer a therapeutic target in diseases such as cancer, diabetic retinopathy, and macular degeneration, where the disruption of sprouting angiogenesis would be desirable (Adini, 2003).
Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. This study demonstrates that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying a nuclear signaling pathway through which Ang-1 modulates gene expression. Microarray analysis was used to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. RNA interference was used to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. These data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability (Daly, 2004).
To elucidate the functions of the serine/threonine kinase Akt/PKB in vivo, mice were generated lacking both akt1 and akt2 genes. Akt1/Akt2 double-knockout (DKO) mice exhibit severe growth deficiency and die shortly after birth. These mice display impaired skin development because of a proliferation defect, severe skeletal muscle atrophy because of a marked decrease in individual muscle cell size, and impaired bone development. These defects are strikingly similar to the phenotypes of IGF-1 receptor-deficient mice and suggest that Akt may serve as the most critical downstream effector of the IGF-1 receptor during development. In addition, Akt1/Akt2 DKO mice display impeded adipogenesis. Specifically, Akt1 and Akt2 are required for the induced expression of PPARgamma, the master regulator of adipogenesis, establishing a new essential role for Akt in adipocyte differentiation. Overall, the combined deletion of Akt1 and Akt2 establishes in vivo roles for Akt in cell proliferation, growth, and differentiation. These functions of Akt were uncovered despite the observed lower level of Akt activity mediated by Akt3 in Akt1/Akt2 DKO cells, suggesting that a critical threshold level of Akt activity is required to maintain normal cell proliferation, growth, and differentiation (Peng, 2003).
Protein kinase B/Akt plays crucial roles in promoting cell survival and mediating insulin responses. The enzyme is stimulated by phosphorylation at two regulatory sites: Thr 309 of the activation segment and Ser 474 of the hydrophobic motif, a conserved feature of many AGC kinases. Analysis of the crystal structures of the unphosphorylated and Thr 309 phosphorylated states of the PKB kinase domain provides a molecular explanation for regulation by Ser 474 phosphorylation. Activation by Ser 474 phosphorylation occurs via a disorder to order transition of the alphaC helix with concomitant restructuring of the activation segment and reconfiguration of the kinase bilobal structure. These conformational changes are mediated by a phosphorylation-promoted interaction of the hydrophobic motif with a channel on the N-terminal lobe induced by the ordered alphaC helix and are mimicked by peptides corresponding to the hydrophobic motif of PKB and potently by the hydrophobic motif of PRK2 (Yang, 2002).
Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Evidence is provided that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1 (Garami, 2003).
The role of subcellular localization in the regulation of protein kinase B (PKB) activation has been investigated. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKBalpha is associated with the plasma membrane of transfected cells, whereas the wild-type kinase is mostly cytosolic. The activity of m/p-PKBalpha is 60-fold higher compared with the unstimulated wild-type enzyme, and can not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32P labeling and mutagenesis has demonstrated that m/p-PKBalpha activity is due to phosphorylation on Thr308 and Ser473, two amino acids that are normally modified on PKB following stimulation of the cells with insulin or insulin-like growth factor-1 (IGF-1). A dominant negative form of phosphoinositide 3-kinase (PI3-K) does not affect m/p-PKBalpha activity. The pleckstrin homology (PH) domain of m/p-PKBalpha is not required for its activation or phosphorylation on Thr308 and Ser473, suggesting that this domain may serve as a membrane-targeting module. Consistent with this view, PKBalpha is translocated to the plasma membrane within minutes after stimulation with IGF-1. This translocation requires the PH domain and is sensitive to wortmannin. These results indicate that PI3-K activity is required for translocation of PKB to the plasma membrane, where its activation occurs through phosphorylation of the same sites that are induced by insulin or IGF-1. Following activation the kinase detaches from the membrane and translocates to the nucleus (Andjelkovic, 1997).
Protein kinase B (PKB) is a proto-oncogene that is activated in signaling pathways initiated by phosphoinositide 3-kinase. Chromatographic separation of brain cytosol reveals a kinase activity that phosphorylates and activates PKB only in the presence of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Phosphorylation occurs exclusively on threonine-308, a residue implicated in activation of PKB in vivo. PtdIns(3,4,5)P3 was determined to have a dual role: its binding to the pleckstrin homology domain of PKB is required to allow phosphorylation by the upstream kinase and it directly activates the upstream kinase (Stokoe, 1997).
Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bind PtdIns(3,4,5)P3 and associate with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism (Stephens, 1998).
Protein kinase B (PKB) is involved in the regulation of apoptosis, protein synthesis and glycogen metabolism in mammalian cells. Phosphoinositide-dependent protein kinase (PDK-1) activates PKB in a manner dependent on phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], which is also needed for the translocation of PKB to the plasma membrane. It has been proposed that the amount of PKB activated is determined exclusively as a result of its translocation, and that a constitutively active pool of membrane-associated PDK-1 simply phosphorylates all the PKB made available. The effects of membrane localization of PDK-1 on PKB activation has been investigated. Ectopically expressed PDK-1 translocated to the plasma membrane in response to platelet-derived growth factor (PDGF) and translocation is sensitive to wortmannin, an inhibitor of phosphoinositide 3-kinase. Translocation of PDK-1 also occurs upon its co-expression with constitutively active phosphoinositide 3-kinase, but not with an inactive form. Overexpression of PDK-1 enhances the ability of PDGF to activate PKB. PDK-1 disrupted in the pleckstrin homology (PH) domain, which does not translocate to the membrane, does not increase PKB activity in response to PDGF, whereas membrane-targeted PDK-1 activates PKB to the extent that it can not be activated further by PDGF. It is concluded that in response to PDGF, binding of Ptdlns (3,4,5)P3 and/or Ptdlns(3,4)P2 to the PH domain of PDK-1 causes its translocation to the plasma membrane where it co-localizes with PKB, significantly contributing to the scale of PKB activation (Anderson, 1998).
Two products of PI 3-kinase activation, PtdIns(3,4,5)P3 and its immediate breakdown product PtdIns(3,4)P2, trigger physiological processes by interacting with proteins possessing pleckstrin homology (PH) domains. One of the best characterized PtdIns(3,4,5)P3/PtdIns(3,4)P2 effector proteins is protein kinase B (PKB), also known as Akt. PKB possesses a PH domain located at its N terminus, and this domain binds specifically to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 with similar affinity. Following activation of PI 3-kinase, PKB is recruited to the plasma membrane by virtue of its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2. PKB is then activated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which like PKB, possesses a PtdIns(3,4,5)P3/PtdIns(3,4)P2 binding PH domain. This study describes the high-resolution crystal structure of the isolated PH domain of PKBalpha in complex with the head group of PtdIns(3,4,5)P3. The head group has a significantly different orientation and location compared to other Ins(1,3,4,5)P4 binding PH domains. Mutagenesis of the basic residues that form ionic interactions with the D3 and D4 phosphate groups reduces or abolishes the ability of PKB to interact with PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The D5 phosphate faces the solvent and forms no significant interactions with any residue on the PH domain, and this explains why PKB interacts with similar affinity with both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (Thomas, 2002).
Full activation of PKB requires phosphorylation on residues Thr308 and Ser473. While the Thr308 kinase, named 3-phosphoinositide-dependent kinase-1 (PDK1), has been extensively characterized, the identity of the Ser473 kinase remains unclear. This study focused on the plasma membrane (PM) fraction because membrane localization is sufficient to activate PKB, and this suggests that PKB upstream kinases are constitutively active at the membrane. A constitutively active PKB Ser473 kinase activity has been identified that is enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity is released from the membrane by high salt, and gel filtration analysis shows that the kinase responsible is present in a large complex of >500 kDa. Two major phosphoproteins and integrin-linked kinase (ILK) were detected in partially purified PKB Ser473 kinase preparations. In contrast to previous observations, however, ILK immunoprecipitates do not retain Ser473 kinase activity. Thus, a novel raft-associated PKB Ser473 kinase has been identifed, implicating a role for lipid rafts in PKB signaling (Hill, 2002).
