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The evolution of phosphatidylinositol
3-kinases as regulators of growth
and metabolism
Jeffrey A. Engelman*‡, Ji Luo* and Lewis C. Cantley*§
Abstract | Phosphatidylinositol 3-kinases (PI3Ks) evolved from a single enzyme
that regulates vesicle trafficking in unicellular eukaryotes into a family of enzymes that
regulate cellular metabolism and growth in multicellular organisms. In this review, we
examine how the PI3K pathway has evolved to control these fundamental processes,
and how this pathway is in turn regulated by intricate feedback and crosstalk
mechanisms. In light of the recent advances in our understanding of the function of
PI3Ks in the pathogenesis of diabetes and cancer, we discuss the exciting therapeutic
opportunities for targeting this pathway to treat these diseases.
*Department of Systems
Biology, Harvard Medical
School and Division of Signal
Transduction, Beth Israel
Deaconess Medical Center,
Boston, Massachusetts
02115, USA.
‡
Massachusetts General
Hospital Cancer Center,
Boston, Massachusetts
02114, USA.
§
Department of Systems
Biology, 77 Avenue Louis
Pasteur, Boston,
Massachusetts 02115, USA.
Correspondence to L.C.C.
e-mail: lewis_cantley@hms.
harvard.edu
doi:10.1038/nrg1879
The phosphatidylinositol 3-kinases (PI3Ks) are members of a unique and conserved family of intracellular
lipid kinases that phosphorylate the 3′-hydroxyl group
of phosphatidylinositol and phosphoinositides. This
reaction leads to the activation of many intracellular
signalling pathways that regulate functions as diverse as
cell metabolism, survival and polarity, and vesicle trafficking. A host of intracellular signalling proteins have
evolved the ability to bind to the lipid products of PI3Ks
and therefore become activated by PI3K signalling. The
most ancient role for this family of enzymes is probably to mark specific cellular membranes for trafficking
events, and this seems to be the primary function of the
single form of PI3K that is found in yeast: Vps34 (vacuolar protein-sorting defective 34). However, in multicellular eukaryotes, additional isoforms of PI3K have
evolved for the dedicated purpose of signal transduction
(BOX 1). Genetic experiments in Caenorhabditis elegans,
Drosophila melanogaster and mice clearly implicate
PI3K as a key component in insulin and growth factor
responses that regulate metabolism and cell growth.
Over the past decade, it has become evident that the
PI3K signalling pathway is one of the most highly
mutated systems in human cancers, underscoring its
central role in human carcinogenesis.
We are now at a pivotal junction in translating our
knowledge of the PI3K signalling pathway into developing therapeutics for the treatment of cancer and diabetes. The intracellular signalling cascades initiated by,
and intersecting with, PI3K are complex and intricate.
A detailed understanding of these circuits is essential
606 | AUGUST 2006 | VOLUME 7
for the successful manipulation of this pathway to
ameliorate disease states.
This review focuses on the evolution of PI3K signalling by examining how additional levels of control in
this pathway have evolved to meet the complex needs of
multicellular organisms. We also discuss several mouse
models of human disease that result from mutations in
components of the pathway. In addition, we examine the
intricate circuitry of PI3K signalling and discuss the many
feedback loops that fine-tune the signalling output. We
bring these concepts together and frame the challenges
involved in designing therapeutics that manipulate PI3K
signalling for the treatment of various diseases.
PI3K classification and signal transduction
PI3Ks are grouped into three classes (I–III) according
to their substrate preference and sequence homology
(reviewed in REF. 1) (BOX 1). Different classes of PI3K
have distinct roles in cellular signal transduction, as do
the different isoforms that can exist within each class.
The next sections describe in more detail the functions
of each class of PI3K, followed by a discussion of the
physiological functions of the various PI3K pathways in
model organisms.
Class I PI3Ks. Class I PI3Ks are divided into two subfamilies, depending on the receptors to which they
couple. Class IA PI3Ks are activated by growth factor
receptor tyrosine kinases (RTKs), whereas class IB PI3Ks
are activated by G-protein-coupled receptors (GPCRs;
reviewed in REF. 2) (FIG. 1).
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Box 1 | Classification of phosphatidylinositol 3-kinase (PI3K) family members
There are three classes (I–III) of PI3K (see panel a in the figure), which show distinct substrate preferences in vitro.
In vivo, class I PI3Ks primarily generate phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol4,5-bisphosphate (PI-4,5-P2), whereas class III PI3Ks generate phosphatidylinositol-3-phosphate (PI-3-P) from
phosphatidylinositol (PI). Class II PI3Ks preferentially generate PI-3-P and phosphatidylinositol-3,4-biphosphate
(PI-3,4-P2) in vitro, and might generate PI-3-P, PI-3,4-P2 and possibly PIP3 in vivo2.
The domain structures of various PI3K isoforms (see panel b in the figure) are discussed below. In mammals,
numerous genes encode different isoforms of PI3Ks101 (TABLE 1). All PI3K isoforms are widely expressed, with the
exception of the class IA p55γ subunit, which is enriched in the brain and the testes, and the p110δ subunit, which is
predominantly expressed in lymphocytes.
Class IA PI3K
Class IA PI3K is a heterodimer that consists of a p85 regulatory subunit and a p110 catalytic subunit. Three genes,
PIK3R1, PIK3R2 and PIK3R3, encode the p85α, p85β and p55γ isoforms of the p85 regulatory subunit, respectively. The
PIK3R1 gene also gives rise to two shorter isoforms, p55α and p50α, through alternative transcription-initiation sites.
The class IA p85 regulatory isoforms have a common core structure consisting of a p110-binding domain (also called
the inter-SH2 domain) flanked by two Src-homology 2 (SH2) domains. The two longer isoforms, p85α and p85β, also
have an extended N-terminal region (dashed outline) containing an Src-homology 3 (SH3) domain and a BCR
homology (BH) domain flanked by two proline-rich (P) regions101.
The p85 regulatory subunit is crucial in mediating the activation of class IA PI3K by receptor tyrosine kinases (RTKs).
The SH2 domains of p85 bind to phospho-tyrosine residues in the sequence context pYxxM on activated RTKs or
adaptor molecules (such as IRS1)102. This binding both relieves the basal inhibition of p110 by p85 and recruits the
p85–p110 heterodimer to its substrate (PI-4,5-P2) at the plasma membrane103,104.
Three genes — PIK3CA, PIK3CB and PIK3CD — encode the highly homologous p110 catalytic subunit isoforms
p110α, p110β and p110δ, respectively101. They possess an N-terminal p85-binding domain that interacts with the
p85 regulatory subunit, a Ras-binding domain (RBD) that mediates activation by the small GTPase Ras, a C2
domain, a phosphatidylinositol kinase homology (PIK) domain and a C-terminal catalytic domain. The PIK and
catalytic domains of p110 are homologous to domains found in a family of protein kinases that includes mTOR
(mammalian target of rapamycin), ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia Rad3 related)
and DNA-PK (DNA-dependent serine/threonine protein kinase), indicating that these proteins share an ancient
evolutionary origin.
Class IB PI3Ks
Class IB PI3K is a heterodimer consisting of a p101 regulatory subunit and a p110γ catalytic subunit. Although p110γ
shares extensive homology with the class IA p110 proteins, p101 is distinct from p85 proteins. Two other regulatory
subunits, p84 and p87PIKAP, have been described recently5,6.
Class II PI3Ks
Members of this class consist of only a p110-like catalytic subunit. The three isoforms of class II PI3Ks — PIK3C2α,
PIK3C2β and PIK3C2γ — are encoded by distinct genes. All three isoforms share significant sequence homology with
the class I p110 subunits. In addition, class II PI3Ks have
an extended divergent N terminus, and additional
a
PX and C2 domains at the C terminus.
Class I, II and III PI3Ks
PI-3-P
PI
Class III PI3Ks
Class III PI3Ks consist of a single member, Vps34
(vacuolar protein-sorting defective 34).
Class I and II PI3Ks
PI-4-P
Class I PI3Ks
PI-4,5-P2 (PIP2)
PI-3,4-P2
PI-3,4,5-P3 (PIP3)
b
Class IA PI3K
SH3
P
BH
p85 binding
P
SH2
Ras binding
p110 binding
C2
PIK
SH2
Regulatory (p85α/p55α/p50α, p85β, p55γ)
Catalytic domain
Catalytic (p110α/p110β, p110δ)
Class IB PI3K
Regulatory (p101, p84, p87PIKAP)
Ras binding
C2
PIK
Catalytic domain
Catalytic (p110γ)
Class II PI3K
Ras binding
C2
PIK
Catalytic domain
PX
C2
Catalytic (PIK3C2α, PIK3C2β, PIK3C2γ)
Class III PI3K
C2
PIK
Catalytic domain
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Catalytic (Vps34)
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RTKs
PIP2 Ras
p110 p85 P
P
Class IA PI3K
P
Adaptors
PIP3
GPCRs
PIP2
P p85 p110
P
PIP3
PTEN
PIP2
P Class IA PI3K
P
PIP2
PTEN
PIP3
PIP2
Gβγ Gα
p101
p110γ
Class IB PI3K
Figure 1 | Mechanisms of class I phosphatidylinositol 3-kinase (PI3K) activation. Class IA PI3Ks are activated by
growth factor receptor tyrosine kinases (RTKs; left). Both insulin and insulin growth factor 1 (IGF1) receptors use the
insulin receptor substrate (IRS) family of adaptor molecules (shown in green, on the right of the RTK) to engage class
IA PI3Ks, whereas other receptors, such as the platelet-derived growth factor (PDGF) receptor, recruit class IA PI3Ks
directly (shown to the left of the RTK). By contrast, class IB PI3Ks are activated by G-protein-coupled receptors
(GPCRs) by binding to Gβγ (right). PTEN (phosphatase and tensin homologue) dephosphorylates phosphatidylinositol3,4,5-trisphosphate (PIP3) and therefore terminates PI3K signalling. The 5′-phosphatase SHIP converts PIP3 to phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2) (REF. 1). Gα, guanine nucleotide binding protein (G protein), α; Gβγ,
guanine nucleotide binding protein (G protein), βγ; p110 and p110γ, catalytic subunits of PI3K; p85 and p101,
regulatory subunits of PI3K; SHIP, SH2-domain-containing inositol-5-phosphatase.
Class IA PI3Ks are heterodimers of a p85 regulatory
subunit and a p110 catalytic subunit. In mammals, there
are numerous isoforms of each subunit (BOX 1; TABLE 1).
Interestingly, the p110β isoform of class IA PI3K is regulated not only by the p85 regulatory subunit but also by
binding to Gβγ subunits of heterotrimeric G proteins.
Therefore, the class IA p110β isoform might integrate
signals from GPCRs as well as RTKs.
Class IB PI3Ks do not have p85 family regulatory subunits and therefore are not regulated by RTKs. They seem
to be exclusively activated by GPCRs through interacting
directly with the Gβγ subunit of trimeric G proteins3.
Similar to class IA PI3Ks, class IB PI3Ks are heterodimers
of regulatory and catalytic subunits. The p101 regulatory
subunit facilitates the activation of the p101–p110γ heterodimer by Gβγ 4. Recently, two laboratories identified
two p101 homologues, termed p84 and p87PIKAP (PI3Kγ
adaptor protein of 87 kDa), indicating that class IB PI3Ks
might have many modes of regulation5,6.
As discussed below, class I PI3Ks are involved in
many important physiological processes. By acting
downstream of an insulin-like hormone, the class IA
PI3K in C. elegans regulates dauer formation, metabolism
and longevity. The class IA PI3K in D. melanogaster is
part of a similar pathway that controls cell growth and
proliferation. In mammals, class I PI3Ks regulate glucose
homeostasis, cell migration, growth and proliferation
(reviewed in REF. 1).
Dauer formation
A developmentally arrested
stage in which Caenorhabditis
elegans larvae remain dormant,
stress-resistant and long-lived.
Dauer larvae can resume a
normal developmental
programme when
environmental conditions
improve.
