Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B

The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.     

Threonine 34 phosphorylation by phosphoinositide-dependent protein kinase 1 facilitates dissociation of Akt from the plasma membrane

Akt is a key mediator of cell proliferation, survival and metabolism. After translocation to the membrane and phosphorylation at T308 and S473, the activated Akt dissociates from the plasma membrane to cytoplasm, which is an important step to phosphorylate its downstream targets. In addition to its central role in regulating the kinase activity, phosphorylation of T308 in the kinase loop has been reported to be necessary for this dissociation process. However, it is not clear whether the membrane detachment requires further mechanisms. In the present report, we demonstrate that membrane dissociation of Akt requires phosphoinositide-dependent protein kinase 1 (PDK1) which directly phosphorylates not only T308 but also T34 in the pleckstrin homology (PH) domain. Like T308, T34 was phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate- and phosphatidylserine-dependent manner. Phosphorylation of T34 also occurred in cells following growth factor stimulation, concurrently with T308 phosphorylation. Moreover, when T34 was mutated to aspartic acid (T34D) to mimic its phosphorylation, Akt-membrane association assessed by surface plasmon resonance spectroscopy was significantly reduced. In cells, this mutation impaired the IGF-induced Akt membrane translocation and subsequent phosphorylation at T308 and S473. Taken together, our results demonstrate that T34 phosphorylation by PDK1 promotes the membrane dissociation of activated Akt for its downstream action through attenuating membrane binding affinity. This membrane dissociation mechanism offers a new insight for Akt activation process and provides a potential new target for controlling the Akt-dependent cellular processes.     

Protein kinase C betaII regulates Akt phosphorylation on Ser-473 in a cell type- and stimulus-specific fashion

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2.     

Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1

Protein kinase B (PKB/Akt) has been well established as an important signaling intermediate, and its deregulation has been implicated in the development of human cancer and diabetes (reviewed in). 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 (reviewed in ), the identity of the Ser473 kinase remains unclear. We have focused our study 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. Here, we report the identification of a constitutively active PKB Ser473 kinase activity enriched in buoyant, detergent-insoluble plasma membrane rafts that are distinct from the cytosolic distribution of PKB and PDK1. This Ser473 kinase activity was released from the membrane by high salt, and gel filtration analysis showed 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 did not retain Ser473 kinase activity. Thus, we have identified a novel raft-associated PKB Ser473 kinase, implicating a role for lipid rafts in PKB signaling.     

Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes

By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt.     

Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.     

A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.     

IKBKE protein activates Akt independent of phosphatidylinositol 3-kinase/PDK1/mTORC2 and the pleckstrin homology domain to sustain malignant transformation

Serine/threonine kinase Akt regulates key cellular processes such as cell growth, proliferation, and survival. Activation of Akt by mitogenic factor depends on phosphatidylinositol 3-kinase (PI3K). Here, we report that IKBKE (also known as IKKε and IKKi) activates Akt through a PI3K-independent pathway. IKBKE directly phosphorylates Akt-Thr308 and Ser473 independent of the pleckstrin homology (PH) domain. IKBKE activation of Akt was not affected by inhibition of PI3K, knockdown of PDK1 or mTORC2 complex. Further, this activation could be inhibited by Akt inhibitors MK-2206 and GSK690693 but not the compounds (perifosine and triciribine) targeting the PH domain of Akt. Expression of IKBKE largely correlates with activation of Akt in breast cancer. Moreover, inhibition of Akt suppresses IKBKE oncogenic transformation. These findings indicate that IKBKE is an Akt-Thr308 and -Ser473 kinase and directly activates Akt independent of PI3K, PDK1, and mTORC2 as well as PH domain. Our data also suggest that Akt inhibitors targeting the PH domain have no effect on the tumors in which hyperactive Akt resulted from elevated IKBKE.     

Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343

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) 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. We now show by in-gel kinase assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry 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.     

Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice

The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.     

Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.     

