Biochem. J. (2008) 415, 333–344 (Printed in Great Britain)
333
doi:10.1042/BJ20081056
REVIEW ARTICLE
The life of a cell: apoptosis regulation by the PI3K/PKB pathway
Vincent DURONIO1
Department of Medicine, University of British Columbia, Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6, and Immunity and Infection Research Centre,
Vancouver Coastal Health Research Institute, 100–2647 Willow Street, Vancouver, BC, Canada, V5Z 3P1
The activation of PI3K (phosphoinositide 3-kinase) family
members is a universal event in response to virtually all cytokines,
growth factors and hormones. As a result of formation of
PtdIns with an added phosphate at the 3 position of the inositol
ring, activation of the protein kinases PDK1 (phosphoinositidedependent kinase 1) and PKB (protein kinase B)/Akt occurs.
The PI3K/PKB pathway impinges upon a remarkable array of
intracellular events that influence either directly or indirectly
whether or not a cell will undergo apoptosis. In this review, the
INTRODUCTION
Historical overview
It is well known that investigations of the pathways that are
regulated following activation of the PI3K (phosphoinositide 3kinase) family of enzymes has been one of the most intensively
studied set of signalling events in recent years. In the mid-tolate 1980s, investigators began observing phosphoinositide kinase
enzymatic activity that modified a specific site on PtdIns, showing
that it was associated with tyrosine-phosphorylated proteins.
Much of this work was initiated in the laboratories of Tom Roberts
and Lewis Cantley [1–3]. In 1988, Whitman et al. [4] provided
biochemical proof that the kinase activity was able to specifically
phosphorylate the 3 position on the inositol ring, thereby initiating
the era of PI3K investigations. Subsequent characterization and
molecular cloning revealed that a p110 catalytic subunit was
bound with a regulatory subunit, p85, containing SH2 (Src
homology 2) domains, which was responsible for the association
with tyrosine-phosphorylated proteins that initiated the activation
of PI3K activity. The p85 subunit, which was later shown to be
one of several forms of regulatory subunit, associated with and
regulated one or more of the several p110 subunits responsible
for the catalytic activity.
Studies over several years revealed that the PI3K family of
PtdIns-modifying enzymes consists of at least nine genes in
mammalian systems, corresponding to various isoforms that are
categorized into class I, II and III. This family has been reviewed
extensively elsewhere [5]. For the subject of this review, the class
I subgroup is of most relevance when discussing growth-factor- or
cytokine-activated PI3K, and its regulation of cell survival. The
mechanism by which PI3K enzymes function, and particularly
many ways in which PI3K/PKB can control these processes are
summarized. Not all of the events described will necessarily play
a role in any one cell type, but a subset of these events is probably
essential for the survival of every cell.
Key words: apoptosis, Bcl-2, nuclear factor κB (NF-κB),
phosphoinositide-dependent kinase 1 (PDK1), phosphoinositide
3-kinase (PI3K), protein kinase B (PKB).
their regulation of downstream kinases such as PKB (protein
kinase B)/Akt (which will be referred to as PKB in this review),
will be briefly described here, and the major emphasis will be the
impact of these events on promotion of cell survival and inhibition
of apoptosis. To help those not familiar with this field, some key
abbreviations are summarized in Table 1.
PI3K/PKB REGULATION
Class I PI3Ks
Unlike some of the other PI3K isoforms, the class I subgroup
of PI3Ks is thought to exclusively phosphorylate PtdIns(4,5)P2
to generate PtdIns(3,4,5)P3 in vivo, even though these enzymes
can utilize other lipids such as PtdIns and PtdIns4P when
assayed in vitro. The class I enzymes consist of p110α, p110β,
p110γ and p110δ catalytic subunits. The α, β and δ are
referred to as class IA and associate with regulatory subunits
of the p85 type, which contain SH2 domains. Following activation of tyrosine kinases, the SH2 domains of the p85 subunits can
mediate activation of PI3K following their binding to a consensus
YXXM motif when the tyrosine residue is phosphorylated [6].
In an alternative pathway of class IA PI3K activation, activated
p21ras has been shown to directly bind and activate the p110
catalytic subunits [7]. The potential importance of the Ras-mediated regulation of PI3K was reinforced recently in Downward’s
laboratory, using a murine model to show that Ras-driven
tumorigenesis requires the Ras-binding site on p110α [8].
The less-well-characterized p110γ isoform of PI3K associates
with a regulatory subunit known as p101, and is activated
in response to GPCR (G-protein-coupled receptor) stimulation
Abbreviations used: BH3, Bcl-2 homology domain 3; DNA-PK, DNA-dependent protein kinase; eIF-4E, eukaryotic initiation factor 4E; 4E-BP, eIF-4Ebinding protein; ER, endoplasmic reticulum; ERK, extracellular-signal-regulated kinase; FoxO3a, forkhead box O 3a; GPCR, G-protein-coupled receptor;
GSK-3, glycogen synthase kinase 3; Hsp27, heat-shock protein 27; IAP, inhibitor of apoptosis protein; IκB, inhibitor of nuclear factor κB; IKK, IκB
kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; Mdm2, murine double minute 2; Mlc-1, myeloid cell leukaemia-1; MLK-3, mixed-lineage kinase
3; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor κB; PDCD4, programmed cell death protein 4; PDGF, platelet-derived growth factor;
PDK1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKB, protein kinase B;
PKC, protein kinase C; PUMA, p53-up-regulated modulator of apoptosis; raptor, regulatory associated protein of mTOR; SH2, Src homology 2; SHIP,
SH2-containing inositol phosphatase; TCL1, T-cell leukaemia 1; TOR, target of rapamycin; TSC, tuberous sclerosis complex.