Class II PI3Ks. Class II PI3Ks consist of only a single
p110-like catalytic subunit2. In vitro, these enzymes
preferentially phosphorylate phosphatidylinositol and,
to a lesser extent, phosphatidylinositol-4-phosphate
(PI-4-P). However, phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) is a poor substrate for these enzymes.
Class II PI3Ks bind clathrin and localize to coated pits,
indicating a function in regulating membrane trafficking and receptor internalization7. In addition, class II
PI3Ks can be activated by RTKs, cytokine receptors and
integrins. Their specific functions in response to these
activators, however, are not well understood2.
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Class III PI3Ks. The yeast class III PI3K, Vps34, was
originally identified in budding yeast as the gene product required for trafficking vesicles from the Golgi
apparatus to the vacuole8. Recently, Vps34 was found
to regulate mammalian target of rapamycin (mTOR)
activity in response to amino-acid availability, and so
this enzyme, similar to the class I PI3Ks, might also
be crucial for controlling cell growth9,10. In addition,
Vps34 has been implicated in autophagy, which is a
cellular response to nutrient starvation11,12.
Relatively little is known about the specific functions of
class II and III PI3Ks. Furthermore, the commonly used
PI3K inhibitors, wortmannin and LY294002, inhibit class I
and class III PI3Ks, and to a lesser extent class II PI3Ks,
as well as other PI3K-like protein kinases13. Therefore,
some of the functions ascribed to class I PI3Ks from
these studies might, in fact, be due to inhibition of the
other PI3Ks. This lack of class specificity precludes
the utility of these inhibitors in identifying the particular
species of PI3K responsible for a particular biological
process. The development of isoform-specific smallmolecule inhibitors, the use of RNAi against specific
isoforms, and the generation of knockout mice for the
various PI3K isoforms should clarify the distinct cellular
functions of the different PI3Ks.
Evolution of the PI3K signalling pathway
Phylogenetic distribution of PI3Ks. The PI3K signalling
pathway is highly conserved in evolution (FIG. 2; TABLE 1).
The class III PI3K Vps34 is present in every eukaryotic
organism that has been examined so far, and has a highly
conserved function in regulating vesicle trafficking. It
seems to be the prototype for other PI3Ks in higher
eukaryotes.
Although both budding and fission yeast have
only a single class III PI3K (Vps34), the social amoeba
Dictyostelium discoideum has evolved a family of
class I PI3Ks. In fact, both class I and class II PI3Ks appear
in multicellular organisms that originated as early as
D. discoideum and C. elegans. Class I PI3Ks regulate growth
and/or metabolism in most of the higher organisms
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Table 1 | Evolution of phosphatidylinositol 3-kinase (PI3K) family members
Class
Type
Vertebrates*
Drosophila
melanogaster
Caenorhabditis
elegans
Dictyostelium
discoideum‡
Saccharomyces
cerevisiae§
Class IA
Catalytic
PIK3CA (p110α)
PIK3CB (p110β)
PIK3CD (p110δ)
Pi3K_92D (Dp110)
AGE-1 (p110)
DdPIK1
DdPIK2
DdPIK3
None
Regulatory
PIK3R1 (p85α/p55α/p50α)
PIK3R2 (p85β)
PIK3R3 (p55γ)
Pik57 (p60)
AAP-1 (p55)
Catalytic
PIK3CG (p110γ)
None
None
Regulatory
PIK3R5 (p101)
p84
PIK3R6 (p87PIKAP)
None
None
Catalytic
PIK3C2α
PIK3C2β
PIK3C2γ
Pi3K_68D
NP_510529§
Catalytic
VPS34
Pi3K_59F (DVps34p)
VPS34
DdPIK5 (Vps34p)
VPS34
Regulatory
PIK3R4 (p150)
Vps15-like
VPS15-like
VPS15-like
VPS15
Class IB
Class II
Class III
‡
§
*Mammals, birds and fish. There is homology between the D. discoideum PI3Ks and vertebrate Class I and II PI3Ks. S. pombe has a hypothetical Vps34 protein (gene
identification (ID) number 2543444). Gene names are given (protein name, if different from gene name, is give in parenthesis). §Although there are no published
reports on class II PI3K in C. elegans, locus NP_510529 is highly homologous to D. melanogaster class II PI3K (4e-157). AAP-1, AGE-1 adaptor protein;
AGE-1, ageing alteration 1; DdPIK1, D. discoideum phosphotidylinositol kinase 1; DdPIK2, D. discoideum phosphotidylinositol kinase 2; DdPIK3, D. discoideum phosphotidylinositol kinase 3; droPIK57, D. melanogaster regulatory subunit of the phosphotidylinositol 3-kinase; PI3K_59F, phosphotidylinositol 3-kinase 59F; PI3K_
68D, phosphotidylinositol 3-kinase 68D; PI3K_92D, phosphotidylinositol 3-kinase; PIK3C2α, phosphotidylinositol 3-kinase, class 2, α-polypeptide; PIK3C2β, phosphotidylinositol 3-kinase, class 2, β-polypeptide; PIK3C2γ, phosphotidylinositol 3-kinase, class 2, γ-polypeptide; PIK3CA, phosphotidylinositol 3-kinase, catalytic,
α-polypeptide; PIK3CB, phosphotidylinositol 3-kinase, catalytic, β-polypeptide; PIK3CD, phosphotidylinositol 3-kinase, catalytic, δ-polypeptide; PIK3R1,
phosphotidylinositol 3-kinase, regulatory subunit, polypeptide 1; PIK3R2, phosphotidylinositol 3-kinase, regulatory subunit, polypeptide 2; PIK3R3, phosphotidylinositol 3-kinase, regulatory subunit, polypeptide 3; PIK3CG, phosphotidylinositol 3-kinase, catalytic, γ-polypeptide; PIK3R4, phosphotidylinositol 3-kinase,
regulatory subunit 4; PIK3R5, phosphotidylinositol 3-kinase, regulatory subunit 5, p101; PIK3R6: p87 phosphotidylinositol 3-kinase γ-adaptor protein; Vps15,
vacuolar protein-sorting defective 15; VPS34, vacuolar protein-sorting defective 34 (a phosphotidylinositol 3-kinase, class III).
that have been examined. The class I PI3Ks in D. discoideum
are crucial for chemotaxis, and have a similar function in
mammalian neutrophils14.
As discussed above, mammals have evolved two
subfamilies of class I PI3Ks — IA and IB — which
vary in their mode of regulation. Both C. elegans and
D. melanogaster have a single class IA PI3K, which controls growth and metabolism downstream of an insulinlike receptor. In fact, the entire insulin–PI3K–v-akt
murine thymoma viral oncogene homologue (AKT)–
forkhead box (FOXO) signalling pathway is highly
conserved from C. elegans to mammals (BOX 2; FIGS 1,2).
Although the function of class II PI3Ks remains
poorly understood, these enzymes are present in both
D. melanogaster and C. elegans as well as in higher eukaryotes. By contrast, class IB PI3Ks are found only in vertebrates
and might therefore have evolved more recently.
Neutrophils
Leukocytes that are a primary
defence against bacterial
infection. Neutrophils show
significant chemotaxis in
response to chemokines.
Budding and fission yeast. The only PI3K in
Saccharomyces cerevisiae, Vps34, regulates Golgi-toendosome vacuolar protein sorting 8. Vps34 localizes
to endosomal membranes and is constitutively associated with the protein serine/threonine kinase Vps15
(REF. 15) . A temperature-sensitive mutant of Vps34
that has defective lipid kinase activity at the restrictive
temperature causes missorting of vacuolar proteins16.
The lipid product of Vps34, phosphatidylinositol
3-phosphate (PI-3-P), mediates the recruitment of
specific cytosolic proteins to the endosome by FYVE
or PX domains17. Mammalian orthologues of Vps34
and Vps15, together with PI-3-P-binding proteins, also
NATURE REVIEWS | GENETICS
regulate endosomal vesicle trafficking18. Given the recent
discovery that mammalian Vps34 mediates mTOR activation in response to amino-acid repletion, it will be
interesting to learn whether Vps34 regulates nutrient
sensing in yeast.
The fission yeast Schizosaccharomyces pombe also
possesses Vps34 as the sole PI3K isoform. Unexpectedly,
S. pombe has been shown to accumulate both phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and
phosphatidylinositol 3,4,-bisphosphate (PI-3,4-P 2 )
on deletion of ptn1, which is its orthologue of mammalian PTEN (phosphatase and tensin homologue)19.
S. pombe ptn1 deletion results in abnormal vacuole morphology and increased osmotic fragility. Although the
biochemical targets of PI-3,4-P2 and PIP3 in S. pombe are
unknown, S. pombe orthologues of the mammalian PIP3binding proteins, PDK1 (3-phosphoinositide-dependent
kinase 1) and GRP1 (general receptor for phosphatidylinositol 1), have PH domains that might
mediate PIP3 responses. It is possible that S. pombe generates PIP3 and PI-3,4-P2 by phosphorylation of PI-3-P
at the 4′ and 5′ positions through a PIP 5-kinase.
It is therefore tempting to speculate that PIP3 signalling might have emerged before the evolution of class I
PI3Ks, and that only later did class I PI3Ks arise as a
dedicated mechanism of synthesizing PIP3 in response to
extracellular signals.
C. elegans. The C. elegans insulin–PI3K signalling pathway represents an early adaptation event in evolution,
whereby PI3K signalling was used to regulate nutrient
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sensing and metabolism in a multicellular organism.
A single class IA PI3K is present in the nematode
C. elegans. The C. elegans p110 was originally identified as age-1, which is a gene involved in the regulation of development and lifespan20. Loss-of-function
mutations in age-1 lead to the constitutive formation
of dauer larvae regardless of environmental cues; by
contrast, less severe hypomorphic mutations in age-1
allow normal development of the worm but result in
increased lifespan and elevated body fat storage in the
adult21. Mutations in the C. elegans AGE-1 adaptor protein (aap-1) gene, which encodes a p55-like regulatory
subunit of PI3K, yield phenotypes similar to those of
age-1 mutations22.
This PI3K signalling pathway operates downstream
of an insulin-like receptor. In fact, genetic experiments
have established that the C. elegans insulin–PI3K signalling pathway — comprising the insulin-like receptor
abnormal dauer formation-2 (DAF-2) and its insulin
receptor substrate (IRS)-like adaptor (IST-1), class
IA PI3K (AGE-1 and AAP-1), AKT and the forkhead
transcription factor (DAF-16) — is a crucial regulator
of development, metabolism and lifespan21 (FIG. 2). As
in mammalian cells, this pathway is antagonized by
the PTEN phosphatase DAF-18 (REF. 23). The increased
lifespan conferred by hypomorphs of this pathway
probably results from slower metabolism and/or
resistance to oxidative stress24.
Canonical
C. elegans
D. melanogaster
Mammals
Insulin-like
receptor
DAF-2
INR
Insulin and IGF1
receptors
IRS adaptor
proteins
IST-1
Chico
IRS
Class IA PI3K
AGE-1/AAP-1
Dp110/p60
p110/p85
PTEN
DAF-18
PTEN
PTEN
AKT
AKT
AKT
AKT
Forkhead
DAF-16
FOXO
FOXO
Dauer formation
Metabolism
Longevity
Growth
Metabolism
Longevity
Growth
Metabolism
Tumorigenesis
Figure 2 | The insulin–phosphatidylinositol 3-kinase (PI3K) signalling pathway is
conserved in eukaryotic evolution. The corresponding orthologues for the
components of PI3K signalling in Caenorhabditis elegans, Drosophila melanogaster
and mammals are illustrated. In both C. elegans and D. melanogaster there is a single
gene for each of the components, whereas mammals have several isoforms of each
component with the possible exception of PTEN (phosphatase and tensin
homologue). In addition, in mammals, metabolism and growth are separately
regulated by the highly homologous insulin and insulin growth factor 1 (IGF1)
receptors, respectively. AGE-1, ageing alteration 1; AAP-1, AGE-1 adaptor protein;
AKT, v-akt murine thymoma viral oncogene homologue 1; DAF-2, abnormal dauer
formation-2; FOXO, forkhead family of transcription factor; INR, insulin receptor;
IRS, substrate; IST-1, insulin receptor substrate (IRS)-like adaptor.
