Current Perspectives on Akt Akt-ivation and Akt-ions

Abstract

The serine/threonine kinase Akt is an effector of PI3K-generated phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and is a principle mediator of growth factor-induced signal transduction. Akt is activated through phosphorylation by specific kinases, and its activity is reduced directly by phosphorylation-site-specific phosphatases. In addition, Akt activity is effectively reduced by the action of phosphatases which dephosphorylate PI(3,4,5)P3, thereby reducing the levels of the essential lipid activators of PDK1 and Akt. The functions of Akt are pleiotropic and include regulation of cellular proliferation, differentiation, protein synthesis, and survival. Akt stimulates protein synthesis through actions on mTOR/p70S6K, and promotes survival by phosphorylating and inactivating pro-apoptotic molecules such as Ask1, Bad, Bax, and FoxO3a. Furthermore, loss of Akt decreases the intracellular ATP:AMP ratio, thus establishing a role for Akt in energy regulation. Three isoforms of Akt have been identified, and although redundant functions between isoforms exist, recent investigations have enumerated unique functions for each. Therefore, targeting specific Akt isozymes in a tissue- and context-specific fashion may lead to a greater understanding of Akt-mediated processes.

Introduction

Akt (also known as protein kinase B) is a serine/ threonine kinase downstream of phosphoinositide-3′-OH kinase (PI3K) and a member of the AGC family of protein kinases. Since first discovered in 1977 as the oncogene in the transforming retrovirus AKT8 (1), Akt has become the subject of intense research aimed at defining its involvement in cancer progression, metabolism, cellular growth and differentiation, and survival. Much has been learned about the regulation and actions of Akt since its cloning and identification of human homologues (2), yet much still remains to be elucidated. For example, there are three isoforms of Akt in mammals, and the functions of each have been shown to be redundant as well as unique depending on the tissue examined, stimulus, outcome (e.g. differentiation) or the conditions within the cellular milieu. Additionally, our knowledge of Akt regulation in terms of both activation and signal suppression is burgeoning, but further work must be accomplished to apply these findings in practice in humans; for instance, to attenuate cancer progression and metastasis, or to delay onset or minimize age-related sarcopenia. This review discusses factors involved in regulating Akt activation, select substrates of Akt involved in survival and growth, and isoform-specific functions of Akt.

Activation of Akt

Members of the PI3K class of enzymes generate phosphoinositol lipids that act as second-messengers in a number of intracellular signaling cascades, including the activation of Akt (3). PI3K catalytic enzymes are categorized into three classes by their structure, substrate specificity, and lipid products (4). Members of the Class IA PI3Ks (α, β, and δ) are heterodimers consisting of a 110 kDa catalytic subunit and an 85, 55, or 50 kDa regulatory subunit (4, 5). PI3K activation leads to proliferation, survival, cell migration, and growth; however, abnormally increased PI3K signaling can promote aberrant proliferative signals that lead to cellular transformation (6–8). When bound to cognate ligands, receptor tyrosine kinases such as the insulin-like growth factor-I receptor undergo autophosphorylation, resulting in the recruitment of adaptor molecules including insulin receptor substrate (IRS) proteins (9, 10). PI3K regulatory subunits complexed to p110 catalytic subunits then bind IRS and convert plasma membrane-associated PI(4,5)P2 to PI(3,4,5)P3 (5). PI(3,4,5)P3 provides a phospholipid binding substrate for signaling effector molecules such as phosphatidylinositol-dependent kinase 1 (PDK1) and Akt (11). At least two identified phosphatases act to reduce PI(3,4,5)P3 levels: phosphatase and tensin homologue deleted on chromosome 10 (PTEN) (12) and SH2-containing inositol 5′-phosphatase (SHIP) (13). PTEN hydrolyzes the phosphate at the D3 position on the inositol ring of PI(3,4,5)P3, thus generating PI(4,5)P2. SHIP proteins remove phosphates from the D5 position, thus generating PI(3,4)P2.