Peroxisome proliferator-activated receptors (PPARs: distantly related to Drosophila Eip75B) are ligand-activated transcription factors that belong to the nuclear hormone receptor family. Three isotypes that have distinct tissue distributions and functions have been found in vertebrates. Important roles of PPARalpha and PPARgamma in lipid homeostasis and in inflammation have been unveiled. Little is known about the exact function of PPARß, although it has been implicated in colon tumorigenesis, and embryonic development. In adult epidermis, PPARß is undetectable in interfollicular keratinocytes. However, the expression of PPARß is reactivated upon proliferative stimuli such as cutaneous injury, suggesting a role of PPARß in regulating keratinocyte proliferation/differentiation processes. Apoptosis, differentiation, and proliferation are cellular responses that play a pivotal role in wound healing. During this process PPARß translates inflammatory signals into prompt keratinocyte responses. PPARß modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPARß also potentiates NF-kappaB activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPARß protects keratinocytes against apoptosis and may contribute to the process of skin wound closure (Di-Poï, 2002).
The signaling pathway comprising Raf, MEK (mitogen-activated protein kinase, or ERK kinase), and ERK (extracellular signal-regulated kinase) lies downstream of the small guanine nucleotide binding protein Ras and mediates several apparently conflicting cellular responses, such as proliferation, apoptosis, growth arrest, differentiation, and senescence, depending on the duration and strength of the external stimulus and on cell type. Another pathway that lies downstream of Ras includes phosphatidylinositol (PI) 3-kinase and Akt (or protein kinase B) and also regulates these cellular responses, acting either synergistically with or in opposition to the Raf pathway. Coordination of the two pathways in a single cellular response may depend on cell type or the stage of differentiation. Akt interacts with Raf and phosphorylates this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibits activation of the Raf-MEK-ERK signaling pathway and shifts the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt. These results demonstrate that Akt antagonizes Raf activity by direct phosphorylation of Ser259. This modification creates a binding site for 14-3-3 protein, a negative regulator of Raf. Similarly, phosphorylation of BAD or the forkhead transcription factor FKHRL1 by Akt also promotes binding of 14-3-3 protein. In all three instances, phosphorylation by Akt inactivates the function of its substrate. Cross talk between the Raf-MEK-ERK and the PI 3-kinase-Akt pathways, mediated by direct interaction of Akt with and its phosphorylation of Raf, may switch the biological response from growth arrest to proliferation, as shown for MCF-7 cells, and may also modulate senescence or differentiation as shown for myoblast differentiation, depending on the cellular system (Zimmermann, 1999).
GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). However, agents that prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3, the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase (Cross, 1995).
Activation of phosphatidylinositide 3'-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 [p90(Rsk)], p70(S6kinase), and also PKB have all been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation, the role of PKB has been investigated by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at expression levels similar to wild-type PKB), because currently no such mutant has been reported. The PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it has been shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB, induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. These results demonstrate that PKB activation is both necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, since both proteins co-immunoprecipitate. This interaction also results in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB (van Weeren 1998).
Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).
The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However, unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular, GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an indirect effect of AKT (Sears, 2000 and references therein).
The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. The insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of 'primed' glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of 'nonprimed' substrates in the Wnt-signaling pathway (Axin and ß-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes (Frame, 2001).
GSK3 is phylogenetically most closely related to the cyclin-dependent protein kinases (CDKs), such as CDK1 (also called cdc2) and CDK2. However, the specificity of GSK3 is unique in that it requires a priming phosphate located at n + 4 (where n is the site of phosphorylation) in order to phosphorylate many of its substrates, such as glycogen synthase. In contrast, the phosphorylation of Axin and ß-catenin in the Wnt signaling pathway is not known to require a priming phosphate and may rely on high-affinity interactions in a multiprotein complex with GSK3. Thus, Axin binds to both GSK3 and ß-catenin, bringing these proteins into close proximity to facilitate their phosphorylation by GSK3. This study presents evidence for a specific site of interaction between the phosphate of the primed substrate and Arg-96 of GSK3. This same phosphate binding site is also occupied by Ser-9 once it becomes phosphorylated by PKB. The existence of this site helps to explain several features of GSK3, such as its unusual substrate specificity requirements and the mechanism by which it becomes inhibited in response to insulin and growth factors. These findings have important implications for drug development in this area (Frame, 2001).