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D. melanogaster. The D. melanogaster class IA PI3K
consists of a catalytic subunit (Dp110) and a p55-like
regulatory subunit (p60). As for C. elegans and mammals, the conserved D. melanogaster insulin–PI3K
signalling pathway — comprising D. melanogaster
insulin receptor (INR), IRS protein (Chico), class IA
PI3K (DP110 and p60) and AKT — crucially regulates
metabolism, growth and lifespan25. As in mammals and
C. elegans, a PTEN orthologue (dPTEN) negatively regulates the pathway. Hypomorphic mutants that decrease
the activity of this pathway accumulate more body fat,
show elevated body sugar content and have an extended
lifespan. Genetic experiments have shown that this pathway also controls cell size, cell number and organ size in
D. melanogaster. For example, mosaic deletion of Dp110
or p60 leads to an autonomous reduction in cell size and
cell number, whereas deletion of the fruitfly PTEN leads
to an increase in cell size and organ size24. Recent studies
also indicate that this pathway coordinates cell growth
with cell differentiation during D. melanogaster organ
development26. Therefore, in D. melanogaster, the PI3K
signalling pathway has evolved to regulate cell growth as
well as metabolism. The regulation of cell size in fruitflies by PI3K is, at least in part, mediated by the crosstalk
between AKT and the D. melanogaster TSC1–TSC2
complex that controls TOR signalling27,28 (BOX 2). The
regulation of cell number by PI3K, however, seems to
depend on the D. melanogaster forkhead transcription
factor FOXO29. Both of these mechanisms are conserved
in mammals.
Control of metabolism: implications for diabetes
Mammals possess all three classes of PI3K (TABLE 1).
The in vivo functions of the various isoforms of PI3K in
mammals have begun to be addressed by gene-deletion
studies in mice. As discussed in detail below, these studies highlight the importance of class IA PI3K signalling
in regulating growth and metabolism. Furthermore,
dysregulation of this pathway is crucial for the pathophysiology of several human diseases: attenuated PI3K
signalling downstream of the insulin receptor is a major
contributor towards type-2 diabetes (also known as
non-insulin-dependent diabetes mellitus), whereas
mutations that lead to the amplification of PI3K signalling are among the most common mutations in
human cancers.
Genetic studies of insulin signalling in mice. In mammals, the PI3K–AKT signalling pathway has a central
role in the regulation of metabolism downstream
of the insulin receptor and IRS adaptor molecules
(reviewed in REF. 30) (BOX 2). For example, insulinmediated glucose uptake and membrane translocation of the glucose transporter GLUT4 (in adipocytes
and myocytes) can be blocked by the PI3K inhibitors
LY294002 and wortmannin, as well as by dominantnegative forms of AKT or by RNAi against AKT2
(REFS 31,32) . In hepatocytes, PI3K–AKT signalling
inhibits the forkhead-mediated transcription of gluconeogenic enzymes and therefore suppresses hepatic
glucose production33 (BOX 2).
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Box 2 | Phosphatidylinositol 3-kinase (PI3K) signalling pathways
Most of our understanding of PI3K signal transduction is based on studies of class I PI3Ks (centre panel of the figure).
Phosphatidylinositol-3,4,5-trisphosphate (PIP3; shown in orange) is a lipid second messenger that activates many
downstream molecules by binding to their pleckstrin-homology (PH) domains17. The protein serine/threonine kinase
AKT (also known as PKB) is a principal target of PIP3 (REFS 105,106). Binding of PIP3 to AKT leads to the membrane
recruitment of AKT and subsequent phosphorylation by the mTOR (mammalian target of rapamycin)–rictor kinase
complex107 and by PDK1 (3-phosphoinositide-dependent kinase)108. This leads to the full activation of AKT, which in
turn phosphorylates many target proteins, thereby regulating a range of cellular functions. An important AKT target is
the forkhead (FOXO) family of transcription factors. AKT-mediated phosphorylation of FOXO proteins leads to their
inactivation through cytoplasmic sequestration by 14-3-3 proteins1. The main biological functions of these signalling
pathways are described below.
Cell metabolism
In muscle and fat, AKT promotes glucose uptake by stimulating the membrane translocation of the glucose transporter
GLUT4 (REF. 33). In addition, AKT activates glycogen synthase through the inhibition of glycogen synthase kinase 3 (GSK3)
(REF. 109) and regulates fatty-acid synthesis by activating ATP citrate lyase (ATP-CL)110. In the liver, AKT inhibits
gluconeogenesis by blocking FOXO-mediated transcription of gluconeogenic enzymes, such as phosphenolpyruvate
carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase)111.
Cell cycle and cell survival
AKT promotes G1–S cell-cycle transition by blocking FOXO-mediated transcription of cell-cycle inhibitors, such as
p27Kip1 and RBL2 (retinoblastoma-like 2) (REF. 112). AKT might also directly phosphorylate and inactivate p27Kip1. In
addition, AKT indirectly stabilizes the cell-cycle protein c-Myc and cyclin D1 through the inhibition of GSK3 (REF. 113).
AKT promotes cell survival by blocking FOXO-mediated transcription of pro-apoptotic proteins such as FasL (Fas-ligand)
and Bim (BCL2-like 11). AKT can also directly phosphorylate the pro-apoptotic protein BAD (BCL2-antagonist of cell
death), causing its inactivation by 14-3-3 binding. Furthermore, AKT can phosphorylate MDM2 (transformed 3T3
cell double-minute 2 p53-binding protein), leading to p53 degradation113.
Protein synthesis
AKT phosphorylates the tuberous sclerosis complex 2 (TSC2) protein tuberin, and therefore inhibits the GTPase-activating
protein (GAP) activity of the TSC1–TSC2 complex towards Rheb (small G protein Ras homologue enriched in brain). This
allows GTP-bound Rheb to accumulate and activate the mTOR–raptor kinase complex, which in turn mediates
phosphorylation of 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) and p70S6Kinase, ultimately leading
to increased protein synthesis114.
Cell polarity and motility
PI3K, together with the small GTPase Rac and Cdc42, regulates cell polarity and motility by controlling actin dynamics in
motile cells14.
Vesicle sorting
Class III PI3K regulates proper intracellular vesicle trafficking8 and may mediate amino-acid-induced mTOR activation9,10.
Amino acids
RTKs, GPCRs
PI
Class III PI3K
Endosomal
proteins
Vesicle sorting
LKB1
PI-3-P
RTKs?
Class I PI3K
Hypoxia
Class II PI3K
PTEN
PIP3
P-Rex
AMP
PI-4-P
PIP2
PTEN?
MTM1
MTMRs
SHIP
PI-3,4-P2
Rictor–TOR
Rac Cdc42
Actin
rearrangement
PDK1
AKT
PHLPP
AMPK REDD1
ATPCL
AS160
TSC1–TSC2
14-3-3
Rheb
GSK3
PDK1
Glycogen
synthase
PEPCK,
G6Pase
Metabolism
FOXO
Raptor–TOR
MDM2 BAD
4EBPs
GLUT4
translocation
S6K
EIF4E
S6
Protein synthesis
FasL,
p53 BCL-XL Bim
Cell survival
p27Kip1,
Myc,
RBL2 cyclin D1
Cell cycle
BCL-XL, B-cell lymphoma-like protein 1; GPCR, G-protein-coupled receptor; LKB1, Ser/Thr protein kinase 11; MTM1,
myotubularin 1; MTMR, MTM-related protein; PI, phosphatidylinositol; REDD1, regulated in development and DNA damage
response, RTP801; RTK, receptor tyrosine kinase; SHIP, SH2-domain-containing inositol-5-phosphatase.
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Knockout and transgenic mice have ascertained the
role of class IA PI3K in mediating insulin signalling in
vivo (reviewed in REF. 2). Mice with germline deletion
of either p110α or p110β die early during embryogenesis, whereas mice that are doubly heterozygous for
p110α and p110β, and mice that are heterozygous
for a kinase-dead knock-in allele of p110α, exhibit glucose
intolerance34,35. In addition, tissue-specific deletion of
PTEN in muscle, fat or liver leads to enhanced insulin
sensitivity in these tissues36.
Given the crucial role of p85 in stabilizing p110 and
recruiting it to receptors or adaptors, it was initially
surprising that mice lacking individual isoforms of
p85 showed enhanced insulin sensitivity (reviewed in
REF. 2). Subsequent studies indicated that, in many cell
types, p85 is stoichiometrically in excess of p110, and
that free p85 can act as a dominant negative to inhibit
PI3K signalling37. Recently, we showed that free p85 can
form a cytoplasmic sequestration complex with tyrosinephosphorylated IRS1 protein, therefore limiting the ability of IRS1 to activate PI3K at the membrane38. These
findings provide a mechanistic explanation for why a
partial reduction in total p85 levels can lead to enhanced
PI3K signalling even under conditions in which total PI3K
bound to IRS1 is reduced.
The relative contribution of class IA PI3K signalling
in individual tissues to whole-body glucose regulation
is now being addressed using mice in which the Pik3r1
gene is deleted in specific tissues. These studies indicate
that the insulin-hypersensitivity phenotype observed
in germline Pik3r1 knockout mice can be attributed to
increased insulin sensitivity in the liver 166.
A near complete loss of class IA PI3K signalling
can be accomplished by crossing mice with tissuespecific loss of Pik3r1 to mice with germline deletion
of Pik3r2. In contrast to mice lacking individual p85
isoforms, mice deficient in all p85 isoforms in either
muscle or the liver exhibit severely impaired insulin
signalling in these tissues39,40. These findings show that
class IA PI3K is essential in mediating insulin action
in vivo. Interestingly, the loss of PI3K signalling in
the muscle results in impaired muscle glucose uptake,
whole-body glucose intolerance, hyperlipidaemia and
adiposity, whereas the loss of PI3K signalling in the
liver leads to unregulated hepatic gluconeogenesis,
hyperglycaemia, hyperinsulinaemia and reduced
circulating lipids.
Taken together, these genetic studies highlight two
interesting aspects of class IA PI3K signalling downstream of the insulin receptor. First, the p85 regulatory
subunit exerts both positive and negative effects on class
IA PI3K signalling. It is required for the stabilization
of p110 and for the activation of PI3K by the insulin
receptor, but free p85 negatively regulates class IA PI3K
signalling. Therefore, a partial reduction in p85 levels
leads to improved PI3K signalling and increased insulin
sensitivity in vivo. Second, PI3K signalling mediates different cellular responses depending on the tissue context,
and defective PI3K signalling in many tissues contributes
collectively to the complex metabolic defects associated
with type-2 diabetes.
612 | AUGUST 2006 | VOLUME 7
Implications for diabetes in humans. Peripheral insulin resistance is thought to occur at the proximal end
of insulin signalling, where the activation of PI3K is
attenuated41,42. Several polymorphisms in the human
PIK3R1 gene are associated with increased risk of type-2
diabetes43,44, although it is unclear how these changes
affect p85 expression or function. Interestingly, in a
mouse model of pregnancy-induced insulin resistance,
human placental growth hormone specifically increased
the level of p85 in muscle without changing the expression of p110, therefore leading to muscle insulin resistance45. This effect was abrogated in mice heterozygous for
the Pik3r1 gene46. Elevated levels of p85 have also been
recently observed in women with pregnancy-induced
insulin resistance47. Similarly, elevated levels of p85, but
not p110, were observed in muscles of type-2 diabetic
individuals48. These findings indicate that increased levels of p85 might contribute to muscle insulin resistance
in diabetes. Another mechanism that attenuates PI3K
signalling is the serine phosphorylation of IRS proteins,
particularly IRS1, by numerous kinases, such as the
c-Jun N-terminal kinase (JNK). Serine phosphorylation of IRS1 is associated with cellular oxidative stress,
which frequently results from obesity. JNK-dependent
serine phosphorylation inhibits the tyrosine phosphorylation of IRS1, thereby uncoupling IRS1 from PI3K
(see below)49.