Akt exists as three isoforms in mammals (Akt1, Akt2, and Akt3), and each is transcribed from separate genes (14–17); additionally, a splice variant of Akt3 has been identified (18). Akt contains an N-terminal pleckstrin homology (PH) domain which interacts with PI(3,4)P2 and PI(3,4,5)P3 (19, 20), and a C-terminal domain that contains a hydrophobic domain (HD) with homology to other AGC kinases (21). Akt is activated by phosphorylation at two sites including one in the activation loop (A-loop) (T308, T309, T305 in Akt1, Akt2, and Akt3, respectively) in the catalytic domain, and one in the C-terminal HD (S473, S474, S472 in Akt1, Akt2, and Akt3, respectively) (21). The C-terminus of the Akt3 splice variant, however, lacks the analogous portion of the HD that contains S473, S474, or S472 present in the other Akt isoforms (Fig. 1). Phosphorylation at the turn motif (TM) at a highly conserved threonine residue in the carboxy-terminus results in increased stability of the molecule (22).

Phosphorylation at the A-loop is sufficient for activation of Akt, however full activation is achieved when both the A-loop and HD sites are phosphorylated (21). Akt can be activated through growth factor stimulation (21) and oxidative stress (23, 24). Phosphorylation of the A-loop in the catalytic domain is accomplished by PDK1 after recruitment to the cell membrane and binding with PI(3,4,5)P3 (11, 19, 25). HD phosphorylation has been shown to be mediated, under certain conditions, through actions of a number of factors including mammalian target of rapamycin complex 2 (mTORC2) (26), DNA-PK (27), lipid raft-associated elements (28), and autophosphorylation of Akt itself (29). However, growth factor-stimulated phosphorylation of Akt at the HD is considered to be mediated principally by mTORC2.

Direct de-phosphorylation of Akt remained an enigma until the discovery of PH domain leucine-rich repeat protein phosphatase 1 (abbreviated as PHLPP1) (30), and more recently, PHLPP2 (31). Both PHLPP1 and PHLPP2 were found to de-phosphorylate the HD site of Akt, but they did so in an Akt isoform-specific manner.

The mechanisms outlined above describe a general scheme for full activation for Akt in response to growth factor stimulation: generation of PI(3,4,5)P3 or PI(3,4)P2 by PI3K results in membrane recruitment of Akt and co-localization of PDK1. PDK1 phosphorylates Akt at the A-loop which results in conformational change of Akt allowing for phosphorylation of the HD by mTORC2. Akt then acts on downstream targets to initiate signaling cascades. Figure 2 presents these processes diagrammatically.

Downstream of Akt: Survival, Growth, and Energy Homeostasis

A number of known and putative Akt targets have been identified thus far by virtue of their containing the essential Akt consensus motif (R-X-R-X-X-S/T-B) where X is any amino acid and B represents a bulky hydrophobic residue (32, 33). Among these targets are several pro-apoptotic molecules that are inactivated when phosphorylated by Akt, thus demonstrating that Akt can act as a survival factor. There are many pro-apoptotic substrates of Akt including apoptosis-signal regulating kinase 1 (Ask1) (34), Bad (35), Bax (36), FoxO transcription factors (37–39), and human caspase-9 (40). Ask1 is a mitogen-activated protein (MAP) kinase kinase kinase that transmits pro-apoptotic signals through activation of downstream stress-activated protein kinases such as c-jun N-terminal kinase (JNK) and p38 MAPK (41). Phosphorylation of 14-3-3 by activated JNK promotes release of 14-3-3-bound Bad, Bax, and FoxO proteins, thus contributing to apoptosis through both mitochondrial-dependent (Bad and Bax) and -independent (FoxO3a) (42, 43). Each of these mechanisms will be explained in the following sections.

Phosphorylation of Ask1 at serine 83 by Akt has been shown to reduce Ask1 activation stimulated by oxidative stress, as well as apoptosis induced by Ask1 overexpression (34), thus providing evidence for a specific pro-survival role of Akt acting on Ask1. Interaction of Bad with outer mitochondrial membrane (OMM)-associated Bcl-2 and Bcl-XL leads to disruption of OMM integrity and cytochrome c release; hence, binding of Bad with Bcl-2 or Bcl-XL promotes apoptosis through the mitochondrial intrinsic death pathway. Phosphorylation of Bad at serine 136 by Akt leads to dissociation from Bcl-2/Bcl-XL and promotes association with cytosolic 14-3-3, thereby reducing pro-death signals (35). Another protein that disrupts OMM membrane integrity is Bax. Bax exists in the cytosol in a conformation permissive for actions by JNK and p38 MAPK (44); once acted upon by apoptotic stimuli, Bax undergoes a conformational change that permits insertion into mitochondrial membranes and oligomerization. However, prior phosphorylation of cytosolic Bax at serine 184 by Akt prevents this conformational change and reduces half-life, thereby impairing Bax-mediated apoptosis (36, 45).

