Biochem. J. (2012) 443, 587–601 (Printed in Great Britain)
587
doi:10.1042/BJ20120008
REVIEW ARTICLE
Regulation and cellular functions of class II phosphoinositide 3-kinases
Marco FALASCA1 and Tania MAFFUCCI
Inositide Signalling Group, Centre for Diabetes, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street,
London E1 2AT, U.K.
Class II isoforms of PI3K (phosphoinositide 3-kinase) are
still the least investigated and characterized of all PI3Ks. In
the last few years, an increased interest in these enzymes
has improved our understanding of their cellular functions.
However, several questions still remain unanswered on their
mechanisms of activation, their specific downstream effectors
and their contribution to physiological processes and pathological
conditions. Emerging evidence suggests that distinct PI3Ks
activate different signalling pathways, indicating that their
functional roles are probably not redundant. In the present
review, we discuss the recent advances in our understanding
of mammalian class II PI3Ks and the evidence suggesting their
involvement in human diseases.
INTRODUCTION
are divided further into two subclasses: IA and IB. Class IA
comprises three distinct catalytic subunits (p110α, p110β and
p11δ), whereas p11γ is the only catalytic subunit within the
class IB subfamily [2]. Together with the PI3K core, all class
I catalytic subunits possess a Ras-binding domain; the class IA
subunits also specifically possess a p85-binding domain, which
mediates interactions with the regulatory subunit. Class IA PI3Ks
are mainly activated by RTKs (tyrosine kinase receptors), whereas
class IB PI3Ks are mainly activated by GPCRs (G-proteincoupled receptors), although activation by GPCRs has also been
reported for the class IA isoform p110β [8]. The main in vivo
lipid product of class I PI3Ks is PtdIns(3,4,5)P3 , the most studied
PI3K effector and a very well established second messenger,
involved in many cellular functions. The class III PI3K hVps
(human vacuolar protein sorting) 34 is a monomer which lacks
the Ras-binding and the regulatory-subunit-binding domains and
catalyses specifically the synthesis of PtdIns3P [9]. The protein
kinase hVps15, which associates with hVps34, has been described
as a regulatory protein, but its precise role in hVps34 regulation
is still not completely defined [9]. In the present review, we report
the most recent data in the literature on the physiological roles
of the mammalian class II isoforms and we discuss evidence of
their role in pathological conditions.
Phosphoinositides are phospholipids comprising two fatty acid
chains linked by a glycerol moiety to a water-soluble inositol
headgroup. The majority of phosphoinositides possess a stearoyl
residue in the 1-position and an arachidonoyl residue in the 2position, but they are differentially phosphorylated at the 3-, 4and 5-hydroxy groups within the myo-inositol ring (Figure 1A).
PI3Ks (phosphoinositide 3-kinases) specifically catalyse
the phosphorylation of the 3-position of the inositol ring
of selected phosphoinositides [1,2], leading to synthesis of
PtdIns3P (phosphatidylinositol 3-phosphate), PtdIns(3,4)P2
(phosphatidylinositol 3,4-bisphosphate) and PtdIns(3,4,5)P3
(phosphatidylinositol 3,4,5-trisphosphate) (Figure 1B). A
fourth 3-phosphorylated phosphoinositide, PtdIns(3,5)P2 (phosphatidylinositol 3,5-bisphosphate), exists, but it is generally
accepted that it derives from phosphorylation of the 5-position
of PtdIns3P by the kinase PIKFyve, therefore its synthesis does
not seem to be catalysed directly by any PI3Ks.
The lipid products of PI3Ks can act as second messengers
within the cell by activating several proteins through regulation of
their intracellular localization or conformational changes. These
proteins, in turn, control many intracellular functions such as
cell proliferation, survival, migration, glucose homoeostasis and
membrane trafficking [1–3]. Deregulation of PI3K-dependent
cellular pathways is associated with several diseases, including
cancer and diabetes [1,2,4–6].
Eight mammalian isoforms of PI3K exist and they are grouped
into three classes on the basis of their substrate specificity and
structure [2,7]. All isoenzymes possess the so-called ‘PI3K core’,
consisting of C2, helical and catalytic domains [2]. Class I PI3Ks
are dimers of one catalytic and one regulatory subunit, and
Key words: cell migration, glucose transport, insulin secretion,
phosphoinositide, phosphoinositide 3-kinase (PI3K).
