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 (phosphoinositide-dependent 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 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.

INTRODUCTION

Historical overview

It is well known that investigations of the pathways that are regulated following activation of the PI3K (phosphoinositide 3-kinase) family of enzymes has been one of the most intensively studied set of signalling events in recent years. In the mid-to-late 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 [13]. 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 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.

Table 1
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 Bcl-2/Bcl-XL-associated death domain protein BH3-only member of the Bcl-2 family 
Bak Bcl-2 homologous antagonist-killer protein Multi-BH domain pro-apoptosis protein 
Bax Bcl-2 associated X protein Multi-BH domain pro-apoptosis protein 
Bcl-2 B-cell lymphoma-2 Defining member of the family; originally characterized as an oncogene 
Bcl-XL Bcl-2-related gene, long form Bcl-XS is a shorter splice variant that is pro-apoptotic 
Bim Bcl-2-interacting mediator of cell death BH3-only member of the Bcl-2 family 
FoxO Forkhead box, class O proteins FKHR (forkhead in rhabdosarcoma); members of the winged helix or forkhead family of transcription factors 
GPCR G-protein-coupled receptor Serpentine receptor; contain seven transmembrane domains 
GSK-3 Glycogen synthase kinase-3 Kinase activity suppressed after phosphorylation by PKB 
IκB Inhibitor of NF-κB Interacts with NF-κB 
IKK B kinase Phosphorylates IκB to promote its degradation 
Mcl-1 Myeloid cell leukaemia-1 Originally identified as up-regulated upon monocyte differentiation of ML-1 cells 
Mdm2 Murine double minute 2 Gene amplified in mouse 3T3DM cells 
NF-κB Nuclear factor-κ type B Originally linked with enhancement of immunoglobulin κ light chain gene 
p27kip1 27 kDa protein/kinase inhibitor protein 1 Member of the family of cyclin-dependent kinase inhibitors (CDKIs) 
p53 53 kDa protein Tumour-suppressor protein 
p70S6K 70 kDa S6kinase Phosphorylates ribosomal S6 subunit 
PI Phosphatidylinositol Five hydroxy groups on the inositol ring can be phosphorylated; PtdIns 
PI3K Phosphoinositide 3-kinase Phosphatidylinositol 3-kinase; PI 3-kinase; PtdIns3K 
PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate Designation for PI phosphorylated at 3,4, and 5 positions of the inositol ring; PtdIns(3,4,5)P3 
PDK1 Phosphoinositide-dependent kinase 1 Like PKB, has a PH domain that binds phosphorylated PI lipids 
PKB Protein kinase B Akt; RACK, or related to A and C kinase; has PH domain 
PUMA p53-up-regulated modulator of apoptosis BH3-only member of the Bcl-2 family 
Rheb Ras homologue enriched in brain Small G-protein required for activation of TOR 
SH2 Srchomology 2 Protein domain that binds phosphorylated tyrosine sites 