The importance of the PI3K pathway in the regulation of metabolism has been confirmed by mutations
of other components of the pathway. For example,
germline deletion of AKT2, which is an AKT isoform
that is abundant in muscle and liver, results in a diabetic
phenotype in the mouse50. The relevance of this model
for human disease is supported by the identification of
a point mutation in AKT2 in a familial form of severe
insulin resistance51.
Over the next several years, it will be imperative
to define the molecular mechanisms by which insulin
resistance occurs in various tissues in the human form
of the disease. The relative contributions of changes in
p85–p110 ratios, IRS coupling to PI3K, genetic mutations or polymorphisms in components of the PI3K
signalling pathway and other mechanisms need to be
assessed to identify important points for intervention. In
addition, the contributions from isoforms of p110 and
AKT need to be identified, to exploit isoform-specific
interventions. Recent studies using isoform-selective
inhibitors of p110 have indicated that p110α is the
main catalytic isoform activated by insulin signalling35,52. Such observations point to potential limitations in the use of p110α-specific inhibitors for the
treatment of cancer (see below).
Control of growth: implications for cancer
Class IA PI3Ks regulate growth and proliferation downstream of growth factor receptors. Whereas C. elegans and
D. melanogaster use the same receptor (a homologue of
insulin and IGF1 receptors) to regulate both metabolism
and development, vertebrates have distinct receptors
for insulin and IGF1, and therefore have the ability to
independently control acute glucose homeostasis and
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REVIEWS
long-term growth and development. Although the IGF1
receptor and insulin receptor are highly homologous and
activate overlapping downstream signalling components
(including IRS proteins and class IA PI3K), the IGF1
receptor primarily regulates growth and development,
and has only a minor function in metabolism53. Mice with
germline deletion of the IGF1 receptor are severely growth
retarded and die soon after birth from respiratory failure54.
It is not clear how the insulin and IGF1 receptors mediate
different cellular responses by using the same PI3K–AKT
signalling pathway, although both distinct tissue distribution and differential activation of downstream signalling
molecules probably contribute to their differential
effects55. In addition to the IGF1 receptor, other growth
factor receptors — such as the platelet-derived
growth factor (PDGF), Met proto-oncogene (Met) and
epidermal growth factor (EGF) receptors — also activate
class IA PI3Ks, thereby illustrating the adoption of this
pathway by other extracellular cues1.
PI3K signalling and cell growth. In post-mitotic cells,
such as myocytes, PI3K signalling controls cell growth
(that is, cell size), as in D. melanogaster. For example,
over-expression of either the IGF1 receptor 56, a constitutively active form of p110α (REF. 57) or a constitutively
active form of AKT (REF. 58) in the heart results in
increased organ and cell size, whereas over-expression of
a dominant-negative form of p110α reduces heart size.
Furthermore, the expression of a dominant-negative
form of p110α suppresses the growth-promoting effect
of the IGF1 receptor in the heart56. Tissue-specific deletion of both Pik3r1 and Pik3r2 in the heart59 or muscle39
results in mice with reduced organ size owing to diminished cell size. Accordingly, tissue-specific deletion of
PTEN leads to increased cell size60–63, whereas mice with
germline deletion of AKT1 or AKT3 show growth retardation (reviewed in REF. 64). It is interesting to speculate
that the increased cell size is due to enhanced activation
of mTOR by PI3K (BOX 2).
Polyoma middle T
A protein produced by the
polyoma virus that can
transform cells by interacting
with crucial cellular proteins,
such as PI3K.
Haploinsufficiency
A gene is haploinsufficient
when loss of one functional
copy in a diploid organism
results in a phenotype,
because a single copy of the
normal gene is incapable of
providing sufficient protein for
normal function.
PI3K signalling and tumorigenesis. Over the past 20
years, several pivotal studies have established the integral
role of PI3K in tumorigenesis. In the 1980s, PI3K was
discovered because of its association with oncoproteins65.
Mutants of polyoma middle T that failed to bind PI3K were
compromised in their ability to transform fibroblasts66
and polyoma middle T-transformed cells had elevated
levels of PIP3 (REF. 67). A few years later, the p110α
catalytic subunit of PI3K was identified as an avian
retrovirus-encoded oncogene that could transform chick
embryo fibroblasts in vitro68.
Genetic analyses of human tumours performed in the
1990s showed that a locus on chromosome 10q23 was
frequently deleted in advanced cancers. The tumoursuppressor gene located in this region was predicted, on
the basis of its sequence, to encode a phosphatase and
was named PTEN (also called MMAC1 and TEP1)69,70.
Two years later, PTEN was shown to function as a PIP3
3′-phosphatase71, indicating that it functioned as a
tumour suppressor because of its ability to turn off the
PI3K pathway. This idea has been further strengthened
NATURE REVIEWS | GENETICS
by the recent discovery of several genetic mutations in
the pathway in human cancers. As illustrated in TABLE 2,
genetic alterations in several proteins in the PI3K signalling pathway — including p85, p110α, PTEN and AKT —
are observed in cancers. Furthermore, mouse-knockout
and transgenic models confirm the tumorigenic potential
of hyperactivation of this pathway. As shown in BOX 2,
PI3K activity induces many downstream pathways to
promote tumorigenesis. In BOX 3, we specifically highlight PI3K–AKT regulation of mTOR, BCL2-antagonist
of cell death (BAD) and FOXO as potential tumorigenic
mechanisms.
Functional genetic studies of PTEN. Germline mutations in the PTEN gene result in Cowden disease and
Bannayan–Riley–Ruvalcaba syndrome (also known as
macrocephaly, multiple lipomas and haemangiomata),
which are two familial diseases characterized by benign
tumours and high risk of cancer72. Somatic mutations,
gene deletion or gene inactivation of PTEN occurs in
a sizeable fraction of glioblastomas, prostate cancers,
breast cancers and melanomas (TABLE 2). As shown in
TABLE 2, the frequency of loss of heterozygosity for PTEN
far exceeds the frequency of biallelic inactivation. This
indicates that haploinsufficiency for PTEN might suffice
in promoting tumorigenesis in certain cellular contexts.
This model is consistent with findings from an elegant
study showing that progressive reduction in Pten gene
dosage results in increasingly aggressive mouse prostate
neoplasia73. This feature distinguishes PTEN from the
more traditional tumour suppressors, such as p53, for
which biallelic inactivation is necessary for tumorigenesis. Epigenetic mechanisms, such as promoter
methylation, have also been implicated in diminished
PTEN protein expression in some tumours. Therefore,
reduced PTEN protein expression without genetic mutations is probably another mechanism of tumorigenesis
that is under-appreciated in studies that assess genetic
alterations in PTEN.
Genetic studies in mice support a role for PTEN
as a tumour suppressor. Mice heterozygous for Pten
exhibit neoplasia in several epithelial tissues, including
the intestine, prostate, endometrium and mammary
gland (reviewed in REF. 74). Tissue-specific homozygous
deletion of PTEN in the prostate epithelium leads to
aggressive prostate carcinoma73,75. In lymphocytes, loss
of PTEN leads to hyper-proliferation and resistance
to apoptosis76,77.
p110α catalytic subunit of PI3K. The PIK3CA (phosphatidylinositol 3-kinase, catalytic, α-polypeptide)
gene that encodes p110α is frequently amplified in
several human cancers, such as head and neck cancers, cervical cancers, gastric cancers and lung cancers
(TABLE 2) . Recently, point mutations in p110α were
identified in significant fractions of brain cancers, colon
cancers, breast cancers and hepatocellular cancers78
(reviewed in REF. 79) (TABLE 2). Interestingly, most point
mutations in p110α cluster around two hotspots: E545
in the helical phosphatidylinositol kinase homology
domain and H1047 near the end of the catalytic domain.
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Table 2 | Frequency of mutations in the PI3K–AKT pathway in cancers
Genetic mutations
Cancer type
Percentage
frequency*
References
Breast
26% (176/684)
Colon
26% (88/337)
78,132
Glioma
8% (14/182)
78,133,
134
Hepatocellular
36% (26/73)
128
Ovarian
10% (35/365)
Lung
2% (4/253)
78,128
Gastric
7% (24/338)
78,128,
132,135
PIK3CA (p110α)
Mutations
Amplifications
Head and neck
42% (54/128)
Thyroid
9% (12/128)
Lung:
78,
126–131
127,130
136,137
138
139,140
Squamous cell
66% (46/70)
Adenocarcinoma
5% (4/86)
Breast
9% (8/92)
126
Gastric
36% (20/55)
141
Oesophageal
adenocarcinoma
6% (5/87)
142
Cervical
69% (11/16)
143
Glioblastoma
54% (98/180)
144–146
Prostate
35% (88/250)
147–151
Breast
23% (37/164)
152,153
Melanoma
37% (53/143)
154–157
PTEN
Loss of
heterozygosity
Mutations‡
Gastric
47% (14/30)
Glioblastoma
28% (122/432)
144–146,
158–160
141
Prostate
12% (26/218)
147–151,
161
Breast
0% (0/164)
152,153
Melanoma
8% (15/185)
154–157,
162
Gastric
0% (0/30)
141
AKT
Amplifications
Ovarian
12% (18/147)
Pancreatic
20% (7/35)
84,163
86
Breast
3% (3/106)
84
Gastric
20% (1/5)
164
Head and neck
30% (12/40)
136
Ovarian
4% (3/80)
165
Colon
2% (1/60)
165
PIK3R1 (p85α)
Mutations
*Raw numbers are presented in parentheses. ‡Includes mutations and homozygous deletions.
AKT, v-akt murine thymoma viral oncogene homologue 1; PIK3CA, phosphatidylinositol
3-kinase, catalytic, α-polypeptide; PIK3R1, phosphatidylinositol 3-kinase, regulatory subunit,
polypeptide 1; PTEN, phosphatase and tensin homologue.
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These mutations increase in vitro PI3K activity of the
holoenzyme 78,80,81. The expression of these p110α
mutants in cells confers AKT activation in the absence
of growth factor stimulation80–82. Significantly, these
mutants can transform fibroblasts and mammary epithelial cells80,81, and drive tumour formation in mouse
xenografts83. Interestingly, mutations in other p110 isoforms have not been identified in cancers, indicating that
p110α harbours more oncogenic potential.
Amplification of AKT2 has also been found in human
cancers, indicating that the tumorigenic effect of aberrant
PI3K signalling is at least partly mediated through AKT84–86.
Recently, AKT2 mutations and IRS2 amplifications
were also observed in colon cancers87.
PI3K negative-feedback regulation
In D. melanogaster, it was noted that the absence of
TSC1–TSC2 led to reduced AKT activity that was subsequently restored on removal of p70S6Kinase, which is
activated by mTOR–raptor88 (BOX 2). This finding was
the first to indicate a negative-feedback mechanism by
which mTOR attenuates PI3K activity (FIG. 3). Subsequent
studies have shown that mTOR and p70S6kinase function at
the level of IRS1 to inhibit PI3K (reviewed in REFS 89,90).
The activated mTOR–raptor complex and p70S6Kinase cause
serine phosphorylation of IRS1, making it a less effective
substrate for insulin and IGF receptors, and a better target for proteosomal degradation. These effects on IRS1
are reversible by prolonged treatment with rapamycin
(6–10 h). In diabetes and obesity, serine phosphorylation
of IRS1 by JNK and PKCθ at rapamycin-insensitive sites
also attenuates the tyrosine phosphorylation of IRS1, and
its ability to activate PI3K91–93. In addition, suppressors
of cytokine signalling (SOCS) proteins are upregulated
by inflammatory signals and have been implicated in
insulin resistance (reviewed in REF. 49).