CLASS II PI3K ISOFORMS
Class II PI3Ks are monomers of high molecular mass first
identified in Drosophila melanogaster [10]. Mammals possess
three class II isoforms: PI3K-C2α, PI3K-C2β and PI3K-C2γ [7].
Human PI3K-C2α, originally cloned from the cell line U937, is
expressed ubiquitously, and it was reported to have the highest
Abbreviations used: β-GK, β-cell glucokinase; CHO-IR cell, Chinese-hamster ovary cell expressing insulin receptor; CMT, Charcot–Marie–Tooth; CRC,
colorectal cancer; EGF, epidermal growth factor; ESCC, oesophageal squamous cell carcinoma; F-actin, filamentous actin; GFP, green fluorescent protein;
GLUT, glucose transporter; GPCR, G-protein-coupled receptor; GSK3β, glycogen synthase kinase 3β; HEK, human embryonic kidney; hGH, human
growth hormone; hVps, human vacuolar protein sorting; IGF-1, insulin-like growth factor 1; IR, insulin receptor; IR-B, B isoform of the insulin receptor;
Lamp, lysosome-associated membrane protein; LDCV, large dense-core vesicle; LPA, lysophosphatidic acid; miRNA, microRNA; MSH2, mutS homologue
2; MTM, myotubularin; MTMR, MTM-related protein; mTOR, mammalian target of rapamycin; PDGF, platelet-derived growth factor; PI3K, phosphoinositide
3-kinase; PH, pleckstrin homology; PX, Phox homology; RTK, tyrosine kinase receptor; SCF, stem cell factor; SH3, Src homology 3; SHR, spontaneously
hypertensive rat; siRNA, small interfering RNA; SSc, systemic sclerosis; TCR, T-cell receptor; Tiam1, T-cell lymphoma invasion and metastasis 1; TRIM27,
tripartite-motif-containing protein 27; VSMC, vascular smooth muscle cell.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
588
M. Falasca and T. Maffucci
Figure 2
Structure of class II PI3K isoforms
The three mammalian class II PI3K isoforms possess a first C2 domain, a helical domain and a
catalytic domain, all of which are present in all other PI3Ks, a Ras-binding domain, also detected
in class I isoforms, and a class II-specific C-terminal region containing a PX domain and a
second C2 domain. The N-terminal region differs between the distinct class II isoforms.
different from the class I isoforms and that, being able to generate
distinct lipid products, they were likely to activate different
downstream effectors.
Although it is generally well accepted that class II PI3Ks
do not catalyse the synthesis of PtdIns(3,4,5)P3 , a general
consensus of the specific lipid product(s) generated by these
enzymes has not yet been reached. This is mostly due to the
fact that original in vitro studies showed that class II PI3Ks can
phosphorylate both PtdIns (phosphatidylinositol) and PtdIns4P
(phosphatidylinositol 4-phosphate), therefore generating both
PtdIns3P and PtdIns(3,4)P2 , and it has been complicated lately by
the fact that some reports since then have simply assumed that the
enzymes generate PtdIns(3,4)P2 without directly demonstrating
it.
In vitro
Figure 1
Structure of PtdIns and lipid products of PI3Ks
(A) PtdIns is the founding member of the family of phosphoinositides. The two acyl chains
and the water-soluble headgroup are indicated. (B) PI3Ks catalyse the synthesis of three
phosphoinositides: PtdIns3P , derived from phosphorylation of PtdIns; PtdIns(3,4)P 2 , derived
from phosphorylation of PtdIns4P ; and PtdIns(3,4,5)P 3 , derived from phosphorylation of
PtdIns(4,5)P 2 .
levels in heart, placenta and ovary [7,11]. Human PI3K-C2β,
isolated from a cDNA library of the breast cancer cell line
MCF7 [12] and then from U937 cells [13], appeared to be highly
expressed in placenta and thymus. Human PI3K-C2γ showed a
more restricted localization in liver, prostate, breast and salivary
glands [14].
Class II PI3Ks possess a Ras-binding domain and the PI3K core,
but they lack the regulatory-subunit-binding domain (Figure 2).
Distinctively, they all possess a C-terminus extension consisting
of a PX (Phox homology) domain and a second C2 domain. The
N-terminus extensions are distinct between the class II isoforms.