1
email [email protected]
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334
Table 1
V. Duronio
Abbreviations
As with any area of signal transduction, three letter abbreviations are the usual means of naming the components. This list attempts to explain the meaning behind the abbreviations.
Abbreviation
Meaning
Comments (or synonyms)
Bad
Bak
Bax
Bcl-2
Bcl-XL
Bim
FoxO
GPCR
GSK-3
IκB
IKK
Mcl-1
Mdm2
NF-κB
p27 kip1
p53
p70 S6K
PI
PI3K
PI(3,4,5)P3
PDK1
PKB
PUMA
Rheb
SH2
SHIP
TOR
TSC
Bcl-2/Bcl-XL -associated death domain protein
Bcl-2 homologous antagonist-killer protein
Bcl-2 associated X protein
B-cell lymphoma-2
Bcl-2-related gene, long form
Bcl-2-interacting mediator of cell death
Forkhead box, class O proteins
G-protein-coupled receptor
Glycogen synthase kinase-3
Inhibitor of NF-κB
IκB kinase
Myeloid cell leukaemia-1
Murine double minute 2
Nuclear factor-κ type B
27 kDa protein/kinase inhibitor protein 1
53 kDa protein
70 kDa S6 kinase
Phosphatidylinositol
Phosphoinositide 3-kinase
Phosphatidylinositol 3,4,5-trisphosphate
Phosphoinositide-dependent kinase 1
Protein kinase B
p53-up-regulated modulator of apoptosis
Ras homologue enriched in brain
Src homology 2
SH2-domain-containing inositol 5-phosphatase
Target of rapamycin
Tuberous sclerosis
BH3-only member of the Bcl-2 family
Multi-BH domain pro-apoptosis protein
Multi-BH domain pro-apoptosis protein
Defining member of the family; originally characterized as an oncogene
Bcl-XS is a shorter splice variant that is pro-apoptotic
BH3-only member of the Bcl-2 family
FKHR (forkhead in rhabdosarcoma); members of the winged helix or forkhead family of transcription factors
Serpentine receptor; contain seven transmembrane domains
Kinase activity suppressed after phosphorylation by PKB
Interacts with NF-κB
Phosphorylates IκB to promote its degradation
Originally identified as up-regulated upon monocyte differentiation of ML-1 cells
Gene amplified in mouse 3T3DM cells
Originally linked with enhancement of immunoglobulin κ light chain gene
Member of the family of cyclin-dependent kinase inhibitors (CDKIs)
Tumour-suppressor protein
Phosphorylates ribosomal S6 subunit
Five hydroxy groups on the inositol ring can be phosphorylated; PtdIns
Phosphatidylinositol 3-kinase; PI 3-kinase; PtdIns3K
Designation for PI phosphorylated at 3,4, and 5 positions of the inositol ring; PtdIns(3,4,5)P 3
Like PKB, has a PH domain that binds phosphorylated PI lipids
Akt; RACK, or related to A and C kinase; has PH domain
BH3-only member of the Bcl-2 family
Small G-protein required for activation of TOR
Protein domain that binds phosphorylated tyrosine sites
Dephosphorylates the 5-phosphate from PI(3,4,5)P3
Protein kinase regulator of downstream kinases, including p70S6K
Tuberous sclerosis complex; TSC1 (hamartin) and TSC2 (tuberin) are genes linked to this disorder
[9,10]. To date, the exact molecular mechanism involved in
regulation by the p101 subunit is not fully defined, but it has
been shown to have sites of interaction with βγ subunits of the
trimeric G-proteins [11]. On the other hand, earlier studies suggested that, in vitro, the βγ subunits may activate p110γ directly
[12]. Knockout studies showed that the function of p110γ was
most important for T-cells and neutrophils, and was essential
for GPCR-mediated generation of PtdIns(3,4,5)P3 in those cells
[13–15]. The p110α and p110β forms of PI3K have been shown to
be essential for normal mammalian development, as has the p85α
gene [16]. Interestingly, loss of the p85β subunit has a milder
phenotype, resulting in increased sensitivity to insulin receptor.
Therefore, although there are clearly unique roles for the various
isoforms of PI3K, it has also become clear that the family of genes
plays a very important role in normal mammalian development.
Key functions for the various PI3K enzymes have been demonstrated in numerous model systems, utilizing cellular responses
to many different agonists. In fact, virtually every hormone and
growth factor that has ever been investigated has been shown
to have some effect on PI3K activity. There are two major lipid
products of class I PI3K: PtdIns(3,4,5)P3 results from phosphorylation of PtdIns(4,5)P2 , whereas PtdIns(3,4)P2 is a product of 5phosphatases, such as SHIP (SH2-containing inositol phosphatase)-1 or SHIP-2, removing one phosphate residue from the 5
position on the inositol ring of PtdIns(3,4,5)P3 [17]. The mechanism by which the lipids could mediate their cellular effects
was clarified by the identification of PH (pleckstrin homology)
domains in a variety of proteins as docking modules for these
lipids [18]. Thus the lipid products of PI3K action can be considered second messengers in a classical sense, being the internal
chemical signal that is generated in response to an external signal.
The basis by which they transmit information is to orchestrate the
c The Authors Journal compilation c 2008 Biochemical Society
recruitment of cytosolic proteins to the plasma membrane where
they can dock to the polyphosphorylated lipids. As will be clear
from the more detailed discussion of PKB regulation below, the
association of an enzyme at the membrane may serve as the means
by which it becomes activated, and further propagation of the
signal can be transmitted to the various substrates of the enzyme,
which may be at the membrane, in the cytosol or in the nucleus.