SHIP SH2-domain-containing inositol 5-phosphatase Dephosphorylates the 5-phosphate from PI(3,4,5)P3 
TOR Target of rapamycin Protein kinase regulator of downstream kinases, including p70S6K 
TSC Tuberous sclerosis Tuberous sclerosis complex; TSC1 (hamartin) and TSC2 (tuberin) are genes linked to this disorder 
Abbreviation Meaning Comments (or synonyms) 
Bad Bcl-2/Bcl-XL-associated death domain protein BH3-only member of the Bcl-2 family 
Bak Bcl-2 homologous antagonist-killer protein Multi-BH domain pro-apoptosis protein 
Bax Bcl-2 associated X protein Multi-BH domain pro-apoptosis protein 
Bcl-2 B-cell lymphoma-2 Defining member of the family; originally characterized as an oncogene 
Bcl-XL Bcl-2-related gene, long form Bcl-XS is a shorter splice variant that is pro-apoptotic 
Bim Bcl-2-interacting mediator of cell death BH3-only member of the Bcl-2 family 
FoxO Forkhead box, class O proteins FKHR (forkhead in rhabdosarcoma); members of the winged helix or forkhead family of transcription factors 
GPCR G-protein-coupled receptor Serpentine receptor; contain seven transmembrane domains 
GSK-3 Glycogen synthase kinase-3 Kinase activity suppressed after phosphorylation by PKB 
IκB Inhibitor of NF-κB Interacts with NF-κB 
IKK B kinase Phosphorylates IκB to promote its degradation 
Mcl-1 Myeloid cell leukaemia-1 Originally identified as up-regulated upon monocyte differentiation of ML-1 cells 
Mdm2 Murine double minute 2 Gene amplified in mouse 3T3DM cells 
NF-κB Nuclear factor-κ type B Originally linked with enhancement of immunoglobulin κ light chain gene 
p27kip1 27 kDa protein/kinase inhibitor protein 1 Member of the family of cyclin-dependent kinase inhibitors (CDKIs) 
p53 53 kDa protein Tumour-suppressor protein 
p70S6K 70 kDa S6kinase Phosphorylates ribosomal S6 subunit 
PI Phosphatidylinositol Five hydroxy groups on the inositol ring can be phosphorylated; PtdIns 
PI3K Phosphoinositide 3-kinase Phosphatidylinositol 3-kinase; PI 3-kinase; PtdIns3K 
PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate Designation for PI phosphorylated at 3,4, and 5 positions of the inositol ring; PtdIns(3,4,5)P3 
PDK1 Phosphoinositide-dependent kinase 1 Like PKB, has a PH domain that binds phosphorylated PI lipids 
PKB Protein kinase B Akt; RACK, or related to A and C kinase; has PH domain 
PUMA p53-up-regulated modulator of apoptosis BH3-only member of the Bcl-2 family 
Rheb Ras homologue enriched in brain Small G-protein required for activation of TOR 
SH2 Srchomology 2 Protein domain that binds phosphorylated tyrosine sites 
SHIP SH2-domain-containing inositol 5-phosphatase Dephosphorylates the 5-phosphate from PI(3,4,5)P3 
TOR Target of rapamycin Protein kinase regulator of downstream kinases, including p70S6K 
TSC Tuberous sclerosis Tuberous sclerosis complex; TSC1 (hamartin) and TSC2 (tuberin) are genes linked to this disorder 