For instance PI3K-C2α specifically possesses a clathrin-binding
region, whereas PI3K-C2β possesses proline-rich sequences
(Figure 2). This suggests specific roles for this region, possibly in
activation of the enzymes.
Although original studies have shown that class II PI3Ks can
phosphorylate PtdIns and PtdIns4P, as mentioned above, they
also showed that PtdIns was indeed the preferential substrate
[10,13,15,16]. For instance, PI3K-C2β was able to phosphorylate
PtdIns and PtdIns4P in the presence of Mg2 + , but the activity
of the enzyme towards PtdIns4P was only 10 % of the activity
towards PtdIns [13]. Interestingly, it was shown that PI3K-C2β
was able to phosphorylate only PtdIns and not PtdIns4P when the
in vitro kinase assay was performed in the presence of Ca2 + as
the divalent cation. The authors reported further that incubation
of the enzyme in the presence of a fixed concentration of MgATP and increasing concentrations of Ca2 + abolished its activity
toward PtdIns4P [13]. A similar selective activity towards PtdIns
in the presence of Ca2 + was also observed for PI3K-C2α [17].
Furthermore, PtdIns3P remains the main product in vitro of PI3KC2α even upon addition of clathrin, which is able to increase its
in vitro activity in particular towards PtdIns4P and PtdIns(4,5)P2
[18]. Taken together, these data indicate that, although class
II PI3Ks can potentially generate PtdIns3P and PtdIns(3,4)P2
in vitro, the monophosphate PtdIns3P is the main in vitro product
of these enzymes and it is the sole product in the presence of Ca2 + .
In vivo
LIPID PRODUCT(S) OF CLASS II PI3Ks
Original studies showed that the in vitro activity towards
PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) was only
1 % of the total [13] and it was detected only in the
presence of phosphatidylserine for PI3K-C2α [11] or in
the presence of phosphatidylcholine, phosphatidylserine or
phosphatidylethanolamine for PI3K-C2β. Therefore very early
evidence already suggested that class II PI3Ks were profoundly
c The Authors Journal compilation c 2012 Biochemical Society
One of the first indications of the in vivo product of class II
PI3Ks came from our studies indicating that insulin could generate
PtdIns3P in a mechanism resistant to high concentrations of
wortmannin and LY294002, a feature of PI3K-C2α [19]. We then
performed a detailed analysis of phosphoinositides by HPLC that
revealed that synthesis of PtdIns3P, but not of PtdIns(3,4)P2 and
PtdIns(3,4,5)P3 , upon insulin stimulation was completely blunted
in L6 cells lacking PI3K-C2α [20]. Consistent with these data, the
insulin-induced translocation of the PtdIns3P-binding probe GFP
Class II phosphoinositide 3-kinases
(green fluorescent protein)–2×FYVEHrs to the plasma membrane
was inhibited upon PI3K-C2α down-regulation. This was the
first demonstration that PtdIns3P is the sole in vivo product of
PI3K-C2α, at least upon insulin stimulation [20]. Supporting
this conclusion further, overexpression of a catalytically inactive
PI3K-C2α in PC12 cells reduced the steady-state levels of
PtdIns3P [21]. It was initially reported that the steady-state
levels of PtdIns(3,4,5)P3 were also inhibited in these cells [21].
However, subsequent studies clearly identified PtdIns3P as the
main product generated by PI3K-C2α activation in LDCVs (large
dense-core vesicles) of PC12 upon stimulation of exocytosis [22]
and demonstrated that this specific phosphoinositide is critical for
PI3K-C2α-dependent functions [22,23].
Recently, it has been reported that transient down-regulation
of PI3K-C2α inhibited the insulin-induced synthesis of
PtdIns(3,4)P2 , but not PtdIns(3,4,5)P3 or levels of PtdIns3P,
in MIN6 pancreatic β-cells [24]. A co-distribution of GFP–
PH (pleckstrin homology) domain from Akt1 [which bind
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 ], but not of GFP–2×FYVEHrs
with IR-B (B isoform of the insulin receptor) was also reported
[24]. Consistent with this, an increase in Akt1 activity was
detected in MIN6 cells overexpressing wild-type, but not
catalytically inactive, PI3K-C2α, and inhibition of the insulininduced Akt1 activation was reported in these cells upon
down-regulation of PI3K-C2α [24]. Several lines of evidence
support the hypothesis that PtdIns3P is also the main in vivo
product of PI3K-C2β. For instance, we showed that downregulation of this enzyme inhibited the LPA (lysophosphatidic
acid)-induced synthesis of PtdIns3P at the plasma membrane,
assessed by analysis of plasma membrane translocation of GFP–
2×FYVEHrs [25]. Similarly, in vitro assays revealed an increase
in radiolabelled PtdIns3P, but not PtdIns4P, PtdIns(4,5)P2 or
PtdIns(3,4,5)P3 , in the nuclei and nuclear envelopes of HL-60
cells progressing into the G2 /M-phase of the cell cycle with
a simultaneous increase in the activity of immunoprecipitated
PI3K-C2β [26], suggesting that PtdIns3P is the main product of
activation of the nuclear PI3K-C2β during cell-cycle progression.