Members of the FoxO family of transcription factors are structurally related proteins whose name is derived from the product of the fork head gene originally identified in D. melanogaster (46). FoxO transcription factors belong to a superfamily of Fox (Forkhead Box) proteins in a class defined as O (Other) which reflects disparities in DNA-binding domain sequences (47, 48). Among the FoxO proteins, FoxO3a has been shown to possess pro-apoptotic functions including regulation of FasL gene expression (49); however, FoxO3a-mediated apoptosis can be attenuated by Akt (39). FoxO3a is phosphorylated at three sites by Akt (T32, S253, S315), although S315 is preferentially phosphorylated by serum- and glucocorticoid-inducible kinase (SGK) (50). When phosphorylated by Akt at target residues, nuclear FoxO3a associates with nuclear 14-3-3; this association masks the nuclear localization sequence, and the complex is exported to the cytosol in an inactive state (51).

In addition to the target molecules described above, Akt can phosphorylate a number of other pro-death molecules to cause their inactivation, as well as oppose apoptosis indirectly by acting on proteins involved in transcription of pro-apoptotic genes (52). Altogether, it is clear that Akt plays a vital role in cell survival and is positioned at a point of convergence of a number of signaling pathways, balancing both pro-survival and pro-death signals.

The mammalian target of rapamycin (mTOR) responds to nutrient or growth factor inputs, depending on its association with one of two adaptor molecules, either Raptor or Rictor (53). One principle effector of mTOR action is p70S6K, an AGC kinase family member whose actions promote growth and survival (54, 55). Raptor-bound mTOR, when also bound in complex with GβL (referred to as mTOR complex 1, or mTORC1) is generally considered the rapamycin-sensitive p70S6K kinase (56), but Rictor-bound mTOR (the complex, in association with GβL and SAPK interacting protein 1 (SIN1) (57), being referred to as mTORC2) has been shown to phosphorylate rapamycin-resistant mutants of p70S6K (58). Current models suggest that Akt positively regulates mTOR by acting on mTOR-inhibitory molecules such as proline-rich Akt substrate of 40 kDa (PRAS40) and tuberous sclerosis complex 2 (TSC2) (53, 59–61). mTORC2 is capable of phosphorylating Akt at the HD site (26, 57), as well as controlling folding and stability of the enzyme (22, 62); thus, there exists positive feedback signaling between mTOR and Akt.

AMP-activated protein kinase (AMPK) acts as an intracellular energy sensor (63) that is sensitive to Akt signaling. In a recent study, decreased ATP levels were observed in Akt1 ^−/− Akt2 ^−/− mouse embryonic fibroblasts (MEFs) as compared to wild-type cells (64), suggesting that depletion of Akt affects energy charge. This reduced energy charge was associated with increased AMPK. Since AMPK can inactivate mTOR through direct phosphorylation of TSC2 (59), these findings suggest the existence of a mechanism to reduce growth and translation during low-energy states secondary to Akt depletion.

Isoform-Specific Actions of Akt

Three Akt isoforms have been identified, and murine gene disruption models demonstrate that each isoform possesses a distinct function (65). Mice lacking Akt1 have reduced body size (66), mice lacking Akt2 show abnormal glucose homeostasis and a diabetic phenotype (67, 68), and mice lacking Akt3 have diminished brain size (69, 70). In mammary tumor cells, Akt1 or Akt2 knockdown had opposing effects on differentiation (71), while in melanoma cells, Akt3 was found to be the principle Akt isoform involved in tumor progression (72). Furthermore, in differentiating skeletal muscle cells, Akt2 mRNA, protein, and activity were found to be upregulated, and overexpression of Akt2 prevented apoptosis in response to serum deprivation, suggesting that Akt2 acts to reduce apoptosis during myogenic differentiation (73). Although these data demonstrate discrete functions for Akt isoforms, there is also accumulating evidence for overlapping actions. For example, each Akt isoform contributed to phosphorylation of GSK3α and GSK3β in 3T3-L1 adipocytes (74). Additionally, knockdown of individual Akt isoforms in H157 cells demonstrated distinct roles for Akt3 acting on p27 and Akt2 acting on GSK3α, but redundant roles for all three Akt isoforms in regulating FoxO1, GSk3β, and TSC2 (31). Moreover, in Akt1/Akt2 double knockout MEFs (DKO MEFs), Akt3 alone was sufficient to prevent cell death until reduced to only 20% of the initial level (75). Reduction of Akt3 through increased concentrations of siRNA, however, potentiated apoptosis in response to several pro-apoptotic stimuli in DKO MEFs. This latter study underscored that very limited amounts of total Akt, even of a single isoform, can promote survival under non-stressed conditions; but that under stress, cells require a higher threshold of Akt to remain viable. Together, these studies illustrate the complexity of Akt regulation and specificity, and demonstrate that Akt isoforms possess both distinct and functionally redundant actions in growth, metabolism, and cell survival.