PI3K, PDK1 (phosphoinositide-dependent kinase 1) and PKB
The initial signalling cascade that occurs following PI3K activation (Figure 1) sets in motion the activation of one of
the key kinases acting downstream of PI3K at the plasma
membrane, PDK1 [19]. Interestingly, PDK1 has a PH domain
that allows it to bind to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 , and
thus to be bound to the plasma membrane at sites of PI3K
activation, but lipid binding to PDK1 does not appear to be
required for activation of the enzyme. PDK1 has an activating
phosphorylation site that is likely to be a site of autophosphorylation [20]. The activity of PDK1 is directed at the activating loop
phosphorylation site of virtually all kinases of the AGC family,
which includes PDK1 itself, as well as PKB, PKC (protein kinase
C) isoforms and p70 S6 kinase. Even before the characterization
of PDK1, the dependence of PKB activity on upstream regulation
by PI3K had been demonstrated in numerous studies [21–27].
Perhaps most critically for PI3K-dependent cell survival, when
both PDK1 and PKB are recruited to the plasma membrane via
their PH domains (Figure 1), the kinase activity of PDK1 was
shown to phosphorylate the Thr308 site on PKB, dependent upon
the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2 [19,28,29].
The AGC kinase family also encodes a hydrophobic motif that
must either be phosphorylated to activate the kinase fully, or, in
Apoptosis regulation by the PI3K/PKB pathway
Figure 1
335
Overview of the key function of PI(3,4,5)P 3 and PtdIns(3,4)P 2 in regulation of PKB and downstream substrates in both cytoplasm and nucleus
TKR, tyrosine kinase-type growth factor receptor.
some cases, has a charged residue at a site corresponding to the
phosphorylation site [27]. The charged site in the hydrophobic
motif, which is the phosphorylated Ser473 residue in PKB, allows
intramolecular binding to occur, resulting in activation of these
kinases. Although earlier studies had suggested that PDK1 might
also serve to phosphorylate Ser473 , many lines of evidence have
now shown that a distinct kinase activity is responsible for what
is often referred to as ‘PDK2’ activity [30]. In particular, it was
shown that in PDK1-knockout cells, Ser473 phosphorylation is
retained [31]. Recent work has demonstrated that TOR (target
of rapamycin), when bound with the adaptor protein rictor
[rapamycin-insensitive companion of mTOR (mammalian TOR)],
which makes it rapamycin-insensitive, functions as a Ser473 kinase
[32,33]. In previous studies, other kinases that may phosphorylate
Ser473 have been proposed, including ILK (integrin-linked kinase)
[34], DNA-PK (DNA-dependent protein kinase) [35] or even PKB
itself acting by autophosphorylation [36]. The initial report of
DNA-PK as a Ser473 kinase did not provide a possible explanation
of the potential physiological functions, but Bozulic et al. [37]
have now reported that PKB is activated in a DNA-PK-dependent
manner following double-strand breaks. Not only was DNAPK required for Ser473 phosphorylation, but also this study
showed that PDK1 was necessary for the observed Thr308 phosphorylation of PKB following irradiation of cells to induce DNA
damage [37]. Intriguingly, the activation of PKB was correlated
with pro-survival function, which could be a necessary event in
allowing repair of damaged DNA to proceed. Another important
aspect of this study is that it may explain the important function
previously attributed to nuclear localized PDK1 [38]. Nuclear
PI3K-generated PtdIns lipids have not been described, and thus
PDK1 and PKB in the nucleus probably function independently
of the PtdIns lipids.
The difficulties in defining the precise events involve in phosphorylation of PKB at the hydrophobic domain also extends to
our understanding of how the PI3K lipid products are involved in
the regulation of that event. The above-mentioned study suggests
that PDK1 and DNA-PK-dependent regulation of PKB, at least
under specific circumstances, may proceed independently of PI3K
activation [37]. However, it is clear that, under most conditions,
inhibition of PI3K potently inhibits phosphorylation of both
Thr308 and Ser473 sites on PKB. In considering the more classical
PI3K-dependent events, our laboratory has demonstrated that,
although Thr308 phosphorylation is most closely tied with the
levels of PtdIns(3,4,5)P3 [or a combination of PtdIns(3,4,5)P3
and PtdIns(3,4)P2 ], phosphorylation of Ser473 is linked to the
levels of PtdIns(3,4)P2 only [39,40]. Furthermore, the levels of
PtdIns(3,4)P2 and Ser473 phosphorylation were found to be more
accurate predictors of PKB activation state than PtdIns(3,4,5)P3
and Thr308 phosphorylation [40]. The crystal structure of PKB has
demonstrated that its PH domain can bind either PtdIns(3,4)P2 or
PtdIns(3,4,5)P3 , since the phosphate at the 5-position does not fit
into the binding pocket [41]. Thus either of the two lipids can serve
to recruit PKB to the plasma membrane. On the other hand, none
of the reported PKB Ser473 kinases, with the exception of PKB
itself, has been linked directly to the level of PtdIns(3,4)P2 . Thus
it is tempting to speculate that PKB autophosphorylation at Ser473
may be regulated either directly or indirectly by PtdIns(3,4)P2
levels, but more work will certainly be required to define the
molecular events involved.