Other lines of evidence supporting the hypothesis that PtdIns3P
is the main product of PI3K-C2β derive from data showing
that several PI3K-C2β-dependent cellular processes can be
inhibited by blockade of PtdIns3P. For instance, overexpression
of 2×FYVEHrs is able to inhibit the PI3K-C2β-dependent cell
migration [25,27]. Similarly, it has been demonstrated that
dialysed PtdIns3P counteracts the inhibition of the KCa3.1
channel activity induced by down-regulation of PI3K-C2β in
human CD4 T-cells [28]. No study so far has investigated the
in vivo products of PI3K-C2γ .
MECHANISMS OF ACTIVATION
Several studies have demonstrated that the activity of PI3KC2α and PI3K-C2β can be regulated by cellular stimulation. In
contrast, no study so far has reported a specific cellular stimulation
able to modulate the activity of PI3K-C2γ . Several stimuli can
activate PI3K-C2α, including hormones such as insulin [20,29],
chemokines such as MCP1 (monocyte chemotactic protein 1)
[30], and cytokines such as leptin and TNFα (tumour necrosis
factor α) [31]. Similarly, PI3K-C2β can be activated by growth
factors such as EGF (epidermal growth factor) and SCF (stem
cell factor) [32], and phospholipids such as LPA [25]. These data
indicate that, as for class I PI3Ks, at least some class II PI3K
isoforms can be activated downstream of RTKs [17,20,29,32] and
GPCRs [25]. The mechanisms of class II PI3K activation are
still not completely defined, but some potential mechanisms that
589
have been proposed for PI3K-C2α and PI3K-C2β are discussed
in the present review. No study has investigated the mechanism
of activation of PI3K-C2γ so far.
Role of protein domains
As mentioned above, in contrast with class I isoforms, class II
PI3Ks do not possess a regulatory subunit but, on the other hand,
they possess several additional protein domains at their N-terminal
and C-terminal regions [7]. It is therefore tempting to speculate
that these domains are involved in regulation of their activity.
The main function of the regulatory subunit within the class I
PI3K subfamily is to maintain the catalytic subunit in a closed
inactive conformation. Association of the regulatory subunit to
the activated RTK not only allows recruitment of the dimer to the
receptor, but also relieves the catalytic subunit from this inhibition.
In the absence of a regulatory subunit, what does control the
catalytic activity of class II PI3Ks and prevent them from
phosphorylating phosphoinositides even in the absence of cellular
stimulation? Evidence suggests that some of the many protein
domains present on class II isoforms can be involved in inhibition
of the ‘basal’ enzymatic activity. For instance, addition of
clathrin in an in vitro kinase assay or removal of an Nterminal region of PI3K-C2α encompassing the clathrin-binding
sites has been reported to increase the activity of the enzyme
[18]. These data suggest a role for clathrin and the clathrinbinding site within PI3K-C2α in regulation of the activity of
the enzyme. Interestingly, although lacking the specific clathrinbinding domain present on PI3K-C2α, PI3K-C2β has also been
shown to be able to bind clathrin [33], suggesting that clathrin
may have a generic role in regulation of class II PI3K activation.
How exactly clathrin regulates class II PI3K activity is still not
clear.
The hypothesis that the N-terminal region of class II
PI3Ks may keep the enzymes in an inactive conformation in
the absence of cellular stimulation is supported further by the
observation that removal of the N-terminal region of PI3KC2β increases the in vitro activity of the enzyme [13]. More
specifically, deletion of the first proline-rich region appeared
to inhibit the kinase activity and recruitment of PI3K-C2β to
the activated EGF receptor, whereas removal of the second and
third proline-rich motifs increased PI3K-C2β kinase activity
[33]. These data suggest that association of factors to the Nterminal region might be able to modulate PI3K-C2β catalytic
activity. Further validation is needed to confirm the specific role
of the N-terminal region in class II PI3K activation upon cellular
stimulation.