Conclusions and Future Outlook

Much has been learned about Akt regulation and function since it was first discovered, yet much still remains to be elucidated. For instance, what elements control intracellular localization of Akt? Akt activation depends on proximity to PI(3,4)P2 and PI(3,4,5)P3 in cell membranes and it is important to define those factors within the cell that are responsible for membrane recruitment. Interaction of the PH domain with phosphoinositide lipids is essential for activation, but identification of the processes and molecules involved in executing this specifically targeted relocation is of importance. It then follows that the location of Akt in unstimulated cells is of interest in order to define movement patterns of Akt after stimuli.

Defining intracellular localization also extends to isoform-specific locations within the cell, as well as isoform-specific activation in response to stimuli. What are the determining factors that contribute to activation of Akt isoforms in response to growth factors? Why is one isoform preferentially activated over another? One answer may be that Akt isoforms are expressed at different levels in a given cell-type. If availability of one isoform is greater than another, then it may be a matter of proximity to membrane-located signaling complexes. For example, if Akt1 is the most highly expressed Akt isoform in a cell, then it would logically follow that there would be more Akt1 available for recruitment to membranes than Akt2 or Akt3. It is also possible that inactive Akt may be localized to specific intracellular compartments or areas within the cell, possibly near the membrane, that contain increased densities of a particular isoform.

In addition to activation of Akt, it is important to further characterize existing known phosphatases as well as to discover new methods to de-activate the enzyme. The identification of isoform-specific de-phosphorylation of Akt by PHLPP1 and PHLPP2 was a groundbreaking discovery, and further research in defining the mechanisms of action and regulation of these two phosphatases is warranted.

An exciting and promising line of research involves the development of Akt isoform-specific small molecule inhibitors. Some inhibitors exert their effects by preventing Akt from being activated due to changes in ternary structure. For instance, an Akt-specific inhibitor commonly referred to as “Akti-1/2” has recently been developed that can bind to the PH domain and/or hinge region on Akt1 or Akt2 thereby preventing activation (76). This inhibitor can also bind to activated Akt and inhibit phosphorylation of substrates. However, Akti-1/2 can also inhibit Akt3 in 3T3-L1 adipocytes and L6 myotubes (77), as well as in C2C12 myoblasts (Matheny and Adamo, unpublished observations) at micromolar concentrations. Although Akti-1/2 is not specific for Akt isoforms in some cell lines at specific concentrations, this compound still possesses great potential under conditions where all three Akt isoforms can act in a redundant manner. In any case, intense research in the development of Akt isoform-specific inhibitory compounds is ongoing (78).

In conclusion, Akt is at the crossroads of a number of intracellular pathways and is a key signaling intermediate in growth and survival. Much has been learned thus far with respect to regulation and signaling of Akt, yet much remains to be discovered; specifically, the localization of Akt isoforms in response to various stimuli in different tissues, as well as isoform-specific actions of Akt on target substrates.

Figure 1.
Comparison of human Akt isoform domain structures. Three isoforms of Akt exist in man that share approximately 80% amino acid sequence homology. Additionally, an alternative splice variant of Akt3 with a truncated hydrophobic domain (designated “Akt3-(γ1)” in this figure) has been identified (see text). All Akt isoforms possess an N-terminal PH domain responsible for phospholipid binding that is tethered to a catalytic region containing a threonine residue (T308 in Akt1) critical for activation of the enzyme. A C-terminal hydrophobic domain (designated “HD” in this figure) follows the catalytic domain and contains a serine residue (S473 in Akt1) important for full activation. Phosphorylation of a threonine residue in the turn motif (T450 in Akt1) by mTORC2 contributes to stability of the molecule. Numbers to the immediate left of the images designate the first amino acid, and numbers to the immediate right of the images represent the number of the most C-terminal residue as determined by comparative sequence analysis. A color version of this figure is available in the online journal.