Although PKB translocates to the plasma membrane following
activation of PI3K, as a result of the elevated levels of 3phosphorylated PtdIns lipids, targets of PKB kinase activity are
present in the cytosol and nucleus, as has been shown in many
studies, and will become evident from the discussion below. PKB
associates with a number of proteins that may regulate its activity
and/or localization, one of which is the TCL1 (T-cell leukaemia 1)
family of proteins, which are small binding proteins with unknown
molecular functions. TCL1 works to enhance PKB activity and
has oncogenic properties that are likely to be related to its function
in regulating PKB. The TCL1–PKB complex forms at the cell
membrane, but it has also been shown to be transferred to the
nucleus [42,43]. The potential importance of TCL1 in nuclear
localization of PKB in early stages of embryo development has
been suggested recently [44]. As noted above, nuclear localization
of PDK1 has also been reported [38], which allows an additional
layer of regulation of PKB and its downstream nuclear targets.
Another recent study proposed yet another potential regulatory
event that may control PKB release from the plasma membrane:
calmodulin/Ca2+ was shown to directly bind to the PH domain of
PKB, which would potentially enhance the release of active PKB
from membranes, or prevent its re-association with membranes
[45].
In summary, we now have a relatively detailed understanding
of the molecular events involved in regulation of these two protein
kinases that are controlled by key lipid products of PI3K action.
Some of the details of PKB phosphorylation, and the complete
details of the full scope of PDK1’s functions, remain to be fully
explored, both at the plasma membrane and in the cytoplasm and
nucleus. Clearly the vast majority of PKB’s targets are not at
the plasma membrane and thus the cytosolic and nuclear targets
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336
Table 2
V. Duronio
Specific phosphorylation sites regulated by PI3K/PKB action
(a) Targets phosphorylated directly by PKB
Target protein
Site(s)
Outcome
Reference(s)
Bad
Bax
FoxO3a
GSK-3α
GSK-3β
IKK-α
Mdm2
TSC2
Ser136
Ser184
Thr32 /Ser253 /Ser315
Ser21
Ser9
Thr23
Ser166 /Ser186
Ser939 /Ser1086 /Ser1088 /Thr1462
Promotes association with 14-3-3
Suppress Bax function
Promote nuclear export and binding to cytosolic 14-3-3 proteins
Inhibits kinase activity
Inhibits kinase activity
Increases IKK activity
Promote nuclear localization of Mdm2 and suppression of p53
Suppresses TSC2 function and thus allows activation of p70S6K
[69–72]
[83]
[91–94]
[125,126]
[125,126]
[117,118]
[163]
[166–168]
(b) Targets phosphorylated downstream of PI3K/PKB action
Target protein
Site
Direct kinase; outcome
Reference(s)
IκB
P70S6K
4E-BP1
4E-BP1
Mcl-1
S6
Ser32 /Ser36 (α), Ser19 /Ser23 (β)
Thr252 /Thr412
Ser65 /Thr70
Thr37 /Thr46
Ser159
Ser235
IKKα/β; promotes degradation
PDK1
mTOR; regulates dissociation from eIF4E
mTOR; priming sites
GSK-3; promotes degradation of Mcl-1
p70S6K ; enhances protein translation
[169–171]
[172]
[147,173]
[174]
[132,133]
[144,149,175]
of PKB that are playing a role in apoptosis regulation will be
discussed in greater detail below.
FUNCTIONS OF PI3K/PKB IN APOPTOSIS REGULATION
It is now well accepted that activation of PI3K, and downstream
activation of PKB, play critical roles in keeping cells alive by
blocking apoptotic pathways. Apoptosis refers to the complex
set of events that can be initiated within a cell that eventually
leads to cellular destruction initiated by proteolytic enzymes
known as caspases [46,47]. The onset of apoptosis has been well
studied, and many of the key pathways are now well understood.
A detailed summary of the many possible ways in which cells
undergo apoptosis is beyond the scope of this review, but has been
reviewed extensively elsewhere [48–51]. As described below,
there are many different regulatory events involved specifically
in promoting cell survival that are modulated as a result of
PI3K/PKB activation. The PI3K/PKB pathway can block many
pro-apoptotic proteins, or have a positive effect on multiple prosurvival components, several categories of which are briefly
summarized in Figure 2. Some of the specific phosphorylation
sites affected by PKB activation are summarized in Table 2.
In one of the simplest explanations of the events that are
regulated by PI3K/PKB, it can be considered that in the absence
of growth factors or cytokines to provide a pro-survival or antiapoptotic signal, numerous cellular systems, including protein
synthesis and cell-cycle regulation, become disrupted. Either as
a direct consequence, or acting in parallel, potential pro-apoptotic proteins may be increased in their level of expression, or
they become activated as a result of the loss of suppressive phosphorylation events. In some cases, maintenance of normal levels
of anti-apoptotic proteins in a healthy living cells requires direct,
or indirect, regulation by the PI3K/PKB pathway. It may also
be obvious that the maintenance of normal levels of protein
synthesis, and/or cell-cycle progression, is required for the cell
to avoid entering a state whereby apoptosis becomes one of the
options to be pursued. In the remaining sections of this review,
the many downstream events regulated by PI3K/PKB will be
summarized, and they will be placed into broad categories of
functional readouts. Although not all of the events occur in all
cell types to the same extent, it is likely that some subset of these
c The Authors Journal compilation c 2008 Biochemical Society
Figure 2 Summary of the key processes regulated in the cytoplasm, at the
mitochondria, in the nucleus, or extracellularly, by the PI3K/PKB pathway in
controlling apoptosis
The green colour denotes pro-survival events that are positively regulated either directly or
indirectly by PI3K/PKB, whereas the red colour denotes pro-apoptosis events that are negatively
regulated.
events must be operating within any given cell in order for that
cell to remain viable.
