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Experimental Biology and Medicine
Ronald W. Matheny, Jr. and Martin L. Adamo
Current Perspectives on Akt Akt-ivation and Akt-ions
doi: 10.3181/0904-MR-138
2009, 234:1264-1270.Experimental Biology and Medicine
http://ebm.rsmjournals.com/content/234/11/1264
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MINIREVIEW
Current Perspectives on Akt Akt-ivation
and Akt-ions
RONALD W. MATHENY JR.AND MARTIN L. ADAMO
1
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio,
Texas 78229; and The Sam and Ann Barshop Institute for Longevity and Aging Studies, University of
Texas Health Science Center at San Antonio, San Antonio, Texas 78229
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. Exp Biol Med
234:1264–1270, 2009
Key words: Akt; growth; apoptosis
Introduction
Akt (also known as protein kinase B) is a serine/
threonine kinase downstream of phosphoinositide-39-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 catego-
rized into three classes by their structure, substrate
specificity, and lipid products (4). Members of the Class
IA PI3Ks (a,b, and d) are heterodimers consisting of a 110
This work was supported by NIA grant R01AG026012 to MLA. RWM was supported
by pre-doctoral award from NIA training grant T32 AG021890-08.
1
To whom correspondence should be addressed at Department of Biochemistry MSC
7760, UT Health Science Center at San Antonio, 7703 Floyd Curl Drive, San
Antonio, TX 78229-3900. E-mail: adamo@biochem.uthscsa.edu
1264
DOI: 10.3181/0904-MR-138
1535-3702/09/23411-1264$15.00
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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 autophos-
phorylation, resulting in the recruitment of adaptor mole-
cules 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 pro-
vides 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 59-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 activa-
tion 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 diagrammati-
cally.
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,
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-(c1)’’ 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.
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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. How-
ever, 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 phos-
phorylated 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 GbL (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 GbL 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
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 (PIP
2
)then
phosphorylates the D3 position of the inositol ring generating
PI(3,4,5)P3 (PIP
3
). PIP
3
then recruits PH-domain-containing mole-
cules including PDK1 and Akt to the lipid bilayer (Akt recruitment not
shown for clarity), allowing for binding to PIP
3
, and phosphorylation of
Akt at the A-loop (T308 in Akt1) by PDK1. Reconversion of PIP
3
to
PIP
2
is promoted by PTEN, which reduces available PIP
3
for PDK1
and Akt binding, thus terminating growth factor signals. Phosphor-
ylation 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.
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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 differ-
entiating 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 GSK3aand GSK3bin 3T3-L1 adipocytes (74). Addi-
tionally, knockdown of individual Akt isoforms in H157
cells demonstrated distinct roles for Akt3 acting on p27 and
Akt2 acting on GSK3a, but redundant roles for all three Akt
isoforms in regulating FoxO1, GSk3b, 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 demon-
strate that Akt isoforms possess both distinct and function-
ally 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 mem-
branes 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
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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.
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