Phosphoinositide 3-Kinase

Synonyms

Phosphatidylinositol 3-kinase; Phosphatidylinositol 3-OH kinase; PI 3-K; PI 3-kinase; PI3K; PI(3)K

Historical Background

Phosphoinositide 3-kinase, commonly abbreviated PI3K, is one of the most well-studied enzymes in the field of signal transduction (Fruman et al. 1998; Vanhaesebroeck et al. 2010). PI3K actually refers to a family of enzymes encoded by eight genes in mammals. Orthologs of one or more PI3K genes exist in all animals as well as in yeast. These enzymes share the ability to phosphorylate the 3′-hydroxyl of the inositol head group of phosphatidylinositol (PtdIns), generating the lipid PtdIns-3-P (Fig. 1). Some members of the PI3K family can act on phosphoinositides, which are phosphorylated derivatives of PtdIns (such as PtdIns-4,5-P₂). Therefore, the family is properly referred to as phosphoinositide 3-kinases rather than simply phosphatidylinositol 3-kinases. The products of PI3Ks, generally termed 3-phosphorylated inositides (3-PIs), serve as membrane recruitment signals for cytoplasmic proteins with selective 3-PI-binding domains. Since the production of 3-PIs is transient and reversed by lipid phosphatases, PI3K activation provides a means to dynamically control assembly of signaling complexes at cellular membranes.

Phosphoinositide 3-Kinase, Fig. 1

Pathways of synthesis and degradation of 3-phosphorylated phosphoinositides (3-PIs). The brown area represents the inner leaflet of the membrane bilayer, viewed en face. The three classes of PI3K are indicated in blue; major phosphatases are in orange. Effectors of class I PI3K (green) bind selectively to PIP₃ and/or PtdIns-3,4-P₂. The colors designated in this figure legend and others refer to the electronic versions of the figures

PI3K was first discovered in the mid-1980s as an enzymatic activity that co-precipitated with activated growth factor receptors and oncoproteins (Cantley et al. 1991). Although controversial at first, it was soon appreciated that activation of PI3K and production of 3-PIs is a signaling response common to a great variety of receptor systems. The genes encoding PI3Ks were cloned starting in the early 1990s. Identification of PI3K inhibitors began at this time and continues to the present day, with pharmaceutical companies and academic laboratories around the world racing to develop the most potent and selective compounds (Marone et al. 2008; Yap et al. 2008; Engelman 2009; Liu et al. 2009; Workman et al. 2010). The reason for this intense activity is that hyperactive PI3K signaling is now recognized as a driving force in human cancer, inflammatory diseases, metabolic disorders, and in many other clinical conditions (Engelman et al. 2006; Fruman and Bismuth 2009). PI3K genes are bona fide oncogenes in that gain-of-function mutations are found in a large fraction of human cancers (Samuels and Ericson 2006; Denley et al. 2008). Some of the most common tumor suppressor genes act by opposing PI3K signaling (Salmena et al. 2008). Gene targeting studies have confirmed the central role of PI3K enzymes and regulatory subunits in cell proliferation and in a diverse set of physiological functions (Deane and Fruman 2004; Fruman and Bismuth 2009; Vanhaesebroeck et al. 2010). Note to reader: The review articles cited in this section are definitive resources that provide references to primary literature. The main text cites additional reviews relevant to specific topics not covered in the above references.