PI3K signalling as an essential anti-apoptotic event
The first evidence suggesting that one of the important functions
of PI3K might be to maintain cell survival, or to avoid undergoing cell death by apoptosis, appeared in two publications in
1995 [52,53]. Yao and Cooper [52] showed that, in rat phaeochromocytoma PC12 cells, blocking PI3K by wortmannin or
LY294002 caused death of cells, and that PDGF (platelet-derived
growth factor)-dependent survival required a PDGF receptor that
could activate PI3K activity. On the other hand, p21ras , which is
another major signal transducer activated by most growth factors
and largely working via the ERK (extracellular-signal-regulated
Apoptosis regulation by the PI3K/PKB pathway
Figure 3
337
Target substrates of PI3K/PKB whose pro-apoptotic activities are suppressed by phosphorylation
As described in the text, the PI3K/PKB pathway can have multiple pro-survival effects that impinge on almost every aspect of apoptosis regulation, including mitochondrial and death receptor
pathways, regulation of cell cycle, regulation of transcriptional and protein translation events. In this schematic diagram, the positive events controlled either directly or indirectly by PI3K/PKB are
indicated by arrowheads, whereas block arrows represent phosphorylation events that have inhibitory effects.
kinase) 1/2 protein kinases, was shown to be required for neuronal
cell differentiation, but not survival of PC12 cells, based on cells
expressing a dominant-negative form of Ras. In our laboratory,
we utilized IL (interleukin)-3-dependent MC/9 cells to show
that inhibition of PI3K caused the death of these cells, although
these cells did not die when they were incubated with PI3K
inhibitors in the presence of GM-CSF (granulocyte/macrophage
colony-stimulating factor) [53]. The difference in responses in
the presence of the two related cytokines may be a peculiarity
of this cell type, but was not due to signalling via the p21ras /ERK
pathway.
PI3K function in promoting cell survival subsequently became
a very important area of research, and cytokine-dependent haemopoietic cells have been used in many of the studies [54,55].
Haemopoietic cell survival (i.e. apoptosis inhibition) had long
been known to depend on specific cytokines [56–58], and thus
these in vitro cell models provided an ideal opportunity to
study processes that were activated by cytokines to promote cell
survival. At the early stages of this research, the role of PI3K
as a survival-promoting event quickly became accepted, but the
specific events being regulated in the pathway were the next focus
of interest.
Recognition of PKB as a key mediator of PI3K-dependent survival
Before identification of PDK1, and concurrent with PI3K being recognized as a key regulator of cell survival, one of the key protein
kinases to be activated in a PI3K-dependent manner was shown
to be PKB or RACK (related to A and C kinase), which was
discovered based on its similarity to both PKA (protein kinase A)
and PKC [59,60]. This protein kinase was discovered independently as a retroviral oncogene referred to as akt [61]. The
connection between PI3K and PKB was established in several independent studies, first using the PI3K inhibitor wortmannin, but
eventually validating the connection by various alternative means
of activating and inhibiting PI3K and/or PKB ([21,22,62,63],
reviewed in [23,24,64]). As noted above, details of PI3K-mediated
regulation of PKB have subsequently been established. The next
obvious direction in this field came with the demonstration that
activation of PKB was a key event in the promotion of cell
survival that was dependent upon PI3K [23,65]. An immediately
obvious consequence of this knowledge was that it at least partially
explained how v-akt could be functioning as an oncogene.
Much of the attention in this field was initially focused on
PKB-mediated phosphorylation of the pro-apoptotic protein Bad.
This was not surprising, since at the same time that PKB was
being recognized as a ‘survival kinase’, Stan Korsmeyer’s group
was delineating the molecular details of Bad’s apoptotic activity
[66,67], and showed that this activity could be suppressed by
phosphorylation [68]. As will be discussed below, Bad is one of a
number of Bcl-2 family proteins that can be regulated, in either a
positive or negative sense, by the PI3K/PKB pathway. However,
there are also many other events regulated by PI3K/PKB that
play a role in apoptosis regulation, as summarized in Figure 3,
and these will be described in more detail in the following
sections.
PI3K/PKB targeting of Bcl-2 family proteins
In a series of publications that appeared within months of each
other, it was shown that PKB could mediate phosphorylation
of Bad at Ser136 , and this phosphorylation served to inactivate
Bad activity by promoting Bad’s association with cytosolic
14-3-3 proteins [69–72]. However, we came to the opposite
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338
V. Duronio
conclusion in studies of endogenous levels of PKB and Bad in
haemopoietic cells. We demonstrated that PI3K/PKB-dependent
survival proceeded independently of the phosphorylation of Bad
[73]. One explanation of this difference might be due to the
focus in most of the early studies on the use of overexpressed
active PKB and/or Bad [69,71,72,74–76], which may not reflect
the normal physiological events. In fact, we have since shown
that Bad could be phosphorylated by PKB in the cell types we
were studying when either PKB or Bad was overexpressed [77].
Thus Bad may not be considered a key PKB target, and others
have come to similar conclusions [78,79]. However, it must
be pointed out that many studies in the literature have also
provided compelling evidence that endogenous levels of Bad
may be regulated by PI3K/PKB-mediated phosphorylation and
thus this pathway may be important in specific cell types. Also,
many other kinases are known to phosphorylate Bad at other
sites, including PKA [80,81] and p90rsk [82], and thus Bad’s
activity in cells is normally very well suppressed. Most recently,
our laboratory has identified the γ isoform of Ca2+ /calmodulindependent protein kinase II (CaMKIIγ ) as yet another regulator of
Bad’s pro-apoptotic activity (P. Hojabrpour, I. Waissbluth and V.
Duronio, unpublished work). When one considers the relatively
limited expression of Bad, and the fact that the Bad-knockout
mice developed normally and had a very mild phenotype, it might
be expected that the profound pro-survival effect of PI3K/PKB
must be functioning through other pathways.