Axon-dendrite polarity is a cardinal feature of neuronal morphology essential for information flow. A differential distribution of GSK-3ß activity is found in the axon versus the dendrites. A constitutively active GSK-3ß mutant inhibits axon formation, whereas multiple axons formed from a single neuron when GSK-3ß activity is reduced by pharmacological inhibitors, a peptide inhibitor, or siRNAs. An active mechanism for maintaining neuronal polarity was revealed by the conversion of preexisting dendrites into axons upon GSK-3 inhibition. Biochemical and functional data show that the Akt kinase and the PTEN phosphatase are upstream of GSK-3ß in determining neuronal polarity. These results demonstrate that there are active mechanisms for maintaining as well as establishing neuronal polarity, indicate that GSK-3ß relays signaling from Akt and PTEN to play critical roles in neuronal polarity, and suggest that application of GSK-3ß inhibitors can be a novel approach to promote generation of new axons after neural injuries (Jiang, 2005).
Serine (Ser) 9 in GSK-3ß can be regulated by multiple kinases, among which Akt is differentially localized in axons. To test for GSK-3ß regulation by the candidate upstream molecules, the same inhibitors for PI3K as those used previously for studies of neuronal polarity were used. LY294002 significantly inhibits Akt phosphorylation at Ser 473 and GSK-3ß phosphorylation at Ser 9. LY294002 does not affect GSK-3ß phosphorylation at Tyr216. In addition to the biochemical evidences, the distribution of GSK-3ß and pGSK-3ß was examined. LY294002 reduces the ratio of pGSK-3ß over GSK-3ß. aPKC is another candidate GSK-3ß regulator, but GSK-3ß phosphorylation is not affected by the aPKC inhibitor bisindolylmaleimide I (Bis), or by the MAPK inhibitor U0126 or the PKA inhibitor KT5720 (Jiang, 2005).
To test for the functional significance of Akt, attempts were made to examine the effect of inhibiting Akt by using short hairpin RNAs (shRNAs), but they caused neuronal death. The effect of increasing Akt activity on neuronal polarity was examined by using Myr-Akt, a constitutively active Akt derived by fusion with the myristoylation signal of Src. The wild-type Akt does not affect neuronal polarity, whereas Myr-Akt causes the formation of multiple axons (Jiang, 2005).
Because GSK-3ß S9A should not be phosphorylated by Akt after the substation of Ser 9, it was possible to test for the relationship between Akt and GSK-3ß by cotransfection of GSK-3ß S9A and Myr-Akt. GSK-3ß S9A could partially, but not completely, reverse the effect of Myr-Akt on the formation of multiple axons. These results are consistent with the idea of Akt being upstream of GSK-3ß. The partial reversal could result from either incomplete overlap in GSK-3ß S9A and Myr-Akt transfection or the possibility that GSK-3ß constitutes a part, but not all, of the output for Akt (Jiang, 2005).
Detailed examination indicates that Akt plays multiple roles in axon and dendrite development. Myr-Akt increases the number of neurites per neuron both in neurons forming multiple axons, and in neurons with single axons. Myr-Akt increases both the numbers of axons and dendrites from the normal total of five neurites to eight. When the number of axons is increased in Myr-Akt neurons, axon length is shorter than normal, perhaps due to limited materials for an increased number of axons. When the number of axons is not increased in Myr-Akt neurons, the single axon is longer than normal, indicating that Akt promotes axon outgrowth. The length of dendrites is not affected by Myr-Akt (Jiang, 2005).
Only the effect of increasing axon number is shared between Akt activation and GSK-3ß inactivation. GSK-3ß is therefore only one downstream component of Akt, specifically mediating the function of Akt in axon-dendrite polarity, but not in neurite number or axon length (Jiang, 2005).
PTEN is a lipid and protein phosphatase that functions in opposition to PI3K by dephosphorylating the lipid product of PI3K, phosphatidylinositol 3,4,5-trisphosphate (PIP3) . The role of PTEN was examined further and the functional relationship of PTEN and GSK-3ß was characterized. Neuronal polarity is lost upon PTEN overexpression. Detailed analysis indicates that PTEN transfection inhibits axon formation without affecting dendrite formation. The inactive PTEN control (PTEN G129R) does not affect neuronal polarity (Jiang, 2005).