On the other hand, interaction of the many specific protein
domains to regulatory/adaptor proteins or to membrane lipids
may promote activation of class II PI3Ks. For instance, although
it is not clear whether the C-terminal PX domain of PI3K-C2α
has a role in activation of the enzyme, results have indicated
that the full-length enzyme and the isolated PX domain have
the same affinity for PtdIns(4,5)P2 -containing membranes and the
same monolayer penetration [34]. A co-operative role of the C2
domain in binding to PtdIns(4,5)P2 -containing membranes has
also been suggested [34]. The possibility that the C2 domains
may be involved in the Ca2 + -induced activation of PI3K-C2α
(see below) is another interesting hypothesis to be tested.
On the other hand, a PI3K-C2β mutant lacking the C-terminal
C2 domain showed an increased in vitro activity compared with
the full-length protein in the presence of Mg2 + (but not in the
presence of Ca2 + ), which led the authors to suggest that the C2
domain may inhibit the kinase activity by competing with the
catalytic domain for PtdIns binding [13]. This study also indicated
c The Authors Journal compilation c 2012 Biochemical Society
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M. Falasca and T. Maffucci
that the C2 domain was not responsible for the localization of the
enzyme.
Plasma membrane translocation
Insulin activates PI3K-C2α [29] and stimulates the synthesis
of PtdIns3P at the plasma membrane of L6 muscle cells
[19,20]. We reported that this occurs through an insulindependent translocation of PI3K-C2α to the plasma membrane
[20], suggesting that recruitment to the plasma membrane is
involved in activation of the enzyme (Figure 3A). Evidence
suggests that the enzyme is specifically recruited to the lipid
raft subdomain of the plasma membrane [20], consistent with
the observation that the PtdIns3P probe GFP–2×FYVEHrs was
also specifically detected in this subcompartment upon insulin
stimulation [19]. The mechanism of insulin-induced PI3KC2α plasma membrane translocation is currently unknown.
The insulin-induced activation of class I PI3Ks occurs through
recruitment of the regulatory subunit to the IR (insulin receptor)
via the insulin receptor substrate adaptor proteins. It is currently
not known whether PI3K-C2α is recruited to the plasma
membrane through a similar mechanism. No phosphorylated
IR was detected in PI3K-C2α immunoprecipitates from insulinstimulated CHO-IR cells (Chinese-hamster ovary cells expressing
IR) [29] and from neurotrophin-3-stimulated 3T3-L1 adipocytes
[35]. However, more recently, an association of PI3K-C2α
with IR-B in MIN6 pancreatic β-cells has been determined
by FRET (Förster resonance energy transfer) analysis and coimmunoprecipitation studies of overexpressed constructs [24].
Even if PI3K-C2α is activated through binding to the IR, this
latter study suggested that the mechanism may still be different
from activation of class I PI3Ks, since class I PI3Ks appeared
to be specifically activated downstream of the A isoform of IR,
whereas PI3K-C2α was specifically activated by IR-B [24].
As discussed below, we showed that the insulin-dependent
activation of PI3K-C2α is regulated by the small GTP-binding
protein TC10 [19] (Figure 3A). However, TC10 does not seem to
be directly involved in recruitment of the enzyme to the plasma
membrane, since no direct association of PI3K-C2α with TC10
has been detected [36].
Translocation of PI3K-C2α to the plasma membrane and to
vesicular compartments at the periphery of the cells also occurs
during dynamin-independent endocytosis, and PI3K-C2α colocalizes with the internalized proteins during internalization [37],
suggesting that intracellular relocation of PI3K-C2α is also crucial
for the enzyme to exert its role in endocytosis (discussed below).
The hypothesis that translocation to the plasma membrane can
represent a generic mechanism of class II PI3Ks activation is
supported by our data showing that LPA stimulation induces
translocation to the plasma membrane of a Myc-tagged PI3KC2β in ovarian and cervical cancer cells [25] (Figure 4A). As
for PI3K-C2α, it is not clear how PI3K-C2β is recruited to the
plasma membrane. Co-localization of PI3K-C2β with F-actin
(filamentous actin) at the level of lamellipodia was also reported
in A431 cells overexpressing the kinase [38].