Figure 2.
Regulation of Akt activation. Tyrosine kinase growth factor receptor-induced activation of Akt is initiated upon growth factor (GF) ligand binding to its cognate receptor. Recruitment of PI3K (p85 regulatory subunit complexed with p110 catalytic subunit) to the receptor at the membrane occurs after binding of adaptor molecules, including IRS docking proteins, to the intracellular subunits of receptors. PI3K, in close proximity to PI(4,5)P2 (PIP₂) then phosphorylates the D3 position of the inositol ring generating PI(3,4,5)P3 (PIP₃). PIP₃ then recruits PH-domain-containing molecules including PDK1 and Akt to the lipid bilayer (Akt recruitment not shown for clarity), allowing for binding to PIP₃, and phosphorylation of Akt at the A-loop (T308 in Akt1) by PDK1. Reconversion of PIP₃ to PIP₂ is promoted by PTEN, which reduces available PIP₃ for PDK1 and Akt binding, thus terminating growth factor signals. Phosphorylation of Akt at the HD (S473 in Akt1) occurs through the actions of the mTORC2 complex, which contains Rictor, SIN1, and mTOR. Activated Akt can then act on substrates in the cytosol to promote survival, growth and energy homeostasis, or translocate to the nucleus and phosphorylate target molecules such as FoxO3a. A color version of this figure is available in the online journal.

Footnotes

This work was supported by NIA grant R01AG026012 to MLA. RWM was supported by pre-doctoral award from NIA training grant T32 AG021890-08.

References

Staal SP, Hartley JW, Rowe WP. Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc Natl Acad Sci U S A 74:3065–3067, 1977.

Staal SP. Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci U S A 84:5034–5037, 1987.

Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253:239–254, 1999.

Hirsch E, Costa C, Ciraolo E. Phosphoinositide 3-kinases as a common platform for multi-hormone signaling. J Endocrinol 194:243–256, 2007.

Geering B, Cutillas PR, Nock G, Gharbi SI, Vanhaesebroeck B. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers. Proc Natl Acad Sci U S A 104:7809–7814, 2007.

Denley A, Kang S, Karst U, Vogt PK. Oncogenic signaling of class I PI3K isoforms. Oncogene 27:2561–2574, 2008.

Kang S, Denley A, Vanhaesebroeck B, Vogt PK. Oncogenic transformation induced by the p110beta, -gamma, and -delta isoforms of class I phosphoinositide 3-kinase. Proc Natl Acad Sci U S A 103: 1289–1294, 2006.

Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, Yan H, Gazdar A, Powell SM, Riggins GJ, Willson JK, Markowitz S, Kinzler KW, Vogelstein B, Velculescu VE. High frequency of mutations of the PIK3CA gene in human cancers. Science 304:554, 2004.

Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34, 1995.

10.

Dey BR, Frick K, Lopaczynski W, Nissley SP, Furlanetto RW. Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10. Mol Endocrinol 10:631–641, 1996.

11.

Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7:261–269, 1997.

12.

Cully M, You H, Levine AJ, Mak TW. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6:184–192, 2006.

13.

Kalesnikoff J, Sly LM, Hughes MR, Buchse T, Rauh MJ, Cao LP, Lam V, Mui A, Huber M, Krystal G. The role of SHIP in cytokine-induced signaling. Rev Physiol Biochem Pharmacol 149:87–103, 2003.

14.

Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 88:4171–4175, 1991.

15.

Jones PF, Jakubowicz T, Hemmings BA. Molecular cloning of a second form of rac protein kinase. Cell Regul 2:1001–1009, 1991.

16.

Konishi H, Kuroda S, Tanaka M, Matsuzaki H, Ono Y, Kameyama K, Haga T, Kikkawa U. Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. Biochem Biophys Res Commun 216:526–534, 1995.

17.

Brodbeck D, Cron P, Hemmings BA. A human protein kinase Bgamma with regulatory phosphorylation sites in the activation loop and in the C-terminal hydrophobic domain. J Biol Chem 274:9133–9136, 1999.

18.

Brodbeck D, Hill MM, Hemmings BA. Two splice variants of protein kinase B gamma have different regulatory capacity depending on the presence or absence of the regulatory phosphorylation site serine 472 in the carboxyl-terminal hydrophobic domain. J Biol Chem 276:29550–29558, 2001.