To investigate the role of endogenous PTEN, PTEN siRNA, a small inhibitor RNA (siRNA) construct designed specifically for PTEN, was used, and PTEN SsiRNA, a scrambled control. PTEN siRNA, but not PTEN SsiRNA, reduces PTEN protein levels in hippocampal neurons. PTEN siRNA increases the number of axons at the expense of dendrites. When the level of PTEN protein was examined, the level of PTEN was reduced by more than 50% in PTEN siRNA neurons with multiple axons, whereas PTEN was reduced less than 20% in PTEN siRNA neurons with single axons. Significant reduction of PTEN expression therefore correlates with the formation of multiple axons (Jiang, 2005).
To test for a functional relationship between PTEN and GSK-3ß, two kinds of experiments were performed. The first was to transfect neurons with PTEN and also treat them with SB415286. SB415286 almost completely reverses the effect of PTEN overexpression on axon-dendrite polarity, resulting in multiple axons at the expense of dendrites. The second kind of experiment was to cotransfect PTEN siRNA and GSK-3ß S9A. The effect of PTEN siRNA on multiple axon formation could be inhibited by an S0A mutant of GSK-3ß in which Ser 9 was replaced with alanine. These results indicate that GSK-3ß is downstream of PTEN in axon-dendrite polarity formation because GSK-3ß manipulations dominate over PTEN manipulations (Jiang, 2005).
One role of PTEN does not seem to be downstream of GSK-3ß. When neurons are cotransfected with PTEN siRNA and GSK-3ß S9A, GSK-3ß S9A could not eliminate axons, suggesting that either PTEN siRNA can antagonize the effect of GSK-3ß S9A in axon formation or that GSK-3ß S9A has to act in a PTEN-dependent pathway to inhibit axon formation. In the latter scenario, GSK-3ß activation could be upstream of PTEN in inhibiting axon formation (Jiang, 2005).
The S/T-protein kinases activated by phosphoinositide 3-kinase (PI3K) regulate a myriad of cellular processes. An approach using a combination of biochemistry and bioinformatics can identify substrates of these kinases. This approach identifies the tuberous sclerosis complex-2 gene product, tuberin (Drosophila homolog Gigas), as a potential target of Akt/PKB. Upon activation of PI3K, tuberin is phosphorylated on consensus recognition sites for PI3K-dependent S/T kinases. Moreover, Akt/PKB can phosphorylate tuberin in vitro and in vivo. S939 and T1462 of tuberin are PI3K-regulated phosphorylation sites and T1462 is constitutively phosphorylated in PTEN-/- tumor-derived cell lines. Finally, a tuberin mutant lacking the major PI3K-dependent phosphorylation sites can block the activation of S6K1, suggesting a means by which the PI3K-Akt pathway regulates S6K1 activity (Manning, 2002).
Class I phosphoinositide 3-kinases (PI3Ks) are activated by many extracellular growth and survival stimuli. These lipid kinases catalyze the production of the second messengers phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5P3). Downstream targets containing specialized domains, such as pleckstrin-homology (PH) domains, that specifically bind to these lipid products of PI3K are then activated. These activated proteins control a wide array of cellular processes, including survival, proliferation, protein synthesis, growth, metabolism, cytoskeletal rearrangements, and differentiation. However, there is still much that is not known about the signaling events leading from activation of PI3K effectors to downstream changes in cell physiology (Manning, 2002).
Serine/threonine (S/T) protein kinases can account for much of the functional diversity of PI3K signaling. Akt/protein kinase B and the 70 kDa-S6 kinase 1 (S6K1) are the best characterized of the PI3K-regulated S/T kinases. The mitogen-stimulated activation of both of these kinases is blocked by PI3K-specific inhibitors. Akt contains a PH domain that is specific to PtdIns-3,4P2 and PtdIns-3,4,5P3. Akt is thereby recruited to these PI3K-generated second messengers and to the PDK1 protein kinase, which also specifically binds to these lipids. PDK1 then phosphorylates and activates Akt (Manning, 2002).