PI3K Enzymes and Products

The eight PI3K catalytic subunits in mammals are grouped into three classes according to structure and substrate preference (Fig. 2). Class I PI3Ks can utilize various substrates, but it is thought that the primary activity of these enzymes is to convert PtdIns-4,5-P₂ to PtdIns-3,4,5-P₃ (PIP₃) (Fig. 1). PIP₃ is nearly undetectable in unstimulated cells but is rapidly produced following engagement of receptors that activate class I PI3K – which include most receptor tyrosine kinases, tyrosine kinase-coupled receptors, and G-protein-coupled receptors (GPCRs). PIP₃ can be converted back to PtdIns-4,5-P₂ by the phosphatase and tensin homolog (PTEN), or dephosphorylated on the 5′-phosphate by SH2-containing inositol phosphatases (SHIP1 or SHIP2) to produce PtdIns-3,4-P₂. The four class I enzymes are customarily subdivided into class IA (p110α, β, δ) and class IB (p110γ), based on their distinct regulatory subunits and upstream activators (Fig. 2). Class IA enzymes form obligate heterodimers with one of five regulatory isoforms (p85α, p55α, p50α, p85β, or p55γ) whereas the class IB PI3K associates with distinct regulatory isoforms (p87 or p101). Each class IA regulatory isoform contains Src homology-2 (SH2) domains that mediate binding to tyrosine-phosphorylated signaling complexes. The class IB regulatory subunits link class IB PI3K to GPCR signaling via heterotrimeric G proteins. However, there is increasing evidence that class IA enzymes can be activated downstream of GPCRs, indicating that the class IA-IB distinction is not absolute. Furthermore, both class IA and class IB isoforms possess domains for binding to Ras, a GTPase activated downstream of tyrosine kinases.

Phosphoinositide 3-Kinase, Fig. 2

Domain structure of PI3K catalytic and regulatory subunits. Among the class IA regulatory subunits, only p85α and p85β possess the domains denoted by the red dashed box

Structural studies have provided considerable insight into the domain topology of class I PI3Ks, their mode of regulation by regulatory subunits and Ras, and their interaction with inhibitor compounds (Williams et al. 2009). The p110γ isoform was the first to be crystallized, followed more recently by p110α, p110β and p110δ. The studies of p110α have provided insight into the mechanisms by which oncogenic mutations in the genes encoding p110α (PIK3CA) and p85α (PIK3R1) cause elevated activity of p85α/p110α dimers.

There are three class II enzymes, each containing a C2 domain and termed PI3K-C2α, PI3K-C2β, and PI3K-C2γ. There is a single class III PI3K that is homologous to the yeast PI3K enzyme Vps34. Class III enzymes associate with the protein Vps15. Class II and III enzymes act on PtdIns to produce PtdIns-3-P, though the class II PI3Ks might also convert PtdIns-4-P to PtdIns-3,4-P₂. It is clear that regulated production of PtdIns-3-P is essential for vesicle trafficking and for the initiation of autophagy. However, compared to the class I PI3Ks, less is known about the function of class II and class III PI3Ks. A recent review article provided a comprehensive review of the available literature on class II and III PI3Ks (Vanhaesebroeck et al. 2010).

PI3K Effectors

The term “PI3K effector” is applied to any protein that contains a domain with selective affinity for 3-PIs, allowing the protein to be recruited to membranes where a PI3K enzyme is active (Fig. 3). There are several types of 3-PI-binding domain, of which the most common are the pleckstrin homology (PH), phox homology (PX), and FYVE domains (Lemmon 2008). PH domains have diverse lipid preferences, with only a subset binding selectively to 3-PIs and, within this group, differing preference for PIP₃, PtdIns-3,4-P₂, etc. Most PX domains are selective for PtdIns-3-P or PtdIns-3,4-P₂, and FYVE domains mostly bind to PtdIns-3-P.