Nuclear translocation
A nuclear localization motif was observed in PI3K-C2α [39].
More recently, it has been reported that EGF stimulation increases
levels of PI3K-C2β in the cytosol and in the nuclei where the
enzyme appears to co-localize with lamin A/C [40]. An increase
in nuclear PI3K-C2β levels and lipid kinase activity was reported
upon EGF stimulation. A nuclear localization motif was identified
c The Authors Journal compilation c 2012 Biochemical Society
Figure 3
Mechanisms of activation and action of PI3K-C2α
(A) Upon insulin stimulation, PI3K-C2α is recruited to the plasma membrane and it is activated
in a mechanism involving the small GTP-binding protein TC10. Synthesis of PtdIns3P at the
plasma membrane participates in modulation of glucose transport by contributing to regulation
of GLUT4 translocation. Alternatively, TC10 can activate Rab5, which can also induce PtdIns3P
synthesis at the plasma membrane and ultimately regulate glucose transport. Whether Rab5 can
have a role in PI3K-C2α activation and whether PtdIns3P modulates glucose transport through
further conversion into PtdIns(3,5)P 2 remain to be addressed (broken arrows). (B) Ca2 + induced activation of PI3K-C2α has been demonstrated in VSMCs and LDCVs, and has been
associated with VSMC contraction and neurosecretory granule release respectively. A role for
PI3K-C2α in Rho activation and 20 kDa myosin light chain phosphorylation has been reported
in VSMCs, whereas the enzyme regulates the exocytosis of neurosecretory granules through
synthesis of PtdIns3P . cAMP and PIKFyve can inhibit the two processes (red lines). (C) In a very
simplified and schematic representation of the complex process of insulin secretion, glucose
enters pancreatic β-cells through GLUT2 and it is converted into glucose 6-phosphate (G6P) by
β-GK. G6P is then metabolized, and this ultimately results in an increase in the ATP/ADP ratio,
and depolarization of the plasma membrane through closure of the KATP channel (ATP-sensitive
K + channel) which allows Ca2 + entry through voltage-dependent Ca2 + channels. Ca2 + influx
then allows fusion of the insulin granules to the plasma membrane and insulin release. It has
been suggested that the released insulin can then activate PI3K-C2α which ultimately regulates
insulin secretion at least in part by up-regulating β-GK and therefore promoting the conversion
of glucose into G6P. Alternatively, PI3K-C2α may directly regulate the secretion of insulin
granules, possibly by facilitating or regulating their fusion to the plasma membrane. Whether
Ca2 + has a role in PI3K-C2α activation in this context remains to be established (broken arrow).
in the C-terminal C2 domain [40]. Whether translocation of the
enzyme to the nucleus is related to activation of the enzyme
remains to be established.
Class II phosphoinositide 3-kinases
591
Other protein associations
Figure 4
Mechanisms of activation and action of PI3K-C2β
(A) Upon LPA stimulation, PI3K-C2β is recruited to the plasma membrane where it catalyses
synthesis of PtdIns3P . Activation of this pathway regulates migration of ovarian and cervical
cancer cells through mechanisms still undefined. A potential target of PtdIns3P is the
guanine-nucleotide-exchange factor Tiam1 which would ultimately regulate Rac1 activity (broken
lines). (B) A multiprotein complex has been detected in A-431 cells overexpressing PI3K-C2β
comprising the lipid kinase, Gbr2, Eps8, Abi1 and Sos1. In this context, PI3K-C2β appears
to control cell speed and migration through regulation of Rac1 activity and cytoskeletal
rearrangements. (C) PI3K-C2β is activated downstream of TCR and it is involved in regulation
of Kca3.1 K + channel activation. A specific role for PtdIns3P has been demonstrated in this
process. Recently, it has been reported that TRIM27 can inhibit PI3K-C2β activity by promoting
ubiquitination of the lipid kinase and this ultimately results in reduced Kca3.1 activation and
cytokine production.
Small GTP-binding proteins
The insulin-induced activation of PI3K-C2α appears to be
mediated by the small GTP-binding protein TC10 [19].