The regulation of S6K1 is much more complex, with both PI3K-dependent and -independent signaling pathways involved in its activation. Several PI3K-regulated effectors are known to participate in the activation of S6K1, including PDK1, PKCzeta/lambda, Cdc42, Rac1, and Akt. However, the molecular mechanism of how these contribute to S6K1 activation remains unclear. In addition to mitogen-regulated signaling to S6K1, the metabolic state of the cell and the availability of nutrients control S6K1 activation through the mammalian target of rapamycin (mTOR, also known as FRAP, RAFT, and RAPT). Recent studies suggest that mTOR is also regulated by mitogenic signals. Interestingly, it has been suggested that the point of convergence of the mitogenic and nutrient-sensing signals in the regulation of S6K1 may be at the level of Akt directly phosphorylating mTOR. However, this phosphorylation does not appear to affect mTOR activity or S6K1 activation. Thus, of the PI3K-regulated effectors thought to participate in S6K1 activation, the molecular basis of how Akt regulates S6K1 remains the least well understood (Manning, 2002).
Akt itself has been implicated in many of the PI3K-regulated cellular events, and several substrates have been shown to be phosphorylated in vitro and/or in vivo by Akt. Therefore, the total cellular effect of PI3K activation and subsequent activation of Akt is mediated through a variety of different targets. However, it seems unlikely that the large array of processes controlled by the PI3K-Akt pathway can be accounted for by the current knowledge of downstream targets (Manning, 2002).
An approach has been developed to screen for substrates of PI3K-dependent S/T kinases, such as Akt. This approach uses phospho-specific antibodies generated against a phosphorylated protein kinase consensus recognition motif in combination with a protein database motif scanning program called Scansite (http://scansite.mit.edu). Scansite is a web-based program that searches protein databases for optimal substrates of specific protein kinases and for optimal binding motifs for specific protein domains with data generated by peptide library screens. The phospho-motif antibody is used to recognize proteins phosphorylated specifically under conditions in which the kinase of interest is active. Scansite is then used to identify candidate substrates of this protein kinase that have the predicted molecular mass of the proteins recognized by the phospho-motif antibody. This approach successfully identifies known substrates of Akt. The tuberous sclerosis complex-2 (TSC2) tumor suppressor gene product, tuberin, is also identified and characterized as an Akt substrate. Furthermore, it is found that overexpression of a tuberin mutant lacking the major Akt phosphorylation sites can inhibit growth factor-induced activation of S6K1. These results provide a biochemical link between the PI3K-Akt pathway and regulation of S6K1 and also indicate a biochemical basis for the disease tuberous sclerosis complex (TSC) (Manning, 2002).
TSC is a common disease affecting an estimated 1 in 6000 individuals and is characterized by the occurrence of widespread benign tumors called hamartomas frequently affecting the brain, skin, kidneys, lungs, eyes, and heart. In approximately 85% of TSC patients, the disease is caused by loss-of-function mutations in one of two tumor suppressor genes, TSC1 and TSC2, which encode hamartin and tuberin, respectively. These two proteins form a complex. Tuberin, which has a region of homology to Rap1 GTPase-activating proteins (GAPs), has been shown to possess in vitro GAP activity toward Rap1. However, the true molecular and cellular functions of the hamartin-tuberin complex have yet to be clearly defined. Furthermore, very little is known about how these tumor suppressor gene products are regulated (Manning, 2002).
Based on genetic studies and the fact that PI3K and Akt are oncogenes while the TSC genes are tumor suppressors, one would predict that the PI3K-Akt-mediated phosphorylation of tuberin would inhibit the function of the tuberin-hamartin complex. In Drosophila, hamartin and tuberin appear to function together to antagonize signaling of the insulin-PI3K-Akt pathway and, thereby, restrict cell growth and proliferation. Most strikingly, loss of just one copy of TSC1 or TSC2 partially rescues the lethality of insulin receptor loss-of-function mutants. This result implies that one of the primary functions of the insulin pathway, at least in Drosophila, is to inhibit the hamartin-tuberin complex. Furthermore, both mouse and Drosophila genetic studies have suggested that the tuberin-hamartin complex functions to inhibit S6K1 (Manning, 2002).