Another important Bcl-2 family protein regulated by the PI3K/
PKB pathway is the pro-apoptotic protein Bax which, when
inserted into the mitochondria, is a key regulator of mitochondrial
permeability leading to apoptosis. Bax was reported to be
phosphorylated at an inhibitory site, Ser184 near the C-terminus, by
PKB, which contributed to suppression of Bax-mediated death of
neutrophils [83]. A subsequent study reported that PP2A (protein
phosphatase 2A)-mediated dephosphorylation at this Ser184 site
on Bax, utilizing several different approaches, contributed to
activation of Bax [84]. On the other hand, the latter group also
showed that phosphorylation of Bax at Ser184 could be mediated by
a PKCζ -dependent event [85]. GSK-3 (glycogen synthase kinase
3), which is inhibited by PKB-mediated phosphorylation (see
below), has also been reported to phosphorylate Ser163 on Bax, a
modification that was reported to promote translocation of Bax
to the mitochondria [86]. In both cases, the mechanism by which
the phosphorylation of Bax may alter either its associations or
its conformation remain unclear, but they point to two possible
ways in which PKB can suppress the apoptotic activity of
Bax: by inhibiting it directly by phosphorylation at Ser184 , or
by suppressing Ser163 phosphorylation due to PKB-mediated
inhibition of GSK-3 activity.
A more recent study investigated the role of the stress-induced
heat-shock protein, Hsp27, in regulating the activity of Bax [87].
It was suggested that PKB may be involved in suppression
of Bax function, based on an effect of stress-induced Hsp27
causing PKB activity to be maintained. Although not providing
a precise molecular explanation for these events, this study has
suggested one means by which Hsp27 may function in apoptosis
regulation. Many previous studies have suggested that stressresponse proteins such as Hsp27 can provide protection against
apoptosis in many other contexts [88–90].
Other Bcl-2 family proteins can also be regulated by the PI3K/
PKB signalling pathway, and these will be highlighted in some
of the following sections. In particular, regulation of the forkhead
pathway can suppress expression of pro-apoptotic members
Bim and PUMA (p53-up-regulated modulator of apoptosis),
whereas the NF-κB (nuclear factor κB) survival pathway can
promote expression of Bcl-XL .
c The Authors Journal compilation c 2008 Biochemical Society
PI3K/PKB and forkhead transcription factors
A very important set of targets of PKB with respect to apoptosis
regulation are the forkhead transcription factors [91–94]. Their
phosphorylation leads to sequestration and degradation in the
cytoplasm [93], thus their activity increases when PI3K/PKB is
not active, causing increases in multiple potential regulators of
cell death, including the Fas death receptor ligand, FasL [95], as
well as the BH3 (Bcl-2 homology domain 3)-only protein Bim,
and the cell cycle inhibitor, p27kip1 [96–99]. As also shown in
a number of studies, increases in p27kip1 and the pro-apoptotic
Bcl-2 family member, Bim, as a result of FoxO3a (forkhead
box O 3a) activation, were key events in apoptosis resulting
from cytokine deprivation [96–99]. More recent studies have
suggested that cytokine-starved T-cells undergo apoptosis as a
result of up-regulation of another apoptotic Bcl-2 family member,
PUMA [100,101]. PUMA is a p53-dependent target, but the study
from Mak’s laboratory suggested it may also be regulated in a
FoxO3a-dependent manner [101]. Even more surprising was
a study suggesting that PUMA was a key mediator of cell death
following IL-3 withdrawal and that PI3K-dependent survival was
independent of any effects on Bim, Bad or PUMA [79]. We have
investigated PUMA expression in our cytokine-dependent cells,
and we found no increase in total PUMA expression following
cytokine starvation (J. Anthony and V. Duronio, unpublished
work), although we have seen FoxO3a-dependent up-regulation
of Bim in the same cells [99]. Thus PUMA may not always be
increased when FoxO3a is activated, or at least not in all cell types.
Bim and PUMA are two of the BH3-only Bcl-2 family proteins
that are thought to promote apoptosis as a result of binding to
the pro-survival family members [102]. However, Bim has also
been reported to mediate direct activation of Bax [50], while
PUMA function has been reported to be due to its association
with Bcl-XL and p53 [103]. In several of the studies cited above
investigating the role of PUMA and Bim in apoptosis due to
cytokine deprivation, much of the evidence came from studies
of cells from knockout animals. Under these developmentally
altered conditions, interpretation of the function of the proteins
may be biased by differential expression of other genes. Thus
analysis of Bim and PUMA using cell lines or primary cells expressing endogenous levels of the proteins, together with cells
altered by overexpression or knockdown, may yield more concrete
answers.
PI3K/PKB and NF-κB
The role of NF-κB in regulating multiple genes involved in cell
survival is now well known [104], and forms the basis upon
which NF-κB alterations may play a role in development of many
types of cancer, as has been reviewed extensively [105,106]. NFκB has been shown to be an important regulator of many antiapoptotic or pro-survival genes, perhaps the most important of
which are the anti-apoptotic proteins Bcl-2 and Bcl-XL [107–
113]. In addition, the inhibitor of caspase 8, FLIP {FLICE [FADD
(Fas-associated death domain)-like IL-1β-converting enzyme]inhibitory protein}, which is a natural inhibitor of the death
receptor pathways, can be induced by NF-κB [114]. Other
inhibitor of apoptosis proteins, or IAPs, have been shown to
function in cell survival by blocking multiple caspases; expression
of several of the IAPs can be regulated by NF-κB [115]. It is
generally accepted that these probably represent only a subset
of the genes induced by NF-κB that may play either a direct or
indirect role in promoting cell survival.