It is possible that these feedback loops evolved in
multicellular organisms to coordinate growth and
nutrient utilization by individual cells with that of the
organism as a whole. The consequences of perturbing
these feedback loops have been illustrated in mouse
models of diabetes and cancer. On a high-fat diet, mice
lacking p70S6Kinase are protected from insulin resistance
and obesity by reduced serine-phosphorylation of
IRS1 and the preservation of robust insulin-stimulated
AKT activity94. Similarly, JNK1- and PKCθ-knockout
mice show increased insulin sensitivity on exposure to
high-fat diets, and increased levels of circulating lipids,
respectively 92,93.
Evidence for the importance of this negative-feedback
loop in controlling cancers comes from studies of mice
heterozygous for Tsc2 (REF. 95). Tsc2+/– mice develop
relatively benign haemangiomas owing to sporadic loss
of the functional Tsc2 allele. AKT is inactive in these
Tsc2–/– haemangiomas owing to feedback suppression of
PI3K signalling by a highly activated mTOR pathway.
However, the additional introduction of Pten heterozygosity was sufficient to circumvent such a negativefeedback loop and restore AKT activation, resulting in
more frequent and aggressive haemangiomas95. These
observations warrant caution when treating certain
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Box 3 | Tumorigenic mechanisms of PI3K–AKT signalling
Many downstream targets of AKT operate together to promote tumorigenesis.
Mammalian target of rapamycin (mTOR)–raptor kinase complex
The mTOR–raptor kinase complex is a crucial regulator of cellular growth and a convergence node of many
signalling pathways; mTOR is regulated by growth-factor-receptor signalling, nutrient availability and the energy
status of the cell.
As also described in BOX 2, mTOR regulates growth partly by promoting protein synthesis through 4E-BP1 and
p70S6Kinase, the latter of which is activated by PDK1 (3-phosphoinositide-dependent kinase). AKT activates mTOR–
raptor by directly phosphorylating the product of the tuberous sclerosis 2 (TSC2) gene product, tuberin, thereby
inhibiting the ability of the tuberin–hamartin complex (TSC1/TSC2) to function as a GTPase-activating protein
(GAP) for Rheb (Ras-like GTP-binding protein)27,28,115. This activation occurs acutely and transiently with stimulation
of receptor tyrosine kinases (RTKs) in normal tissues, but can occur chronically when PTEN (phosphatase and
tensin homologue) or PIK3CA (phosphatidylinositol 3-kinase, catalytic, α-polypeptide) are mutated (in cancers) or
when tuberin or hamartin are defective (in tuberous sclerosis patients). The importance of mTOR signalling in
promoting PI3K-dependent tumorigenesis is highlighted by the finding that mTOR–raptor inhibitors, rapamycin
and its analogues, abrogate AKT-driven prostate intraepithelial neoplasia in a transgenic mouse model116, and
inhibit tumorigenesis in PTEN-deficient cancer cell lines117,118.
Intracellular energy levels also regulate mTOR signalling. LKB1, which is a serine/threonine kinase that is
mutated and inactivated in the familial Peutz–Jegher syndrome and lung cancers, phosphorylates and activates
AMP kinase (AMPK) when AMP levels are high (that is, low energy states). AMPK in turn phosphorylates tuberin,
but, unlike phosphorylation by AKT, this activates its GAP activity, leading to inhibition of mTOR activity (reviewed
in REF. 119). However, in LKB1-deficient cells, AMPK cannot be activated, and mTOR remains constitutively active
under conditions of energy stress.
mTOR is also regulated by amino-acid availability, and recent studies suggest that Vps34 (human vacuolar
protein-sorting defective 34), the class III PI3K, mediates this activation9,10.
The regulation of mTOR by growth-factor-receptor signalling, nutrient availability and the energy status of the
cell ensures that a cell normally commits to growth after receiving appropriate stimuli and environmental cues.
Transformed cells, however, bypass one or more of these control mechanisms to grow without restraint.
Pro-apoptotic proteins
Another AKT target that probably contributes to tumorigenesis is BAD (pro-apoptotic protein BCL2-antagonist of
cell death)120,121. Phosphorylation of these sites causes BAD to bind to 14-3-3, sequestering it in the cytoplasm and
preventing its pro-apoptotic effects122,123.
FOXO proteins
AKT phosphorylates and inactivates the forkhead (FOXO) family of transcription factors. In lower metazoans,
Foxo proteins promote the expression of pro-apoptotic genes, such as Bim and Fas. In mammalian cells, FOXO
also promotes expression of p27Kip1 and RBL2 (retinoblastoma-like 2) to inhibit cell-cycle entry (reviewed in
REF. 124). However, mice deficient for individual FOXO family members do not show increased tumour
incidence. It is likely that the many members of this family have redundant functions, and that more than one
isoform must be deleted for tumours to arise. Interestingly, translocations involving FOXO1, FOXO2 and FOXO4
that result in dominant negatives for FOXO function have been observed in some rhabdomyosarcomas and
leukaemias125.
AKT possesses other targets that are likely to be important in promoting tumorigenesis (for example, the p53 E3
ligase, MDM2). Furthermore, it is probable that effectors of phosphatidylinositol-3,4,5-trisphosphate (PIP3) other
than AKT are also important in tumorigenesis.
AMP (low energy)
AMPK
TSC1/TSC2
Rheb
Amino acids
Class III
PI3K
RTKs
PIK3CA mutations
PTEN loss
PIP3
LKB1
AKT
PDK1
mTOR–raptor
14-3-3
4EBPs
S6K
MDM2
BAD
EIF4E
S6
p53
BCL-XL
FasL,
Bim
FOXO
GSK3
p27Kip1,
RBL2
c-myc,
cyclin D1
BCL-XL, B-cell lymphoma-like protein 1; FasL, Fas-ligand; GSK3, glycogen synthase kinase 3.
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cancer patients with rapamycin: a subset of tumours
in which AKT activity is suppressed by mTOR might
respond to rapamycin, paradoxically, with increased
PI3K–AKT signalling and a more aggressive phenotype.
In fact, this feedback has now been observed in certain
cancers treated with rapamycin analogues96.
Therapeutic approaches
Cancer. Genetic analyses of tumours have shown that
aberrant PI3K signalling is a crucial oncogenic stimulus
in several cancer types. How can this information be used
for treating human cancers? As we have learned from the
successful response of lung cancers to EGFR inhibitors,
of breast cancers to antibodies directed against HER2
(human epidermal growth factor receptor 2) and of leukaemias to ABL (Abelson) tyrosine kinase inhibitors, the
genetic mutations of individual cancers point to effective
therapeutic strategies. Therefore, it is plausible that cancers harbouring mutations in the PIK3CA gene or PTEN
are ‘addicted’ to PI3K signalling and will be particularly
sensitive to inhibitors of this pathway. Indeed, it will be
interesting to learn whether rapamycin analogues, which
are currently in clinical trials, are effective treatments for
cancers with these mutations. The rapamycin-induced
activation of AKT might be less detrimental in cancers
with PIK3CA or PTEN mutations given that their PI3K/
AKT signalling is already highly active. By contrast,
rapamycin analogues might be least effective (or even
detrimental) in those cancers in which PI3K activation
is attenuated by mTOR.
Receptor
PI3K
Adaptor
AKT
TSC1/2
mTOR
Cell survival
Therapeutic index
A measure of the benefit of a
drug compared with its toxic
effects. Quantitatively, it is the
ratio of the dose required to
produce the toxic effect and
the therapeutic dose.
Floxed allele
A genetically engineered gene
(or part of a gene) that is
flanked by loxP sites; on
exposure to the Cre
recombinase, the loxP sites
recombine, resulting in
permanent loss of the
intervening DNA. This
methodology is often used to
conditionally knock out a gene
spatially or temporally.
Cell growth
S6 kinase
As many classes and isoforms of PI3K control distinct
cellular processes, isoform-specific inhibitors might be less
toxic and have a higher therapeutic index. Isoform-specific
inhibitors of p110α will be of great interest for treating
cancers that have PIK3CA mutations. However, as p110α
has recently been implicated as a key mediator of insulin
signalling35,52, the use of p110α inhibitors could be limited
by their diabetic effects. Additionally, if these inhibitors
have activity against other isoforms, such as p110γ and
p110δ, they might cause other untoward effects, such
as immunosuppression. Isoform-specific inhibitors of
AKT might provide effective anti-tumour activity with
fewer toxic side effects. For example, AKT1 inhibition
might shrink some tumours with minimal impact on
glucose homeostasis, which is regulated primarily by
AKT2. Furthermore, targeting upstream mechanisms
that lead to PI3K activation might also prove effective in
treating cancer cells and circumvent the metabolic side
effects of PI3K inhibitors. For example, our laboratory
recently showed that non-small-cell lung cancer (NSCLC)
cell lines that use ERBB3 (v-erb-b2 erythroblastic
leukaemia viral oncogene homologue 3) to couple to
class IA PI3K are uniquely sensitive to EGFR inhibitors97. However, these approaches are less likely to be
successful in cancers in which PTEN is deleted or PI3K
is mutated.
Diabetes. As discussed above, there is a significant
body of evidence indicating that attenuated PI3K activation is a key defect in type II diabetes. Agents that
improve the activation of PI3K by IRS protein should
improve peripheral insulin sensitivity. In particular,
obesity-induced insulin resistance is at least partly due
to p70S6Kinase- and JNK-mediated serine phosphorylation of IRS1. Therefore, inhibitors of these kinases
might be effective treatments, as illustrated in mice98.
Furthermore, inhibitors that target PKCθ might restore
insulin-induced activation of PI3K in obese and diabetic
conditions98. As increased levels of monomeric p85 are
associated with insulin-resistant conditions, agents that
specifically downregulate monomeric p85 expression or
upregulate p110α or p110β might also lead to improved
insulin sensitivity.
Rapamycin
Future directions
The tremendous progress made in our understanding
Figure 3 | Rapamycin-sensitive feedback on phosphaof the diverse roles of PI3K signalling over the past
tidylinositol 3-kinase (PI3K) signalling. PI3K is
20 years now puts us in a position to manipulate this
activated by binding to tyrosine-phosphorylated
pathway therapeutically for the treatment of cancers and
receptors or adaptors. Aberrant activation of PI3K
diabetes. As isoform-selective inhibitors of PI3Ks
results in increased cell growth and survival, in part by
activation of AKT. AKT phosphorylates and turns off
and downstream effectors (such as AKT) become availtuberin (TSC2), leading to the activation of mTOR
able both as research tools and as therapeutics, it is
(mammalian target of rapamycin)–raptor. mTOR
crucial to prudently assess their in vivo effects.
contributes to cell growth, and other targets of AKT
Highly specific inhibitors are likely to have fewer
contribute to cell survival and cell-cycle entry. Recently,
side effects than pan-PI3K inhibitors. Independent
it has been found that persistent activation of mTOR–
controls that involve the use of genetic approaches such
raptor results in a negative-feedback loop that turns off
as RNAi, acute deletion of a floxed allele or the induction
PI3K activators (dashed line). Therefore mTOR–raptor
inhibitors (for example, rapamycin) might, in some cases, of a dominant-negative protein function are required
as comparisons to determine the in vivo specificity of
limit tumour growth, but in other cases result in
these agents. As the complete removal of an enzyme
hyperactivation of PI3K and AKT signalling, and so
paradoxically promote tumour growth.
might have more severe consequences than its kinase
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© 2006 Nature Publishing Group
REVIEWS
inactivation (as illustrated by the case of p110γ in the heart,
reviewed in REF. 99) owing to the kinase-independent
functions of the protein, additional methods (such as
the acute replacement of the endogenous enzyme with a
catalytically inactive form) are more likely to mimic the
consequences of introducing a catalytic-site inhibitor. A
crucial control for the off-target effects of an inhibitor
is to show its inactivity in cells or animals expressing
a mutant form of the enzyme that remains functional
but is resistant to the inhibitor — for example, EGFR
T790M in the case of gefitinib (Iressa; AstraZeneca)100.