Expression in human cells of the tuberinS939A/T1462A mutant, which lacks the major PI3K-dependent phosphorylation sites, at levels comparable to endogenous tuberin leads to a decrease in growth factor-induced S6K1 phosphorylation and activity. This phosphorylation and subsequent activation of S6K1 has been previously demonstrated to be dependent on PI3K. These results, along with those from genetic studies in other systems, are consistent with a model in which growth factors activate PI3K leading to the phosphorylation of tuberin by Akt. This phosphorylation inhibits the tuberin-hamartin complex, thereby relieving its inhibition of S6K1. In this model, expression of the tuberinS939A/T1462A mutant, which would not be phosphorylated and inhibited, would have a dominant effect over endogenous tuberin and block growth factor-induced S6K1 activation. It will be of great interest to determine the molecular nature of S6K1 inhibition by the tuberin-hamartin complex in the absence of mitogenic stimuli. It is possible that the complex does so upstream of mTOR, because the constitutive activation of S6K1 in TSC1-/- MEFs is sensitive to rapamycin. Alternatively, mTOR might regulate S6K1 in a nutrient-sensitive pathway parallel to the mitogen-sensitive PI3K-Akt-tuberin pathway (Manning, 2002).
Recent studies have suggested that S6K1 activation can occur independent of PI3K and Akt. These studies demonstrate the existence of multiple pathways regulating S6K1 and that the tuberin-hamartin complex might integrate signals from many different inputs. The identification of tuberin as a direct downstream target of the PI3K-Akt pathway provides the missing link between this signaling cascade and control of S6K1 activity (Manning, 2002).
The identification of this biochemical relationship between the mammalian TSC tumor suppressor gene products and the oncogenic PI3K-Akt pathway could have important implications in human diseases. For instance, in approximately 10%-15% of patients diagnosed with TSC, mutations in TSC1 or TSC2 have not been detected. Based on the findings of this study, it is possible that mutations leading to aberrant activation of the PI3K-Akt pathway, such as PTEN mutations, could inhibit the function of the tuberin-hamartin complex by causing constitutive phosphorylation of tuberin. It will be interesting to examine the phosphorylation state of tuberin within hamartomas from such TSC patients. Indeed, in PTEN-/- cell lines derived from both glioblastoma and prostate tumors, growth factor-independent phosphorylation of tuberin on the PI3K-dependent T1462 site is detected (Manning, 2002).
Germline mutations in either PTEN or the TSC genes cause autosomal dominant diseases that are characterized by the occurrence of widespread hamartomas due to loss of heterozygosity at these loci. However, the tissue distribution of these benign tumors varies between patients with loss of PTEN and those with TSC. These differences might be explained by a model in which the tuberin-hamartin complex is the primary growth-inhibiting target of the PI3K-Akt pathway in tissues affected in TSC patients. In other tissues, such as those affected in patients with PTEN mutations, this complex might be one of many targets of the PI3K-Akt pathway. Interestingly, though, recent studies have suggested that mTOR activity is essential for oncogenic transformation of cells by activated PI3K or Akt and for growth of PTEN-/- tumors. Therefore, aberrant phosphorylation and inhibition of the tuberin-hamartin complex, and subsequent increased activity of mTOR and/or S6K1, would likely contribute to tumorigenesis caused by mutations that activate the PI3K-Akt pathway. Future studies using crosses between PTEN and TSC knockout mice should help determine the contribution of the tuberin-hamartin complex in prevention of the variety of tumors caused by uncontrolled signaling through the PI3K-Akt pathway. Finally, the elucidation of a PI3K-Akt-tuberin pathway controlling S6K1 activity will have important implications in the understanding and treatment of the prevalent TSC disease (Manning, 2002).
Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27(kip1), thereby promoting CDK2 activity and cellular proliferation. These results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function (Dan, 2002).
TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. These data indicate a molecular mechanism for TSC2 in insulin signalling, tumor suppressor functions and in the inhibition of cell growth (Inoki, 2002).
See Akt1: Evolutionary homologs part 2/2 |
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