The regulation of NF-κB transcription factor activity occurs
primarily by regulation of its associated inhibitory molecule, IκB
Apoptosis regulation by the PI3K/PKB pathway
(inhibitor of NF-κB) [116]. IκB is phosphorylated by a specific
kinase complex consisting of IKK (IκB kinase) α and IKKβ,
which targets the protein for ubiquitination and degradation,
thereby releasing the active transcription factor. IKKα can be
activated by multiple kinases, one of which is PKB [117,118].
Thus IKK-mediated phosphorylation of I-κB promotes release of
the IκB from the complex of p50 and p65 subunits of NF-κB, and
IκB is degraded in a proteasome-dependent manner. The PI3K/
PKB pathway thus provides one of the multiple ways in which
NF-κB activity can be elevated in cells.
PI3K/PKB/GSK-3
A well known target of PKB is the kinase GSK-3, a component of
the Wnt pathway [119–121]. GSK-3α and GSK-3β activity have
been shown to be negatively regulated by numerous signalling
pathways, with involvement in multiple regulatory endpoints
[122]. One of the best characterized effects of GSK-3 in regulation
of cellular function is via its phosphorylation and inhibition
of β-catenin. When GSK-3 is active, it promotes degradation of
β-catenin and thus inhibits its transcriptional effects, which are
mediated by its co-activating role on the Lef (lymphoid enhancer
factor)/Tcf (T-cell factor) transcription factors, and include effects
on many growth-promoting genes [123]. In addition, GSK-3
activity appears to play a central role in signalling pathways
regulating developmental processes, as well as EMT (epithelial–
mesenchymal transition), which is known to be involved in
development of some cancers [124].
The activity of GSK-3 is normally suppressed in proliferating
cells by phosphorylation mediated by PKB [125,126], or a
number of other possible kinases [127,128], at an N-terminal
serine residue (Ser21 in GSK-3α, and Ser9 in GSK-3β). Therefore
loss of PKB activity may allow GSK-3 activity to be increased. As
a result, it may not be surprising that increased levels of apoptosis
can be observed in cells in which active forms of GSK-3 are expressed, whereas a dominant-negative form of GSK-3 can inhibit apoptosis, as shown by Pap and Cooper [129]. Since that
initial report, there have been several possible ways in whichGSK-3 has been suggested to contribute to cell death. One was
in regulation of pathways leading to JNK (c-Jun N-terminal
kinase), via activation of the mixed-lineage kinase, MLK-3 [130].
In neuronal cells deprived of growth factors, MLK-3 phosphorylation by GSK-3 correlated with JNK activation and
cell death. Inhibition of GSK-3 has also been shown to
block apoptosis in growth-factor-deprived neuronal precursor
cells [131], again supporting the pro-apoptotic effect of
GSK-3 activity. A recent report suggested that GSK-3 could
also be involved in promoting death of IL-3-dependent FL-5
haemopoietic cells due to its ability to phosphorylate Mcl-1 (myeloid cell leukaemia-1) and promote Mcl-1 degradation [132],
and similar events were also described in fibroblast cells [133].
Interestingly, in our laboratory, we have compared the ability of
an inhibitor of GSK-3 activity to block apoptosis in FL-5 cells
(as shown by Maurer et al. [132]) with the effects in FDC-P1
cells and primary murine macrophages. Although the FL-5 cells
responded as expected, with the GSK-3 inhibitor blocking
starvation-induced apoptosis, there was no effect of GSK-3
inhibition on apoptosis, nor on the stability of Mcl-1, in the FDCP1 cells, and only a partial effect in the macrophages (S. Jamil,
S. W. Wang and V. Duronio, unpublished work). Interestingly, we
had shown in an earlier study that, in the factor-dependent cells
we had tested, phosphorylation of GSK-3 was not dependent
on PI3K/PKB [134]. Therefore this pro-survival effect of PKB,
acting to maintain Mcl-1 expression by inhibition of GSK-3
activity, may only operate in specific cell types.
339
PI3K/PKB regulation of protein synthesis
The importance of PI3K in cell growth and cancer is now well established [135–138] and is also supported by the key role of PTEN
(phosphatase and tensin homologue deleted on chromosome 10),
the PI3K antagonist, that acts as a tumour-suppressor gene
[139,140]. Many studies support a role for PI3K signalling in cell
growth, including protein synthesis regulation and/or cell size via
TSC (tuberous sclerosis complex) 1/2–Rheb–TOR–raptor (regulatory associated protein of mTOR)–p70S6K [141]. Previous
studies have solidified the placement of TSC1/2 as direct
downstream targets of PKB, which lie directly upstream of TOR,
in a pathway originally described in Drosophila [142]. In this
complex set of regulatory events (reviewed in [143,144]), TSC1
and TSC2 (known as hamartin and tuberin respectively) form
a dimer that has a Rheb GTPase activation function that
inhibits the Rheb GTPase. Rheb activity is critical for activating
the kinase activity of TOR, when complexed to its associated
protein, raptor. Thus inhibitory phosphorylation of TSC2 by
PKB allows Rheb to activate TOR and p70S6K . Interestingly, a
recent report has suggested that PKB-mediated phosphorylation
of TSC2 can regulate nuclear/cytoplasmic localization of these
proteins and thus there may be a role for regulation of nuclear as
well as cytoplasmic p70S6K [145].