Indeed, ‘knock-in’ mice that are engineered to express
such mutant kinases will be highly valuable for defining
the off-target effects of an inhibitor.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
Cantley, L. C. The phosphoinositide 3-kinase pathway.
Science 296, 1655–1657 (2002).
Katso, R. et al. Cellular function of phosphoinositide
3-kinases: implications for development, homeostasis,
and cancer. Annu. Rev. Cell Dev. Biol. 17, 615–675
(2001).
Wymann, M. P. et al. Phosphoinositide 3-kinase γ:
a key modulator in inflammation and allergy. Biochem.
Soc. Trans. 31, 275–280 (2003).
Stephens, L. R. et al. The Gβγsensitivity of a PI3K is
dependent upon a tightly associated adaptor, p101.
Cell 89, 105–114 (1997).
Suire, S. et al. p84, a new Gβγ-activated regulatory
subunit of the type IB phosphoinositide 3-kinase
p110γ. Curr. Biol. 15, 566–570 (2005).
Voigt, P., Dorner, M. B. & Schaefer, M. Characterization
of p87PIKAP, a novel regulatory subunit of
phosphoinositide 3-kinase γ that is highly expressed in
heart and interacts with PDE3B. J. Biol. Chem. 281,
9977–9986 (2006).
Gaidarov, I., Smith, M. E., Domin, J. & Keen, J. H. The
class II phosphoinositide 3-kinase C2α is activated by
clathrin and regulates clathrin-mediated membrane
trafficking. Mol. Cell 7, 443–449 (2001).
Odorizzi, G., Babst, M. & Emr, S. D. Phosphoinositide
signaling and the regulation of membrane trafficking
in yeast. Trends Biochem. Sci. 25, 229–235 (2000).
Byfield, M. P., Murray, J. T. & Backer, J. M. hVps34 is
a nutrient-regulated lipid kinase required for activation
of p70 S6 kinase. J. Biol. Chem. 280, 33076–33082
(2005).
Nobukuni, T. et al. Amino acids mediate mTOR/raptor
signaling through activation of class 3
phosphatidylinositol 3OH-kinase. Proc. Natl Acad. Sci.
USA 102, 14238–14243 (2005).
Wurmser, A. E. & Emr, S. D. Novel PtdIns(3)P-binding
protein Etf1 functions as an effector of the Vps34
PtdIns 3-kinase in autophagy. J. Cell Biol. 158,
761–772 (2002).
Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y.
Two distinct Vps34 phosphatidylinositol 3-kinase
complexes function in autophagy and
carboxypeptidase Y sorting in Saccharomyces
cerevisiae. J. Cell Biol. 152, 519–530 (2001).
Stein, R. Prospects of phosphoinositide 3-kinase
inhibition as a cancer treatment. Endocr. Relat.
Cancer 8, 237–248 (2001).
Van Haastert, P. & Devreotes, P. Chemotaxis:
signalling the way forward. Nature Rev. Mol. Cell Biol.
5, 626–634 (2004).
Herman, P. K., Stack, J. H., DeModena, J. A. & Emr, S. D.
A novel protein kinase homolog essential for protein
sorting to the yeast lysosome-like vacuole. Cell 64,
425–437 (1991).
Stack, J. H., Herman, P. K., Schu, P. V. & Emr, S. D.
A membrane-associated complex containing the
Vps15 protein kinase and the Vps34 PI 3-kinase is
essential for protein sorting to the yeast lysosome-like
vacuole. EMBO J. 12, 2195–2204 (1993).
DiNitto, J. P., Cronin, T. C. & Lambright, D. G.
Membrane recognition and targeting by lipid-binding
domains. Sci. STKE 2003, re16 (2003).
Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. &
Balch, W. E. Role for phosphatidylinositol 3-kinase in
the sorting and transport of newly synthesized
lysosomal enzymes in mammalian cells. J. Cell Biol.
130, 781–796 (1995).
To make real-time non-invasive assessments of the
efficacy of new therapeutics in vivo, there is an increased
need to develop imaging technologies that can visualize
the activity of signalling pathways in vivo. For example,
if an isoform-specific inhibitor of p110α is ineffective
against a particular model of tumorigenesis in vivo, it is
important to determine whether inhibition of p110α
is an ineffective strategy or whether the drug simply fails
to inhibit p110α in the tumour.
Therapeutics that target the PI3K signalling network
hold great promise for many diseases, such as diabetes
and cancer. It is therefore with well-grounded optimism
that we anticipate that agents targeting this pathway will
show their promise in clinical trials in the near future.
19. Mitra, P. et al. A novel phosphatidylinositol(3,4,5)P3
pathway in fission yeast. J. Cell Biol. 166, 205–211
(2004).
20. Friedman, D. B. & Johnson, T. E. A mutation in the
age-1 gene in Caenorhabditis elegans lengthens life
and reduces hermaphrodite fertility. Genetics 118,
75–86 (1988).
21. Kenyon, C., Chang, J., Gensch, E., Rudner, A. &
Tabtiang, R. A C. elegans mutant that lives twice as
long as wild type. Nature 366, 461–464 (1993).
22. Wolkow, C. A., Munoz, M. J., Riddle, D. L. & Ruvkun, G.
Insulin receptor substrate and p55 orthologous
adaptor proteins function in the Caenorhabditis
elegans daf-2/insulin-like signaling pathway. J. Biol.
Chem. 277, 49591–49597 (2002).
23. Ogg, S. & Ruvkun, G. The C. elegans PTEN homolog,
DAF-18, acts in the insulin receptor-like metabolic
signaling pathway. Mol. Cell 2, 887–893 (1998).
24. Guarente, L. & Kenyon, C. Genetic pathways that
regulate ageing in model organisms. Nature 408,
255–262 (2000).
25. Oldham, S. & Hafen, E. Insulin/IGF and target of
rapamycin signaling: a TOR de force in growth control.
Trends Cell Biol. 13, 79–85 (2003).
26. Bateman, J. M. & McNeill, H. Temporal control of
differentiation by the insulin receptor/tor pathway in
Drosophila. Cell 119, 87–96 (2004).
27. Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K. L. TSC2 is
phosphorylated and inhibited by Akt and suppresses
mTOR signalling. Nature Cell Biol. 4, 648–657
(2002).
28. Potter, C. J., Pedraza, L. G. & Xu, T. Akt regulates
growth by directly phosphorylating Tsc2. Nature Cell
Biol. 4, 658–665 (2002).
References 27 and 28, along with the study in
reference 115, show that AKT regulates mTOR
activity by directly phosphorylating tuberin.
29. Junger, M. A. et al. The Drosophila forkhead
transcription factor FOXO mediates the reduction in
cell number associated with reduced insulin signaling.
J. Biol. 2, 20 (2003).
30. Taniguchi, C. M., Emanuelli, B. & Kahn, C. R. Critical
nodes in signalling pathways: insights into insulin
action. Nature Rev. Mol. Cell Biol. 7, 85–96 (2006).
31. Katome, T. et al. Use of RNA interference-mediated
gene silencing and adenoviral overexpression to
elucidate the roles of AKT/protein kinase B isoforms in
insulin actions. J. Biol. Chem. 278, 28312–28323
(2003).
32. Zhou, Q. L. et al. Analysis of insulin signalling by
RNAi-based gene silencing. Biochem. Soc. Trans. 32,
817–821 (2004).
33. Thong, F. S., Dugani, C. B. & Klip, A. Turning signals on
and off: GLUT4 traffic in the insulin-signaling highway.
Physiology (Bethesda) 20, 271–284 (2005).
34. Brachmann, S. M., Ueki, K., Engelman, J. A., Kahn, R. C.
& Cantley, L. C. Phosphoinositide 3-kinase catalytic
subunit deletion and regulatory subunit deletion have
opposite effects on insulin sensitivity in mice. Mol.
Cell. Biol. 25, 1596–1607 (2005).
35. Foukas, L. C. et al. Critical role for the p110α
phosphoinositide-3-OH kinase in growth and
metabolic regulation. Nature 441, 366–370 (2006).
36. Kurlawalla-Martinez, C. et al. Insulin hypersensitivity
and resistance to streptozotocin-induced diabetes in
mice lacking PTEN in adipose tissue. Mol. Cell. Biol.
25, 2498–2510 (2005).
NATURE REVIEWS | GENETICS
37. Ueki, K. et al. Positive and negative roles of p85α
and p85β regulatory subunits of phosphoinositide
3-kinase in insulin signaling. J. Biol. Chem. 278,
48453–48466 (2003).
38. Luo, J., Field, S. J., Lee, J. Y., Engelman, J. A. &
Cantley, L. C. The p85 regulatory subunit of
phosphoinositide 3-kinase down-regulates IRS-1
signaling via the formation of a sequestration
complex. J. Cell Biol. 170, 455–464 (2005).
39. Luo, J. et al. Loss of class IA PI3K signaling in muscle
leads to impaired muscle growth, insulin response,
and hyperlipidemia. Cell Metab. 3, 355–366
(2006).
40. Taniguchi, C. M. et al. Divergent regulation of hepatic
glucose and lipid metabolism by phosphoinositide
3-kinase via Akt and PKCλ/ζ. Cell Metab. 3, 343–353
(2006).
41. Saltiel, A. R. & Kahn, C. R. Insulin signalling and the
regulation of glucose and lipid metabolism. Nature
414, 799–806 (2001).
42. Shulman, G. I. Unraveling the cellular mechanism of
insulin resistance in humans: new insights from
magnetic resonance spectroscopy. Physiology
(Bethesda) 19, 183–190 (2004).
43. Hansen, T. et al. Identification of a common amino
acid polymorphism in the p85α regulatory subunit of
phosphatidylinositol 3-kinase: effects on glucose
disappearance constant, glucose effectiveness, and
the insulin sensitivity index. Diabetes 46, 494–501
(1997).
44. Barroso, I. et al. Candidate gene association study in
type 2 diabetes indicates a role for genes involved
in β-cell function as well as insulin action. PLoS Biol. 1,
e20 (2003).
45. Barbour, L. A. et al. Human placental growth hormone
increases expression of the p85 regulatory unit of
phosphatidylinositol 3-kinase and triggers severe
insulin resistance in skeletal muscle. Endocrinology
145, 1144–1150 (2004).
46. Barbour, L. et al. Increased p85α is a potent negative
regulator of skeletal muscle insulin signaling and
induces in vivo insulin resistance associated with
growth hormone excess. J. Biol. Chem. 280,
37489–37494 (2005).
47. Kirwan, J. P. et al. Reversal of insulin resistance
postpartum is linked to enhanced skeletal muscle
insulin signaling. J. Clin. Endocrinol. Metab. 89,
4678–4684 (2004).
48. Bandyopadhyay, G., Yu, J., Ofrecio, J. & Olefsky, J.
Increased p85/55/50 expression and decreased
phosphotidylinositol 3-kinase activity in insulinresistant human skeletal muscle. Diabetes 54,
2351–2359 (2005).
49. Gual, P., Le Marchand-Brustel, Y. & Tanti, J. Positive
and negative regulation of insulin signaling through
IRS-1 phosphorylation. Biochimie 87, 99–109
(2005).
50. Cho, H. et al. Insulin resistance and a diabetes
mellitus-like syndrome in mice lacking the protein
kinase Akt2 (PKBβ). Science 292, 1728–1731
(2001).
51. George, S. et al. A family with severe insulin resistance
and diabetes due to a mutation in AKT2. Science 304,
1325–1328 (2004).
52. Knight, Z. A. et al. A pharmacological map of the
PI3-K family defines a role for p110α in insulin
signaling. Cell 125, 733–747 (2006).
VOLUME 7 | AUGUST 2006 | 617
© 2006 Nature Publishing Group
REVIEWS
53. LeRoith, D., Werner, H., Neuenschwander, S., Kalebic, T.
& Helman, L. J. The role of the insulin-like growth
factor-I receptor in cancer. Ann. NY Acad. Sci. 766,
402–408 (1995).
54. Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J. &
Efstratiadis, A. Mice carrying null mutations of the
genes encoding insulin-like growth factor I (Igf-1) and
type 1 IGF receptor (Igf1r). Cell 75, 59–72 (1993).