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)P3in 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 [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 [1315]. 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 5-phosphatases, 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 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 [2127]. 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].

Overview of the key function of PI(3,4,5)P3 and PtdIns(3,4)P2 in regulation of PKB and downstream substrates in both cytoplasm and nucleus

Figure 1
Overview of the key function of PI(3,4,5)P3 and PtdIns(3,4)P2 in regulation of PKB and downstream substrates in both cytoplasm and nucleus

TKR, tyrosine kinase-type growth factor receptor.

Figure 1
Overview of the key function of PI(3,4,5)P3 and PtdIns(3,4)P2 in regulation of PKB and downstream substrates in both cytoplasm and nucleus

TKR, tyrosine kinase-type growth factor receptor.

The AGC kinase family also encodes a hydrophobic motif that must either be phosphorylated to activate the kinase fully, or, in 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 DNA-PK 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 3-phosphorylated 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 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 [4851]. 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.

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

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.

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.

Table 2
Specific phosphorylation sites regulated by PI3K/PKB action

(a) Targets phosphorylated directly by PKB

Target protein Site(s) Outcome Reference(s) 
Bad Ser136 Promotes association with 14-3-3 [6972
Bax Ser184 Suppress Bax function [83
FoxO3a Thr32/Ser253/Ser315 Promote nuclear export and binding to cytosolic 14-3-3 proteins [9194
GSK-3α Ser21 Inhibits kinase activity [125,126
GSK-3β Ser9 Inhibits kinase activity [125,126
IKK-α Thr23 Increases IKK activity [117,118
Mdm2 Ser166/Ser186 Promote nuclear localization of Mdm2 and suppression of p53 [163
TSC2 Ser939/Ser1086/Ser1088/Thr1462 Suppresses TSC2 function and thus allows activation of p70S6K [166168
(b) Targets phosphorylated downstream of PI3K/PKB action 
Target protein Site Direct kinase; outcome Reference(s) 
IκB Ser32/Ser36 (α), Ser19/Ser23 (β) IKKα/β; promotes degradation [169171
P70S6K Thr252/Thr412 PDK1 [172
4E-BP1 Ser65/Thr70 mTOR; regulates dissociation from eIF4E [147,173
4E-BP1 Thr37/Thr46 mTOR; priming sites [174
Mcl-1 Ser159 GSK-3; promotes degradation of Mcl-1 [132,133
S6 Ser235 p70S6K; enhances protein translation [144,149,175
Target protein Site(s) Outcome Reference(s) 
Bad Ser136 Promotes association with 14-3-3 [6972
Bax Ser184 Suppress Bax function [83
FoxO3a Thr32/Ser253/Ser315 Promote nuclear export and binding to cytosolic 14-3-3 proteins [9194
GSK-3α Ser21 Inhibits kinase activity [125,126
GSK-3β Ser9 Inhibits kinase activity [125,126
IKK-α Thr23 Increases IKK activity [117,118
Mdm2 Ser166/Ser186 Promote nuclear localization of Mdm2 and suppression of p53 [163
TSC2 Ser939/Ser1086/Ser1088/Thr1462 Suppresses TSC2 function and thus allows activation of p70S6K [166168
(b) Targets phosphorylated downstream of PI3K/PKB action 
Target protein Site Direct kinase; outcome Reference(s) 
IκB Ser32/Ser36 (α), Ser19/Ser23 (β) IKKα/β; promotes degradation [169171
P70S6K Thr252/Thr412 PDK1 [172
4E-BP1 Ser65/Thr70 mTOR; regulates dissociation from eIF4E [147,173
4E-BP1 Thr37/Thr46 mTOR; priming sites [174
Mcl-1 Ser159 GSK-3; promotes degradation of Mcl-1 [132,133
S6 Ser235 p70S6K; enhances protein translation [144,149,175

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 anti-apoptotic 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 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 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 [5658], 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.

Target substrates of PI3K/PKB whose pro-apoptotic activities are suppressed by phosphorylation

Figure 3
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.

Figure 3
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.

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 [6972]. However, we came to the opposite 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 [69,71,72,7476], 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+/calmodulin-dependent 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 stress-response proteins such as Hsp27 can provide protection against apoptosis in many other contexts [8890].

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.

PI3K/PKB and forkhead transcription factors

A very important set of targets of PKB with respect to apoptosis regulation are the forkhead transcription factors [9194]. 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 [9699]. 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 [9699]. 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]. NF-κB has been shown to be an important regulator of many anti-apoptotic or pro-survival genes, perhaps the most important of which are the anti-apoptotic proteins Bcl-2 and Bcl-XL [107113]. 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 (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 [119121]. 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 which GSK-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 FDC-P1 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.

PI3K/PKB regulation of protein synthesis

The importance of PI3K in cell growth and cancer is now well established [135138] 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 [146148]. 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 pro- and 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 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].

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 drug- or 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 14-3-3 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/PKB-dependent 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 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.

Abbreviations

     
  • BH3

    Bcl-2 homology domain 3

  •  
  • DNA-PK

    DNA-dependent protein kinase

  •  
  • eIF-4E

    eukaryotic initiation factor 4E

  •  
  • 4E-BP

    eIF-4E-binding 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

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Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum
J. Biol. Chem.
1993
, vol. 
268
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4530
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4533
)