There are multiple targets within the protein translational machinery that are phosphorylated by p70S6K and PKB. Perhaps the best
known of these are eIF-4E (eukaryotic initiation factor 4E) and
the 4E-BPs (eIF4E-binding proteins), which inhibit eIF4E, a key
factor in cap-dependent protein translation [146–148]. Although
phosphorylation of eIF-4E is primarily regulated by the ERKs,
4E-BP1, one of the binding proteins that inhibit the function of
eIF-4E, is phosphorylated in a rapamycin-sensitive manner. When
phosphorylated, the binding protein is released to allow eIF-4E to
function. The target of p70S6K , the ribosomal S6 protein, is also
important for 5 -TOP (terminal oligopyrimidine tract)-dependent
translation [144,149]. Together, these events result in an increased
level of protein synthesis that is at least partially dependent on
activation of the PI3K/PKB pathway, and downstream targets
TOR and p70S6K .
A recently described protein that is elevated in cells undergoing
apoptosis is PDCD4 (programmed cell death protein 4)
[150]. Interestingly, PDCD4 has an inhibitory effect on protein
synthesis. Expression of PDCD4, which acts by inhibition of
protein translation, has also been reported to be elevated in
response to differentiation agents, and it is regulated in a
PI3K/PKB/mTOR-dependent manner [150].
There are also many other proteins whose regulation may be
critical for cell survival, and for which protein translation regulation by PI3K/PKB may be important. These could include several
of the pro-survival Bcl-2 proteins, the IAPs which serve as intracellular inhibitors of caspase activity, as well as cell-cycle-regulatory proteins such as cyclins. A recent review that highlighted the importance of regulation of protein translation
in TRAIL (tumour-necrosis-factor-related apoptosis-inducing
ligand)-induced apoptosis [151] pointed out that, on balance,
many of the anti-apoptosis proteins, including Mcl-1, FLIP, XIAP
(X-linked IAP) and survivin, have short half-lives (under 2 h),
whereas many of the pro-apoptotic Bcl-2 family proteins and
some caspases are much longer-lived. Thus the balance of proand anti-apoptotic proteins can be disrupted in favour of apoptosis
if there is a loss of protein translation.
Some specific studies have also highlighted ways in which
apoptotic regulatory proteins can be specifically regulated at the
translational level by PI3K/PKB. For example, we and others have
shown that PI3K activity contributes to increased translation of
c The Authors Journal compilation c 2008 Biochemical Society
340
V. Duronio
the pro-survival Bcl-2 family protein, Mcl-1 [152,153], which had
earlier been shown to be transcriptionally regulated by cytokines
as well [154]. Maintenance of normal protein synthesis can also
be considered an essential event in cell survival, based on the fact
that ER (endoplasmic reticulum) stress resulting from blocked
protein synthesis can lead to apoptosis [155,156]. In particular,
PKB-mediated events have also been implicated in regulation
of the unfolded protein response that occurs during ER stress
[157,158]. In specific cases, blocking protein synthesis has been
linked to induction of apoptosis as a result of decreased translation
of specific anti-apoptotic proteins, such as Mcl-1 [159].
that research in this field, and other related fields, will have to take
to be able to create a framework that can explain biological end
points, based on a network of interconnected molecular events.
Research in my laboratory has been supported by grants from the Canadian Institutes of
Health Research, BC Lung Association, Cancer Research Society, National Cancer Institute,
Heart and Stroke Foundation of BC and Yukon, and the Michael Smith Foundation for
Health Research. I thank Christopher Duronio for his help with the preparation of the
Figures.
REFERENCES
PI3K/PKB and p53
Yet another pathway utilized by PI3K/PKB to block apoptotic
events is in the regulation of the well-known tumour suppressor,
p53. As has been shown in numerous studies, p53 can regulate apoptosis, most notably when chromosomal aberrations due to drugor radiation-induced DNA damage are detected, by a combination of events including up-regulation of pro-apoptotic molecules
such as Bax and PUMA [160,161]. In addition, it may be
somewhat surprising that p53, best known as a transcriptional
regulator, may also have apoptotic effects that are independent of
its transcriptional activity and resulting from its function at the
mitochondria [162].
Activity of p53 is negatively controlled by a molecule called
Mdm2 (murine double minute 2), which can translocate to the
nucleus and promote the ubiquitination and subsequent inactivation of p53. Several years ago, it was shown that PKB could
mediate phosphorylation of Mdm2, which promoted its translocation into the nucleus [163]. More recently, Mdm2 has been shown
to be associated with MdmX and it has been suggested that MdmX
is a direct target of PKB, and resultant association with a 143-3 protein stabilizes Mdm2 [164]. On the other hand, Mdm2
association with 14-3-3σ has been shown to be involved in its
destabilization [165]. Although the exact molecular details may
still need to be worked out, it seems clear that PKB activity is important in keeping Mdm2 active and thus suppressing p53 activity.
SUMMARY
The above descriptions of interconnections among the PI3K/PKB
signalling pathway and numerous apoptosis regulatory events
highlight the complexity and the universal importance of this
system as a vast signalling network present in all cell types. It is
difficult to discern which specific subset of events can be considered ‘crucial’, although the literature contains many examples
of studies that report what might be considered at the time as the
‘essential new link’ that explains how PI3K/PKB may be controlling cell survival. Certainly, one has to consider the specific
cellular context of each of the myriad events that have been
discussed. One can argue that, in any one cell type, there is a complex fingerprint of molecular features that determine whether a cell
will survive and thus not undergo apoptosis. This fingerprint may
represent a subset of all of the events that have been described
to date, but a different cell type may utilize only a partially overlapping subset of events. At present, we remain a long way from
knowing the exact combination of events that is either sufficient
or required for maintenance of cell survival in a PI3K/PKBdependent manner. Such global descriptions may be accessible
by emerging systems biology approaches using genomics and/or
proteomics. Unfortunately, we are not yet at the stage where such
analyses can be done easily, particularly in comparing multiple
different normal cell types. However, this may be the direction
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