55. Dupont, J. & LeRoith, D. Insulin and insulin-like
growth factor I receptors: similarities and differences
in signal transduction. Horm. Res. 55 (Suppl. 2),
22–26 (2001).
56. McMullen, J. R. et al. The insulin-like growth factor 1
receptor induces physiological heart growth via the
phosphoinositide 3-kinase(p110α) pathway. J. Biol.
Chem. 279, 4782–4793 (2004).
57. Shioi, T. et al. The conserved phosphoinositide
3-kinase pathway determines heart size in mice.
EMBO J. 19, 2537–2548 (2000).
58. Shioi, T. et al. Akt/protein kinase B promotes organ
growth in transgenic mice. Mol. Cell. Biol. 22,
2799–2809 (2002).
59. Luo, J. et al. Class IA phosphoinositide 3-kinase
regulates heart size and physiological cardiac
hypertrophy. Mol. Cell. Biol. 25, 9491–9502 (2005).
60. Backman, S. A. et al. Deletion of Pten in mouse brain
causes seizures, ataxia and defects in soma size
resembling Lhermitte–Duclos disease. Nature Genet.
29, 396–403 (2001).
61. Groszer, M. et al. Negative regulation of neural stem/
progenitor cell proliferation by the Pten tumor
suppressor gene in vivo. Science 294, 2186–2189
(2001).
62. Kwon, J. et al. Reversible oxidation and inactivation of
the tumor suppressor PTEN in cells stimulated with
peptide growth factors. Proc. Natl Acad. Sci. USA 101,
16419–16424 (2004).
63. Crackower, M. A. et al. Regulation of myocardial
contractility and cell size by distinct PI3K–PTEN
signaling pathways. Cell 110, 737–749 (2002).
64. Yang, Z. Z. et al. Physiological functions of protein
kinase B/Akt. Biochem. Soc. Trans. 32, 350–354
(2004).
65. Sugimoto, Y., Whitman, M., Cantley, L. C. & Erikson, R. L.
Evidence that the Rous sarcoma virus transforming
gene product phosphorylates phosphatidylinositol and
diacylglycerol. Proc. Natl Acad. Sci. USA 81,
2117–2121 (1984).
66. Whitman, M., Kaplan, D. R., Schaffhausen, B.,
Cantley, L. & Roberts, T. M. Association of
phosphatidylinositol kinase activity with polyoma
middle-T competent for transformation. Nature 315,
239–242 (1985).
67. Serunian, L. A., Auger, K. R., Roberts, T. M. &
Cantley, L. C. Production of novel
polyphosphoinositides in vivo is linked to cell
transformation by polyomavirus middle T antigen.
J. Virol. 64, 4718–4725 (1990).
68. Chang, H. W. et al. Transformation of chicken cells by
the gene encoding the catalytic subunit of PI 3-kinase.
Science 276, 1848–1850 (1997).
69. Li, J. et al. PTEN, a putative protein tyrosine
phosphatase gene mutated in human brain, breast,
and prostate cancer. Science 275, 1943–1947
(1997).
70. Steck, P. A. et al. Identification of a candidate tumour
suppressor gene, MMAC1, at chromosome 10q23.3
that is mutated in multiple advanced cancers. Nature
Genet. 15, 356–362 (1997).
References 69 and 70 identify PTEN as a
candidate tumour suppressor.
71. Maehama, T. & Dixon, J. E. The tumor suppressor,
PTEN/MMAC1, dephosphorylates the lipid second
messenger, phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem. 273, 13375–13378 (1998).
This study shows that the PTEN tumour suppressor
functions as a lipid phosphatase that
dephosphorylates PIP3.
72. Sansal, I. & Sellers, W. R. The biology and clinical
relevance of the PTEN tumor suppressor pathway.
J. Clin. Oncol. 22, 2954–2963 (2004).
73. Trotman, L. C. et al. Pten dose dictates cancer
progression in the prostate. PLoS Biol. 1, e59 (2003).
74. Parsons, R. Human cancer, PTEN and the PI-3 kinase
pathway. Semin. Cell Dev. Biol. 15, 171–176 (2004).
75. Wang, S. et al. Prostate-specific deletion of the murine
Pten tumor suppressor gene leads to metastatic
prostate cancer. Cancer Cell 4, 209–221 (2003).
76. Di Cristofano, A. et al. Impaired Fas response and
autoimmunity in Pten+/– mice. Science 285,
2122–2125 (1999).
77. Suzuki, A. et al. T cell-specific loss of Pten leads to
defects in central and peripheral tolerance. Immunity
14, 523–534 (2001).
78. Samuels, Y. et al. High frequency of mutations of the
PIK3CA gene in human cancers. Science 304, 554
(2004).
Reports the discovery that somatic mutations in the
PIK3CA gene are a common event in human cancers.
79. Bader, A. G., Kang, S., Zhao, L. & Vogt, P. K.
Oncogenic PI3K deregulates transcription and
translation. Nature Rev. Cancer 5, 921–929 (2005).
80. Kang, S., Bader, A. G. & Vogt, P. K.
Phosphatidylinositol 3-kinase mutations identified in
human cancer are oncogenic. Proc. Natl Acad. Sci.
USA 102, 802–807 (2005).
81. Isakoff, S. J. et al. Breast cancer-associated PIK3CA
mutations are oncogenic in mammary epithelial cells.
Cancer Res. 65, 10992–11000 (2005).
82. Samuels, Y. et al. Mutant PIK3CA promotes cell
growth and invasion of human cancer cells. Cancer Cell
7, 561–573 (2005).
83. Zhao, J. J. et al. The oncogenic properties of p110α
and p110β phosphatidylinositol 3-kinases in human
mammary epithelial cells. Proc. Natl Acad. Sci. USA
102, 18443–18448 (2005).
84. Bellacosa, A. et al. Molecular alterations of the AKT2
oncogene in ovarian and breast carcinomas. Int. J.
Cancer 64, 280–285 (1995).
85. Cheng, J. et al. Amplification of AKT2 in human
pancreatic cells and inhibition of AKT2 expression and
tumorigenicity by antisense RNA. Proc. Natl Acad. Sci.
USA 93, 3636–3641 (1996).
86. Ruggeri, B., Huang, L., Wood, M., Cheng, J. & Testa, J.
Amplification and overexpression of the AKT2
oncogene in a subset of human pancreatic ductal
adenocarcinomas. Mol. Carcinog. 21, 81–86 (1998).
87. Parsons, D. W. et al. Colorectal cancer: mutations in a
signalling pathway. Nature 436, 792 (2005).
88. Radimerski, T., Montagne, J., Hemmings-Mieszczak, M.
& Thomas, G. Lethality of Drosophila lacking TSC
tumor suppressor function rescued by reducing dS6K
signaling. Genes Dev. 16, 2627–2632 (2002).
89. Manning, B. D. Balancing Akt with S6K: implications
for both metabolic diseases and tumorigenesis. J. Cell
Biol. 167, 399–403 (2004).
90. Harrington, L. S., Findlay, G. M. & Lamb, R. F.
Restraining PI3K: mTOR signalling goes back to the
membrane. Trends Biochem. Sci. 30, 35–42 (2005).
91. Aguirre, V., Uchida, T., Yenush, L., Davis, R. & White, M.
The c-Jun NH2-terminal kinase promotes insulin
resistance during association with insulin receptor
substrate-1 and phosphorylation of Ser307. J. Biol.
Chem. 275, 9047–9054 (2000).
92. Hirosumi, J. et al. A central role for JNK in obesity and
insulin resistance. Nature 420, 333–336 (2002).
93. Kim, J. et al. PKC-β knockout mice are protected from
fat-induced insulin resistance. J. Clin. Invest. 114,
823–827 (2004).
94. Um, S. H. et al. Absence of S6K1 protects against
age- and diet-induced obesity while enhancing insulin
sensitivity. Nature 431, 200–205 (2004).
95. Manning, B. D. et al. Feedback inhibition of Akt
signaling limits the growth of tumors lacking Tsc2.
Genes Dev. 19, 1773–1778 (2005).
96. O’Reilly, K. E. et al. mTOR inhibition induces upstream
receptor tyrosine kinase signaling and activates Akt.
Cancer Res. 66, 1500–1508 (2006).
97. Engelman, J. A. et al. ErbB-3 mediates
phosphoinositide 3-kinase activity in gefitinib-sensitive
non-small cell lung cancer cell lines. Proc. Natl Acad.
Sci. USA 102, 3788–3793 (2005).
98. Kaneto, H. et al. Possible novel therapy for diabetes
with cell-permeable JNK-inhibitory peptide. Nature
Med. 10, 1128–1132 (2004).
99. Alloatti, G., Montrucchio, G., Lembo, G. & Hirsch, E.
Phosphoinositide 3-kinase γ: kinase-dependent and
-independent activities in cardiovascular function and
disease. Biochem. Soc. Trans. 32, 383–386 (2004).
100. Kobayashi, S. et al. EGFR mutation and resistance of
non-small-cell lung cancer to gefitinib. N. Engl. J. Med.
352, 786–792 (2005).
101. Fruman, D. A., Meyers, R. E. & Cantley, L. C.
Phosphoinositide kinases. Annu. Rev. Biochem. 67,
481–507 (1998).
102. Songyang, Z. et al. SH2 domains recognize specific
phosphopeptide sequences. Cell 72, 767–778 (1993).
103. Yu, J. et al. Regulation of the p85/p110
phosphatidylinositol 3′-kinase: stabilization and
inhibition of the p110α catalytic subunit by the p85
regulatory subunit. Mol. Cell. Biol. 18, 1379–1387
(1998).
618 | AUGUST 2006 | VOLUME 7
104. Yu, J., Wjasow, C. & Backer, J. M. Regulation of the
p85/p110α phosphatidylinositol 3′-kinase. Distinct
roles for the N-terminal and C-terminal SH2 domains.
J. Biol. Chem. 273, 30199–30203 (1998).
105. Franke, T. F., Kaplan, D. R., Cantley, L. C. & Toker, A.
Direct regulation of the Akt proto-oncogene product
by phosphatidylinositol-3,4-bisphosphate. Science
275, 665–668 (1997).
106. Klippel, A., Kavanaugh, W. M., Pot, D. & Williams, L. T.
A specific product of phosphatidylinositol 3-kinase
directly activates the protein kinase Akt through its
pleckstrin homology domain. Mol. Cell. Biol. 17,
338–344 (1997).
References 105 and 106 report the finding that
AKT is activated on binding to the lipid products of
PI3K.
107. Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini,
D. M. Phosphorylation and regulation of Akt/PKB by
the rictor-mTOR complex. Science 307, 1098–1101
(2005).
Identifies the mTOR/rictor complex as the kinase
that phosphorylates Ser473 of AKT.
108. Mora, A., Komander, D., van Aalten, D. M. &
Alessi, D. R. PDK1, the master regulator of AGC
kinase signal transduction. Semin. Cell Dev. Biol. 15,
161–170 (2004).
109. Cohen, P. & Frame, S. The renaissance of GSK3.
Nature Rev. Mol. Cell Biol. 2, 769–776 (2001).
110. Berwick, D., Hers, I., Heesom, K., Moule, S. & Tavare, J.
The identification of ATP-citrate lyase as a protein
kinase B (Akt) substrate in primary adipocytes. J. Biol.
Chem. 277, 33895–33900 (2002).
111. Barthel, A., Schmoll, D. & Unterman, T. G. FoxO
proteins in insulin action and metabolism. Trends
Endocrinol. Metab. 16, 183–189 (2005).
112. Burgering, B. M. & Medema, R. H. Decisions on life
and death: FOXO forkhead transcription factors are in
command when PKB/Akt is off duty. J. Leukoc. Biol.
73, 689–701 (2003).
113. Vivanco, I. & Sawyers, C. L. The phosphatidylinositol
3-kinase AKT pathway in human cancer. Nature Rev.