Phosphoinositide 3-Kinase, Fig. 3

Examples of PH domain-containing effectors of class I PI3K. Each of the proteins shown (PDK-1, AKT, pREX-1, Tec family kinases) bind selectively to PIP₃. Key downstream events controlled by these PI3K effectors are shown

PH domains selective for PIP₃ or PtdIns-3,4-P₂ are present in a number of important signaling enzymes and small G protein modifiers (Fig. 3). PDK-1 (phosphoinositide-dependent kinase-1) is a constitutively active serine/threonine kinase whose PH domain allows the enzyme to interact with membrane-associated substrates. Most notable of these are the AKT family of serine/threonine kinases (also known as protein kinase B). The three AKT proteins (AKT1, 2, 3) possess PH domains and are phosphorylated by PDK-1-on a threonine residue in the activation loop (T308 in AKT1). Further AKT activation is achieved by phosphorylation of a C-terminal hydrophobic motif (S473 in AKT1). In many cellular contexts, the target of rapamycin (TOR) complex-2 (TORC2) is responsible for AKT-S473 phosphorylation. Active AKT phosphorylates numerous substrates that regulate cell survival, proliferation, nutrient uptake, and metabolism (Manning and Cantley 2007).

PIP₃-selective PH domains are also present in most tyrosine kinases of the Tec family, including ITK and BTK that control Ca²⁺ mobilization and other crucial signaling events downstream of antigen receptors in lymphocytes and mast cells. PH domains in guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) link PI3K activation to regulation of some small GTPases. One well-known example is pREX-1, a GEF for Rac family GTPases that acts downstream of p110γ in neutrophils.

Many important signaling components and transcription factors are activated (or inactivated) indirectly by PI3K signaling, via the direct effectors described in the preceding paragraphs. Space constraints do not allow a comprehensive summary of PI3K-regulated cellular components. The discussion here will focus on one crucial downstream network controlled by TOR complex-1 (TORC1) (Fig. 4). The enzyme TOR (also known as mTOR) is a serine/threonine kinase that exists in two complexes with distinct components, TORC1 and TORC2 (Foster and Fingar 2010; Sparks and Guertin 2010). While TORC2 acts upstream on AKT as mentioned above, TORC1 acts downstream of AKT. When fully active, TORC1 promotes biosynthetic events that drive cell proliferation, including protein and lipid synthesis; TORC1 also regulates metabolism by increasing glycolytic rates and inhibiting autophagy. TORC1 actually integrates signals from many sources: growth factor signaling pathways (AKT and others), nutrient availability, and stress response pathways. AKT promotes TORC1 activity by phosphorylating and inhibiting two negative regulators of TORC1. One AKT substrate, TSC2, is part of the tuberous sclerosis complex (TSC) that has GAP activity toward the Rheb GTPase upstream of TORC1. Proline-rich AKT substrate of 40kDa (PRAS40) is part of the TORC1 complex and its phosphorylation by AKT relieves PRAS40-mediated suppression of TORC1 activity. Active TORC1 phosphorylates ribosomal S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBPs) to promote mRNA translation, and other cellular substrates to regulate autophagy and other processes. It has become increasingly clear, however, that compensatory pathways can maintain TORC1 activity in the absence of detectable PI3K/AKT signaling, particularly in cancer cells. The implications of this will be discussed in the section “PI3K Pharmacology.”

Phosphoinositide 3-Kinase, Fig. 4

Simplified diagram of the TOR signaling network. The target of rapamycin (denoted “mTOR” in this figure, in keeping with previous literature in which the protein is called “mammalian” or “mechanistic” target of rapamycin) is a single kinase that exists in two cellular complexes, TORC1 and TORC2. The subunit composition differs among these complexes, as do the activation mechanisms and downstream functions as indicated. Key points are that TORC2 functions upstream of AKT, TORC1 functions downstream of AKT, and TORC1 also feeds back through S6 kinases to dampen PI3K and AKT activity

PI3K Genetics

Gene targeting and transgenesis experiments have greatly added to the understanding of PI3K gene function in model organisms. The discussion that follows will focus on studies of class I PI3K genes in mice. For each of the catalytic subunits, null “knockout” (KO) alleles as well as kinase-dead “knock-in” (KI) alleles have been reported. p110α (Pik3ca) KO or KI results in an embryonic lethal phenotype in homozygotes. Mice heterozygous for p110αKI are viable but display severe defects in organismal metabolism, including insulin resistance, hyperglycemia and adiposity. At the molecular level, p110α-deficient cells show reduced signaling downstream of receptors that couple to insulin receptor substrate (IRS) proteins. Together with pharmacological studies, this finding illustrates that p110α has a nonredundant function in cellular responses to insulin and insulin-like growth factor. Other studies of p110αKI mice have revealed a role for this isoform in angiogenesis. A distinct knock-in strain carrying a mutation in the Ras-binding domain of p110α provided evidence that p110α is a Ras effector important for lymphangiogenesis and cellular transformation.