19.

Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq JM, Hemmings BA. Role of translocation in the activation and function of protein kinase B. J Biol Chem 272: 31515–31524, 1997.

20.

Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665–668, 1997.

21.

Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551, 1996.

22.

Facchinetti V, Ouyang W, Wei H, Soto N, Lazorchak A, Gould C, Lowry C, Newton AC, Mao Y, Miao RQ, Sessa WC, Qin J, Zhang P, Su B, Jacinto E. The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C. EMBO J 27: 1932–1943, 2008.

23.

Van der Kaay J, Beck M, Gray A, Downes CP. Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses. J Biol Chem 274: 35963–35968, 1999.

24.

Wang X, McCullough KD, Franke TF, Holbrook NJ. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 275:14624–14631, 2000.

25.

Stokoe D, Stephens LR, Copeland T, Gaffney PR, Reese CB, Painter GF, Holmes AB, McCormick F, Hawkins PT. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277:567–570, 1997.

26.

Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101, 2005.

27.

Bozulic L, Surucu B, Hynx D, Hemmings BA. PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 30:203–213, 2008.

28.

Hill MM, Feng J, Hemmings BA. Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1. Curr Biol 12:1251–1255, 2002.

29.

Toker A, Newton AC. Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem 275:8271–8274, 2000.

30.

Gao T, Furnari F, Newton AC. PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell 18:13–24, 2005.

31.

Brognard J, Sierecki E, Gao T, Newton AC. PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol Cell 25:917–931, 2007.

32.

Alessi DR, Caudwell FB, Andjelkovic M, Hemmings BA, Cohen P. Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase. FEBS Lett 399:333–338, 1996.

33.

Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell 129:1261–1274, 2007.

34.

Kim AH, Khursigara G, Sun X, Franke TF, Chao MV. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol 21:893–901, 2001.

35.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241, 1997.

36.

Gardai SJ, Hildeman DA, Frankel SK, Whitlock BB, Frasch SC, Borregaard N, Marrack P, Bratton DL, Henson PM. Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils. J Biol Chem 279:21085–21095, 2004.

37.

Zheng WH, Kar S, Quirion R. Insulin-like growth factor-1-induced phosphorylation of the forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells. J Biol Chem 275:39152–39158, 2000.

38.

Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398:630–634, 1999.

39.

Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857–868, 1999.

40.

Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC. Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318–1321, 1998.

41.

Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K, Gotoh Y. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275:90–94, 1997.

42.

Tsuruta F, Sunayama J, Mori Y, Hattori S, Shimizu S, Tsujimoto Y, Yoshioka K, Masuyama N, Gotoh Y. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins. EMBO J 23:1889–1899, 2004.

43.

Sunayama J, Tsuruta F, Masuyama N, Gotoh Y. JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3. J Cell Biol 170: 295–304, 2005.

44.

Kim BJ, Ryu SW, Song BJ. JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells. J Biol Chem 281:21256–21265, 2006.

45.

Xin M, Deng X. Nicotine inactivation of the proapoptotic function of Bax through phosphorylation. J Biol Chem 280:10781–10789, 2005.

46.

Weigel D, Jurgens G, Kuttner F, Seifert E, Jackle H. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell 57:645–658, 1989.

47.

Huang H, Tindall DJ. Dynamic FoxO transcription factors. J Cell Sci 120:2479–2487, 2007.

48.

Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 14:142–146, 2000.

49.

Wang X, Finegan KG, Robinson AC, Knowles L, Khosravi-Far R, Hinchliffe KA, Boot-Handford RP, Tournier C. Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress. Cell Death Differ 3:2099–2108, 2006.

50.

Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21: 952–965, 2001.

51.

Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni JV, Dalal SN, DeCaprio JA, Greenberg ME, Yaffe MB. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. J Cell Biol 156:817–828, 2002.

52.

Parcellier A, Tintignac LA, Zhuravleva E, Hemmings BA. PKB and the mitochondria: AKTing on apoptosis. Cell Signal 20:21–30, 2008.

53.

Pende M. mTOR, Akt, S6 kinases and the control of skeletal muscle growth. Bull Cancer 93:E39–43, 2006.

54.

Harada H, Andersen JS, Mann M, Terada N, Korsmeyer SJ. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD. Proc Natl Acad Sci U S A 98:9666–9670, 2001.

55.