Endoplasmic reticulum is a main localization site of mTORC2

The Akt kinase is a critical effector in growth factor signaling. Activation of Akt driven by the growth factor dependent PI3K (phosphatidylinositol-3-OH kinase) is coupled to the plasma membrane translocation and phosphorylation of Akt on two sites by PDK1 (phosphoinositide-dependent protein kinase-1) on Thr-308 and by mTORC2 (mammalian Target of Rapamycin Complex 2) on Ser-473. In our study we examined the sub-cellular localization of mTORC2 and identified that this kinase complex predominantly resides on endoplasmic reticulum (ER). Our immunostaining analysis did not show a substantial co-localization of the mTORC2 component rictor with Golgi, lysosome, clathrin-coated vesicles, early endosomes, or plasma membrane but indicated a strong co-localization of rictor with ribosomal protein S6 and ER marker. Our biochemical study also identified the mTORC2 components rictor, SIN1, and mTOR as the highly abundant proteins in the ER fraction, whereas only small amount of these proteins are detected in the plasma membrane and cytosolic fractions. We found that growth factor signaling does not alter the ER localization of mTORC2 and also does not induce its translocation to the plasma membrane. Based on our study we suggest that the mTORC2-dependent phosphorylation of Akt on Ser-473 takes place on the surface of ER.     

Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.     

Disruption of the interface between the pleckstrin homology (PH) and kinase domains of Akt protein is sufficient for hydrophobic motif site phosphorylation in the absence of mTORC2

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.     

The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.     

Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation

3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.     

Essential role of PDK1 in regulating endothelial cell migration

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1(-/-) embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.     

A Small Molecule Inhibits Akt through Direct Binding to Akt and Preventing Akt Membrane Translocation

The Akt pathway is frequently hyperactivated in human cancer and functions as a cardinal nodal point for transducing extracellular and intracellular oncogenic signals and, thus, presents an exciting target for molecular therapeutics. Here we report the identification of a small molecule Akt/protein kinase B inhibitor, API-1. Although API-1 is neither an ATP competitor nor substrate mimetic, it binds to pleckstrin homology domain of Akt and blocks Akt membrane translocation. Furthermore, API-1 treatment of cancer cells results in inhibition of the kinase activities and phosphorylation levels of the three members of the Akt family. In contrast, API-1 had no effects on the activities of the upstream Akt activators, phosphatidylinositol 3-kinase, phosphatidylinositol-dependent kinase-1, and mTORC2. Notably, the kinase activity and phosphorylation (e.g. Thr(P)³⁰⁸ and Ser(P)⁴⁷³) levels of constitutively active Akt, including a naturally occurring mutant AKT1-E17K, were inhibited by API-1. API-1 is selective for Akt and does not inhibit the activation of protein kinase C, serum and glucocorticoid-inducible kinase, protein kinase A, STAT3, ERK1/2, or JNK. The inhibition of Akt by API-1 resulted in induction of cell growth arrest and apoptosis selectively in human cancer cells that harbor constitutively activated Akt. Furthermore, API-1 inhibited tumor growth in nude mice of human cancer cells in which Akt is elevated but not of those cancer cells in which it is not. These data indicate that API-1 directly inhibits Akt through binding to the Akt pleckstrin homology domain and blocking Akt membrane translocation and that API-1 has anti-tumor activity in vitro and in vivo and could be a potential anti-cancer agent for patients whose tumors express hyperactivated Akt.     

Akt1 Intramitochondrial Cycling Is a Crucial Step in the Redox Modulation of Cell Cycle Progression

Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser⁴⁷³ by mTORC2 and Thr³⁰⁸ by PDK1. On these bases, we investigated the mechanistic connection of H₂O₂ yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H₂O₂ entails the entrance of cytosolic P-Akt1 Ser⁴⁷³ to mitochondria, where it is further phosphorylated at Thr³⁰⁸ by constitutive PDK1. Phosphorylation of Thr³⁰⁸ in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H₂O₂, Akt1-PDK1 association is disrupted and P-Akt1 Ser⁴⁷³ accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H₂O₂ effects on Akt1-PDK interaction depend on the selective oxidation of Cys³¹⁰ to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.