Mice with germline deletion of p110β (Pik3cb) die early in embryogenesis, but p110βKI mice are viable. Together with other results this finding establishes kinase-independent functions of the p110β isoform. Kinase-dependent functions of p110β have also been identified through analysis of p110βKI mice as well as strains with conditional p110βKO in specific tissues. Key observations include the following: (1) p110β plays an important role in the malignant phenotype of cancer cells with loss of PTEN, suggesting that p110β mediates growth factor-independent “basal” production of PIP₃ that is normally hydrolyzed by PTEN. (2) p110β has a key function downstream of GPCRs including receptors for lysophosphatidic acid and chemokines, a finding supported by pharmacological approaches. It remains unclear how p110β activity or localization is modulated by GPCR signaling.

Unlike p110α and p110β, p110δ and p110γ are not ubiquitously expressed, and germline KO mice are viable. The most dramatic phenotypes of p110δ (Pik3cd) and p110γ (Pik3cg) KO mice are in the immune system. Notable examples include, for p110δKO and KI, impaired B cell development and function, and defective T cell differentiation and trafficking. In p110γKO and KI mice, there are major defects in migration and function of innate immune cells (i.e., macrophages and dendritic cells). Combined inactivation of p110δ and p110γ produces an early block in T cell development and affects natural killer (NK) cell function. Mast cell function and allergic responses are impaired in the absence of either p110δ or p110γ.

There are three genes encoding class IA regulatory subunits: Pik3r1 encodes p85α, p55α, and p50α; Pik3r2 encodes p85β; and Pik3r3 encodes p55γ. Although inactivation of the Pik3r3 gene has not been reported, the Pik3r1 and Pik3r2 genes have been targeted in various ways and in combination. These investigations have shown complex biology of the class IA regulatory subunits. There are unique functions of individual isoforms in some contexts and redundant functions in others. The functions of class IA regulatory subunits in insulin signaling are particularly complex, in that individual deletion of p85α and p85β enhances insulin sensitivity whereas combined deletion reduces insulin sensitivity. p85α and p85β each have diverse functions in immune cells. Studies of insulin-responsive cells and lymphocytes have also provided evidence for “scaffolding” functions of p85α and p85β, independent of the associated catalytic subunit. There are two genes encoding class IB regulatory subunits (Pik3r5 encodes p101, Pik3r6 encodes p87). Knockout of Pik3r5 has shown a nonredundant role for p101 in neutrophil signaling and functional responses to inflammatory stimuli.

Gain-of-function alleles of class I catalytic subunits have been tested in transgenic models and in tissue culture. Increased p110 activity can be accomplished by overexpression of wild-type or oncogenic mutants, including naturally occurring p110α alleles (see below) and engineered mutations that target p110 proteins constitutively to the membrane. Cell culture experiments were the first to demonstrate transforming activity of p110 proteins, and have shown that any of the four class I enzymes can transform fibroblasts when mutated and/or overexpressed. In vivo, activated alleles of p110α and p110β are oncogenic in mice. Analyses of mice with targeted deletion of PTEN have confirmed the tumor suppressor function of this lipid phosphatase and have shown gene dosage-dependent tumor penetrance. In addition, PTEN heterozygous mice develop lymphoproliferative disease. Similarly, mice expressing constitutively active AKT in T cells develop lymphoproliferation and thymic lymphomas. A truncation mutant of p85α (p65) that causes elevated PI3K signaling also disrupts T cell homeostasis and has oncogenic potential.