Interestingly, it has been demonstrated that TC10 forms a
complex with the guanine-nucleotide-exchange factor GAPEX5 which in turn can activate the small GTPase Rab5 in 3T3-L1
adipocytes [36]. This TC10/Rab5 pathway ultimately regulates
the insulin-induced synthesis of PtdIns3P at the plasma membrane
(Figure 3A). Although in this study the authors ruled out
the potential involvement of PI3K-C2α in this pathway, this
conclusion was mainly based on the observation that the enzyme
was not able to directly interact with TC10. It remains to
be determined whether activation of PI3K-C2α can still occur
downstream of this pathway in the absence of direct association
between the enzyme and TC10. Similarly, further studies are
required to investigate whether other GTPases can have a role
in the activation of PI3K-C2α or other class II PI3K isoforms.
Although association of PI3K-C2α with a 160 kDa protein was
reported upon insulin stimulation [29], the possibility that this
association might be involved in activation of the enzyme has not
yet been investigated further.
Association of PI3K-C2β to the EGF receptor and PDGF
(platelet-derived growth factor) receptor has been detected,
suggesting that this interaction may be involved in the activation
of the enzyme. It was also reported that Grb2, Eps8, Abi1, Sos1
and a Myc-tagged PI3K-C2β formed a multiprotein complex
in A431 cells in a lipid kinase activity-independent manner
[38] (Figure 4B). Although EGF did not appear to stimulate
PI3K-C2β activity in Grb2 immunocomplexes, increased activity
was detected in Eps8 and Abi1 immunoprecipitates upon
EGF stimulation [38]. Similarly, EGF was reported to induce
association of Myc-tagged PI3K-C2β with p45/p52Shc and to
increase class II PI3K activity in Shc immunocomplexes [38].
Association of endogenous PI3K-C2β, but not PI3K-C2α, with
Eps8 was also detected in HEK (human embryonic kidney)293 cells [38]. Consistent with this, the N-terminal and Cterminal SH3 (Src homology 3) domains of Gbr2 were shown to
associate with PI3K-C2β, but not PI3K-C2α [38], supporting the
hypothesis of a specific mechanism of regulation of PI3K-C2β.
Finally, association of PI3K-C2β to c-Kit (the SCF receptor) was
reported, although this did not appear to be modulated by cellular
stimulation [13].
PI3K-C2β was identified as a binding partner for the protein
intersectin in a yeast two-hybrid screen [41]. GST (glutathione
transferase)-pull-down experiments and co-immunoprecipitation
studies using overexpressed proteins revealed further that the
proline-rich sequences at the N-terminal region of PI3K-C2β were
sufficient to bind the SH3 domains of intersectin [41]. An increase
in the association of overexpressed intersectin with PI3K-C2β was
reported upon EGF stimulation in A431 cells, and in vitro kinase
assays suggested that overexpression of intersectin increased the
activity of PI3K-C2β, in particular upon EGF stimulation.
Post-translational modifications
A potential phosphorylation of PI3K-C2α upon insulin
stimulation was originally proposed in 1999 [29], although
this has not been investigated further. Tyrosine phosphorylation
of PI3K-C2β upon SCF, hepatic growth factor, insulin and
fibroblast growth factor-2 stimulation was also reported in the
SCLC cell line H-209 [32]. Similarly EGF appeared to induce
tyrosine phosphorylation of PI3K-C2β in HEK-293 cells. This
phosphorylation event did not seem to mediate binding of PI3KC2β to the receptors, since for instance association with c-Kit
and, to a lesser extent, with c-Met and IGF-1 (insulin-like growth
factor 1) receptor was already detectable under resting conditions
and did not change upon cellular stimulation. Whether tyrosine
phosphorylation of the enzyme is required for its activation is still
not clear.
An interesting potential mechanism of activation was observed
in platelets from patients with SSc (systemic sclerosis) that
displayed nitrotyrosylation of PI3K-C2β [42]. This posttranslational modification increased the activity of the enzyme and
the activity was also higher in lysates from platelets of patients
with SSc compared with normal controls [42]. No other evidence
of nitrotyrosylation of class II enzymes has appeared so far, and
it is not clear whether this modification is indeed involved in
regulation of class II PI3K activity in vivo and how this may
occur.
c The Authors Journal compilation c 2012 Biochemical Society
592
M. Falasca and T. Maffucci
Calpains
Proteolysis of PI3K-C2β upon cellular stimulation has been
reported in few studies, leading to the hypothesis that this process
may be involved in activation of the enzyme. For instance, PI3KC2β seems to be activated during G2 /M-phase of the cell cycle,
as assessed by in vitro assays on the enzyme immunoprecipitated
from nuclei and nuclear envelopes. Under the same conditions,
a gel shift of 18 kDa was detected in Western blot analysis of
the enzyme. Results supported a role for calpains in PI3K-C2β
proteolysis, specifically during G2 /M-phase of the cell cycle [26].