Cancer 2, 489–501 (2002).
114. Richardson, C. J., Schalm, S. S. & Blenis, J. PI3-kinase
and TOR: PIKTORing cell growth. Semin. Cell Dev. Biol.
15, 147–159 (2004).
115. Manning, B. D., Tee, A. R., Logsdon, M. N., Blenis, J. &
Cantley, L. C. Identification of the tuberous sclerosis
complex-2 tumor suppressor gene product tuberin as
a target of the phosphoinositide 3-kinase/akt pathway.
Mol. Cell 10, 151–162 (2002).
116. Majumder, P. K. et al. mTOR inhibition reverses Aktdependent prostate intraepithelial neoplasia through
regulation of apoptotic and HIF-1-dependent
pathways. Nature Med. 10, 594–601 (2004).
117. Neshat, M. S. et al. Enhanced sensitivity of PTENdeficient tumors to inhibition of FRAP/mTOR. Proc.
Natl Acad. Sci. USA 98, 10314–10319 (2001).
118. Podsypanina, K. et al. An inhibitor of mTOR reduces
neoplasia and normalizes p70/S6 kinase activity in
Pten+/– mice. Proc. Natl Acad. Sci. USA 98,
10320–10325 (2001).
119. Sarbassov dos, D., Ali, S. M. & Sabatini, D. M.
Growing roles for the mTOR pathway. Curr. Opin. Cell
Biol. 17, 596–603 (2005).
120. Datta, S. R. et al. Akt phosphorylation of BAD couples
survival signals to the cell-intrinsic death machinery.
Cell 91, 231–241 (1997).
121. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R.
& Nunez, G. Interleukin-3-induced phosphorylation of
BAD through the protein kinase Akt. Science 278,
687–689 (1997).
122. Datta, S. R. et al. 14-3-3 proteins and survival kinases
cooperate to inactivate BAD by BH3 domain
phosphorylation. Mol. Cell 6, 41–51 (2000).
123. Zha, J., Harada, H., Yang, E., Jockel, J. &
Korsmeyer, S. J. Serine phosphorylation of death
agonist BAD in response to survival factor results in
binding to 14-3-3 not BCL-XL. Cell 87, 619–628
(1996).
124. Accili, D. & Arden, K. C. Foxos at the crossroads of
cellular metabolism, differentiation, and
transformation. Cell 117, 421–426 (2004).
125. So, C. W. & Cleary, M. L. Common mechanism for
oncogenic activation of MLL by forkhead family
proteins. Blood 101, 633–639 (2003).
126. Wu, G. et al. Somatic mutation and gain of copy
number of PIK3CA in human breast cancer. Breast
Cancer Res. 7, R609–R616 (2005).
127. Levine, D. A. et al. Frequent mutation of the PIK3CA
gene in ovarian and breast cancers. Clin. Cancer Res.
11, 2875–2878 (2005).
www.nature.com/reviews/genetics
© 2006 Nature Publishing Group
REVIEWS
128. Lee, J. W. et al. PIK3CA gene is frequently mutated in
breast carcinomas and hepatocellular carcinomas.
Oncogene 24, 1477–1480 (2005).
129. Bachman, K. E. et al. The PIK3CA gene is mutated
with high frequency in human breast cancers. Cancer
Biol. Ther. 3, 772–775 (2004).
130. Campbell, I. G. et al. Mutation of the PIK3CA gene
in ovarian and breast cancer. Cancer Res. 64,
7678–7681 (2004).
131. Saal, L. H. et al. PIK3CA mutations correlate with
hormone receptors, node metastasis, and ERBB2, and
are mutually exclusive with PTEN loss in human breast
carcinoma. Cancer Res. 65, 2554–2559 (2005).
132. Velho, S. et al. The prevalence of PIK3CA mutations
in gastric and colon cancer. Eur. J. Cancer 41,
1649–1654 (2005).
133. Hartmann, C., Bartels, G., Gehlhaar, C., Holtkamp, N.
& von Deimling, A. PIK3CA mutations in glioblastoma
multiforme. Acta Neuropathol. (Berl.) 109, 639–642
(2005).
134. Knobbe, C. B., Trampe-Kieslich, A. & Reifenberger, G.
Genetic alteration and expression of the
phosphoinositol-3-kinase/Akt pathway genes PIK3CA
and PIKE in human glioblastomas. Neuropathol. Appl.
Neurobiol. 31, 486–490 (2005).
135. Li, V. S. et al. Mutations of PIK3CA in gastric
adenocarcinoma. BMC Cancer 5, 29 (2005).
136. Pedrero, J. M. et al. Frequent genetic and biochemical
alterations of the PI 3-K/AKT/PTEN pathway in head
and neck squamous cell carcinoma. Int. J. Cancer 114,
242–248 (2005).
137. Woenckhaus, J. et al. Genomic gain of PIK3CA and
increased expression of p110α are associated with
progression of dysplasia into invasive squamous cell
carcinoma. J. Pathol. 198, 335–342 (2002).
138. Wu, G. et al. Uncommon mutation, but common
amplifications, of the PIK3CA gene in thyroid tumors.
J. Clin. Endocrinol. Metab. 90, 4688–4693 (2005).
139. Massion, P. P. et al. Genomic copy number analysis of
non-small cell lung cancer using array comparative
genomic hybridization: implications of the
phosphatidylinositol 3-kinase pathway. Cancer Res.
62, 3636–3640 (2002).
140. Bjorkqvist, A. M. et al. DNA gains in 3q occur
frequently in squamous cell carcinoma of the lung, but
not in adenocarcinoma. Genes Chromosomes Cancer
22, 79–82 (1998).
141. Byun, D. S. et al. Frequent monoallelic deletion of
PTEN and its reciprocal association with PIK3CA
amplification in gastric carcinoma. Int. J. Cancer 104,
318–327 (2003).
142. Miller, C. T. et al. Gene amplification in esophageal
adenocarcinomas and Barrett’s with high-grade
dysplasia. Clin. Cancer Res. 9, 4819–4825 (2003).
143. Ma, Y. Y. et al. PIK3CA as an oncogene in cervical
cancer. Oncogene 19, 2739–2744 (2000).
144. Chiariello, E., Roz, L., Albarosa, R., Magnani, I. &
Finocchiaro, G. PTEN/MMAC1 mutations in primary
glioblastomas and short-term cultures of malignant
gliomas. Oncogene 16, 541–545 (1998).
145. Wang, S. I. et al. Somatic mutations of PTEN in
glioblastoma multiforme. Cancer Res. 57,
4183–4186 (1997).
146. Smith, J. S. et al. PTEN mutation, EGFR amplification,
and outcome in patients with anaplastic astrocytoma
and glioblastoma multiforme. J. Natl Cancer Inst. 93,
1246–1256 (2001).
147. Cairns, P. et al. Frequent inactivation of PTEN/MMAC1
in primary prostate cancer. Cancer Res. 57,
4997–5000 (1997).
148. Feilotter, H. E., Nagai, M. A., Boag, A. H., Eng, C. &
Mulligan, L. M. Analysis of PTEN and the 10q23
region in primary prostate carcinomas. Oncogene 16,
1743–1748 (1998).
149. Pesche, S. et al. PTEN/MMAC1/TEP1 involvement in
primary prostate cancers. Oncogene 16, 2879–2883
(1998).
150. Gray, I. C. et al. Mutation and expression analysis
of the putative prostate tumour-suppressor
gene PTEN. Br. J. Cancer 78, 1296–1300
(1998).
151. Wang, S. I., Parsons, R. & Ittmann, M. Homozygous
deletion of the PTEN tumor suppressor gene in a
subset of prostate adenocarcinomas. Clin. Cancer Res.
4, 811–815 (1998).
152. Feilotter, H. E. et al. Analysis of the 10q23
chromosomal region and the PTEN gene in human
sporadic breast carcinoma. Br. J. Cancer 79,
718–723 (1999).
153. Freihoff, D. et al. Exclusion of a major role for the
PTEN tumour-suppressor gene in breast carcinomas.
Br. J. Cancer 79, 754–758 (1999).
154. Pollock, P. M. et al. PTEN inactivation is rare
in melanoma tumours but occurs frequently in
melanoma cell lines. Melanoma Res. 12, 565–575
(2002).
155. Birck, A., Ahrenkiel, V., Zeuthen, J., Hou-Jensen, K.
& Guldberg, P. Mutation and allelic loss of the
PTEN/MMAC1 gene in primary and metastatic
melanoma biopsies. J. Invest. Dermatol. 114,
277–280 (2000).
156. Celebi, J. T., Shendrik, I., Silvers, D. N. & Peacocke, M.
Identification of PTEN mutations in metastatic
melanoma specimens. J. Med. Genet. 37, 653–657
(2000).
157. Reifenberger, J. et al. Allelic losses on chromosome
arm 10q and mutation of the PTEN (MMAC1)
tumour suppressor gene in primary and metastatic
malignant melanomas. Virchows Arch. 436,
487–493 (2000).
158. Duerr, E. M. et al. PTEN mutations in gliomas and
glioneuronal tumors. Oncogene 16, 2259–2264
(1998).
159. Rasheed, B. K. et al. PTEN gene mutations are seen in
high-grade but not in low-grade gliomas. Cancer Res.
57, 4187–4190 (1997).
160. Liu, W., James, C. D., Frederick, L., Alderete, B. E. &
Jenkins, R. B. PTEN/MMAC1 mutations and EGFR
amplification in glioblastomas. Cancer Res. 57,
5254–5257 (1997).
NATURE REVIEWS | GENETICS
161. Whang, Y. E. et al. Inactivation of the tumor
suppressor PTEN/MMAC1 in advanced human
prostate cancer through loss of expression.
Proc. Natl Acad. Sci. USA 95, 5246–5250
(1998).
162. Tsao, H., Zhang, X., Benoit, E. & Haluska, F. G.
Identification of PTEN/MMAC1 alterations in
uncultured melanomas and melanoma cell lines.
Oncogene 16, 3397–3402 (1998).
163. Cheng, J. Q. et al. AKT2, a putative oncogene
encoding a member of a subfamily of protein-serine/
threonine kinases, is amplified in human ovarian
carcinomas. Proc. Natl Acad. Sci. USA 89,
9267–9271 (1992).
164. Staal, S. P. Molecular cloning of the akt oncogene
and its human homologues AKT1 and AKT2:
amplification of AKT1 in a primary human gastric
adenocarcinoma. Proc. Natl Acad. Sci. USA 84,
5034–5037 (1987).
165. Philp, A. J. et al. The phosphatidylinositol 3′-kinase
p85α gene is an oncogene in human ovarian and
colon tumors. Cancer Res. 61, 7426–7429
(2001).
166. Taniguchi, C. M. et al. Phosphoinositide 3-kinase
regulatory subunit p85a suppresses insulin action via
positive regulation of PTEN. Proc. Natl Acad. Sci. USA
(in the press).
Acknowledgements
We are grateful to members of the Cantley, Soltoff and
Carpenter laboratories for thoughtful discussions and insights
regarding PI3K signalling. We apologize to the many authors
whose work we could not cite directly because of space limitations. Work in the Cantley laboratory is supported by the US
National Institutes of Health.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
aap-1 | age-1 | PIK3CA | PIK3R1 | Tsc2
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
breast cancers | cervical cancers | Cowden disease | gastric
cancers | lung cancers | macrocephaly, multiple lipomas and
haemangiomata | non-insulin-dependent diabetes mellitus |
Peutz–Jegher syndrome | prostate cancers | tuberous sclerosis
UniProtKB: http://ca.expasy.org/sprot
AKT2 | ATM | ATR | BAD | CHICO | DAF-18 | G6Pase | GLUT4 |
IRS1 | IRS2 | JNK1 | MDM2 | P26KIP1 | pdk1 | PTEN | VSP15 |
VSP34
FURTHER INFORMATION
The Cantley laboratory: http://sysbio.med.harvard.edu/
faculty/cantley
Access to this links box is available online.
VOLUME 7 | AUGUST 2006 | 619
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