PI3K in Disease

Many human diseases have been associated with aberrant control of PI3K signaling. The most prominent correlation is between elevated cellular PI3K/AKT activity and cancer. It is now accepted that a large majority of human cancers possess mutations that drive elevated PIP₃ production. This can occur through activated tyrosine kinases (e.g., EGFR, BCR-ABL) or oncogenic Ras upstream of PI3K, loss or inactivation of PTEN, or mutations in PI3K genes themselves (Fig. 5). In epithelial cancers, gain-of-function mutations in PIK3CA (p110α) are common. Mutations in PIK3R1 (p85α, p55α, and p50α) that derepress the associated catalytic subunit are found in glioblastoma at a frequency similar to PIK3CA mutations. Elevated p110δ activity has been associated with certain hematologic malignancies. Gene targeting studies support the model that class I PI3K activity is essential for efficient transformation by BCR-ABL or Ras. It is thought that AKT is the major PI3K effector responsible for enhanced proliferation, survival, and altered metabolism in cancer cells. Indeed, some tumor samples display mutation or overexpression of AKT genes. The activity of both TOR complexes, TORC1 and TORC2, also appears to be important for the establishment and maintenance of cellular transformation. Notably, some cancer cells can maintain TORC1 signaling in the absence of PI3K/AKT activity through inputs from the Ras-ERK pathway and other mechanisms. Also, humans with heterozygous mutations in TSC1 or TSC2 develop benign tumors whose growth is driven by elevated TORC1 activity.

Phosphoinositide 3-Kinase, Fig. 5

Various mechanisms identified in cancer cells that result in elevated cellular PIP₃ levels. Components in green represent protein products of oncogenes that display gain-of-function in cancer. Components in red represent tumor suppressors that display loss-of-function in cancer. FOXO proteins are transcription factors that promote cell cycle arrest and apoptosis

Inflammatory conditions and autoimmune diseases are associated with increased PI3K/AKT/TOR signaling in immune cells. This phenomenon is generally secondary to other environmental or genetic conditions that maintain a heightened state of activation in specific immune cells. Nevertheless, the correlation between immune cell activation and PI3K/AKT/TOR signaling has placed this pathway at the center of worldwide drug discovery efforts for inflammatory diseases.

Insulin-resistance syndromes are a growing health epidemic. By definition, insulin resistance is associated with reduced signaling responses to insulin – including dampened PI3K/AKT activation. Agents that increase PI3K activity or block hydrolysis of PIP₃ could be useful therapies for insulin resistance.

PI3K Pharmacology

As summarized in the previous section, altered PI3K output is involved in the pathogenesis of many human diseases. Consequently, there has been intense and sustained research and development of compounds that modulate PI3K/AKT/TOR signaling for therapeutic benefit. The section that follows will briefly discuss five general strategies (summarized in Table 1) and their potential applicability to human cancer and inflammatory diseases.

Phosphoinositide 3-Kinase, Table 1 Strategies to target PI3K/AKT/TOR activity for human disease therapy

One approach is to develop inhibitors of all class I PI3Ks. Until recently, the available PI3K inhibitors were nonselective and suppressed activity of all PI3K classes as well as other cellular enzymes. Now, compounds are available for both preclinical and clinical studies that are highly selective for class I enzymes (“pan-class I inhibitors”). A well-studied example is GDC-0941, a compound developed by Genentech that is in phase I trials for cancer. Other companies and academic laboratories have synthesized compounds with a comparable target profile. A second approach is to develop isoform-selective PI3K inhibitors. An inhibitor of p110δ, CAL-101, has entered trials for blood cancers, and other p110δ or dual p110δ/p110γ inhibitors are under consideration for inflammatory diseases. The relative benefits of pan-class I inhibitors versus isoform-selective inhibitors in cancer have not yet been established, and might vary among tumor subtypes. Inhibitors selective for p110α might be particularly effective against cancers with PIK3CA mutations, while avoiding toxicities associated with pan-PI3K inhibition. Likewise, inhibitors of p110β might find utility in cancers with loss of PTEN.