Thomas G. The S6 kinase signaling pathway in the control of development and growth. Biol Res 35:305–313, 2002.

56.

Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175, 2002.

57.

Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127:125–137, 2006.

58.

Ali SM, Sabatini DM. Structure of S6 kinase 1 determines whether raptor-mTOR or rictor-mTOR phosphorylates its hydrophobic motif site. J Biol Chem 280:19445–19448, 2005.

59.

Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4: 648–657, 2002.

60.

Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, Carr SA, Sabatini DM. PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25:903–915, 2007.

61.

Vander Haar E, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 9:316–323, 2007.

62.

Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J 27:1919–1931, 2008.

63.

Carling D. The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem Sci 29:18–24, 2004.

64.

Hahn-Windgassen A, Nogueira V, Chen CC, Skeen JE, Sonenberg N, Hay N. Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. J Biol Chem 280:32081–32089, 2005.

65.

Dummler B, Hemmings BA. Physiological roles of PKB/Akt isoforms in development and disease. Biochem Soc Trans 35:231–235, 2007.

66.

Cho H, Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ. Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 276:38349–38352, 2001.

67.

Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB 3rd, Kaestner KH, Bartolomei MS, Shulman GI, Birnbaum MJ. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science 292:1728–1731, 2001.

68.

Garofalo RS, Orena SJ, Rafidi K, Torchia AJ, Stock JL, Hildebrandt AL, Coskran T, Black SC, Brees DJ, Wicks JR, McNeish JD, Coleman KG. Severe diabetes, age-dependent loss of adipose tissue, and mild growth deficiency in mice lacking Akt2/PKB beta. J Clin Invest 112: 197–208, 2003.

69.

Easton RM, Cho H, Roovers K, Shineman DW, Mizrahi M, Forman MS, Lee VM, Szabolcs M, de Jong R, Oltersdorf T, Ludwig T, Efstratiadis A, Birnbaum MJ. Role for Akt3/protein kinase Bgamma in attainment of normal brain size. Mol Cell Biol 25:1869–1878, 2005.

70.

Tschopp O, Yang ZZ, Brodbeck D, Dummler BA, Hemmings-Mieszczak M, Watanabe T, Michaelis T, Frahm J, Hemmings BA. Essential role of protein kinase B gamma (PKB gamma/Akt3) in postnatal brain development but not in glucose homeostasis. Development 132:2943–2954, 2005.

71.

Maroulakou IG, Oemler W, Naber SP, Klebba I, Kuperwasser C, Tsichlis PN. Distinct roles of the three Akt isoforms in lactogenic differentiation and involution. J Cell Physiol 217:468–477, 2008.

72.

Stahl JM, Sharma A, Cheung M, Zimmerman M, Cheng JQ, Bosenberg MW, Kester M, Sandirasegarane L, Robertson GP. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res 64: 7002–7010, 2004.

73.

Fujio Y, Mitsuuchi Y, Testa JR, Walsh K. Activation of Akt2 inhibits anoikis and apoptosis induced by myogenic differentiation. Cell Death Differ 8:1207–1212, 2001.

74.

Sale EM, Hodgkinson CP, Jones NP, Sale GJ. A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase. Biochemistry 45:213–223, 2006.

75.

Liu X, Shi Y, Birnbaum MJ, Ye K, De Jong R, Oltersdorf T, Giranda VL, Luo Y. Quantitative analysis of anti-apoptotic function of Akt in Akt1 and Akt2 double knock-out mouse embryonic fibroblast cells under normal and stressed conditions. J Biol Chem 281:31380–31388, 2006.

76.

Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones RE, Kahana JA, Kral AM, Leander K, Lee LL, Malinowski J, McAvoy EM, Nahas DD, Robinson RG, Huber HE. Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J 385:399–408, 2005.

77.

Green CJ, Goransson O, Kular GS, Leslie NR, Gray A, Alessi DR, Sakamoto K, Hundal HS. Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B/Akt in the insulin-dependent regulation of glucose and system A amino acid uptake. J Biol Chem 283:27653–27667, 2008.

78.

Wu Z, Hartnett JC, Neilson LA, Robinson RG, Fu S, Barnett SF, Defeo-Jones D, Jones RE, Kral AM, Huber HE, Hartman GD, Bilodeau MT. Development of pyridopyrimidines as potent Akt1/2 inhibitors. Bioorg Med Chem Lett 18:1274–1279, 2008.