A third approach is to inhibit AKT. Various compounds have been developed that are either ATP-competitive or act through allosteric inhibition of enzyme activity or membrane localization. Considering that AKT inactivation has more profound effects on cellular and organismal metabolism than does PI3K inactivation, there is some concern about the therapeutic window for AKT inhibitors. Another concern about both PI3K and AKT inhibitors in general is that they will not prevent TORC1 activation through other mechanisms.

A fourth approach is to use allosteric inhibitors of TOR, exemplified by rapamycin and its analogs RAD001 and CCI-779. Rapamycin (sirolimus) has been a clinically approved immunosuppressant since 1997. RAD001 (everolimus) and CCI-779 (temsirolimus) are approved for the treatment of advanced renal cell carcinoma. However, the results of oncology trials with rapalogs have been disappointing overall. Rapamycin and rapalogs have mechanistic drawbacks. They do not completely inhibit TORC1 activity and do not bind to TORC2 or inhibit TORC2 activity acutely in cells. Furthermore, rapamycin disables a negative feedback loop by which S6K1 dampens upstream PI3K/AKT and Ras signaling. Therefore, cancer cells treated with rapalogs generally display elevated PI3K/AKT and Ras activity, enhancing survival even when proliferation is suppressed.

A fifth approach is to use ATP-competitive TOR inhibitors. These bind in the active site of the TOR enzyme in both complexes, and fully inhibit phosphorylation of all known TORC1 and TORC2 substrates. There are two classes of competitive TOR inhibitors. One class is not TOR-selective and inhibits PI3K enzymes (which are structurally related to TOR) at equivalent concentrations. These are termed panPI3K/TOR inhibitors, exemplified by the compounds BEZ235 and XL675. The other class, which was developed only recently, is highly selective active-site TOR inhibitors (asTORi) that do not directly inhibit PI3K. Examples of asTORi are PP242, Torin-1, AZD8055, WYE-354, and INK128. The evidence thus far indicates that asTORi provide anticancer efficacy that is equivalent to panPI3K/TOR inhibitors, and better than rapamycin and rapalogs. asTORi are also less immunosuppressive than panPI3K/TOR inhibitors or rapamycin. Further preclinical and clinical studies are needed to identify the optimal target profile that balances efficacy and tolerability in different cancer patient populations.

Although some genetic events that activate PI3K/AKT are sufficient to transform cells in vitro and promote tumorigenesis in animal models, patient-derived human cancers generally carry multiple additional genetic lesions. Thus, it is likely that inhibitors of this pathway will be most effective when combined with other targeted therapies directed to upstream or parallel pathways. Combined targeting of PI3K/AKT/TOR with Ras/RAF/MEK/ERK is emerging as a useful strategy in some settings.

Summary

PI3K enzymes generate 3-phosphorylated inositol lipids that coordinate many aspects of cellular physiology. In response to extracellular signals, class I PI3K produces the second messenger PIP₃ that recruits numerous cytoplasmic proteins to the plasma membrane to propagate key signaling pathways. In general, PIP₃ production promotes cell proliferation and survival, and also plays a key role in cellular and organismal metabolism. Most cancer cells carry mutations that trigger constitutive elevation of PIP₃ levels, and activated immune cells depend on PI3K signaling to carry out specialized functions. Gene targeting in mice has revealed unique and complex functions of individual PI3K catalytic and regulatory isoforms. Based on the role of aberrant PI3K activation in human disease, PI3K drug discovery has been a priority and will continue to produce inhibitor molecules of interest to basic and translational researchers.

Cross-References

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