Table 1 Intracellular functions regulated by class II PI3Ks assessed by
in vitro studies
Enzyme
Cellular function
Reference(s)
PI3K-C2α
Glucose transport
Neurosecretory granule release
Insulin secretion
Endocytosis
VSMC contraction
Cell growth and survival
Cell migration
K + channel activation
Cell growth and survival
Cell-cycle progression
Homing of leukaemic cells
[20]
[21,22]
[24,45]
[18,37]
[43,55,56]
[46,57,58,59,60]
[25,27,38]
[28,47]
[38,65]
[26]
[66]
PI3K-C2β
Calcium
Several lines of evidence indicate that the activity of PI3KC2α can be enhanced by increasing concentrations of Ca2 +
and that stimuli able to trigger intracellular Ca2 + increase can
modulate the PI3K-C2α-dependent synthesis of PtdIns3P in vitro
and in vivo (Figure 3B). In vitro kinase assays performed on
endogenous protein immunoprecipitated from de-endothelialized
rabbit aortic vascular smooth muscle revealed activation of PI3KC2α upon treatment with KCl, noradrenaline or ionomycin, all
able to increase intracellular Ca2 + [43]. Moreover it was reported
that cAMP inhibited the Ca2 + -induced activation of PI3K-C2α
and related signalling pathway [44]. Similarly, in vitro kinase
assays performed using immunoprecipitated recombinant PI3KC2α showed that increasing Ca2 + concentration enhanced the
activity of the enzyme in a dose-dependent manner, as well
as the synthesis of PtdIns3P in vitro using purified chromaffin
cells [22] (Figure 3B). Increasing the Ca2 + concentration also
recruited the PtdIns3P-binding protein GFP–2×FYVE to the
LDCVs in PC12 cells [22]. Ca2 + may also play a role in PI3KC2α activation during insulin granule exocytosis induced by
membrane depolarization [45], but this hypothesis has not yet
been investigated (Figure 3C).
Interestingly, it has been demonstrated that PI3K-C2α and
PI3K-C2β, but not p110α, can utilize Ca2 + as a cofactor for
phosphate transfer in in vitro kinase assays [13,17]. Under
these conditions, the enzymes appear to lose their already
low activity towards PtdIns4P in vitro [17]. One interesting
hypothesis is that a local increase in Ca2 + , as for instance
during exocytosis, specifically activates class II PI3Ks to generate
PtdIns3P. However, it is still not clear how Ca2 + activates PI3KC2α, and the most likely scenario is that this occurs through the
two C2 domains within the enzyme.
Novel mechanisms of regulation of class II PI3Ks
Evidence is currently emerging suggesting novel potential
mechanisms of regulation of class II PI3Ks. For instance, it has
been demonstrated that the expression levels of PI3K-C2α can
be regulated by miR-30e-3p in CRC (colorectal cancer) cells and
this may have direct consequences in the control of cell growth,
as discussed below [46]. More recently, a yeast two-hybrid screen
identified the protein TRIM27 (tripartite-motif-containing protein
27) as able to bind to PI3K-C2β [47]. Co-immunoprecipitation
of the two proteins was observed using FLAG-tagged TRIM27
and GFP–PI3K-C2β and also between endogenous proteins in
HEK-293 cells [47]. Importantly, the authors showed that coexpression of TRIM27 with PI3K-C2β increased ubiquitination
of the lipid kinase. Specifically, TRIM27 was shown to mediate
Lys48 polyubiquitination of PI3K-C2β and to inhibit its enzymatic
activity without inducing degradation of the enzyme (Figure 4C).
This ultimately resulted in inhibition of the K + channel KCa3.1
and CD4 T-cell activation [47]. It is important to mention that,
although a very weak association between FLAG-tagged TRIM27
c The Authors Journal compilation c 2012 Biochemical Society
PI3K-C2γ
and GFP–PI3K-C2α was also detected, no ubiquitination of PI3KC2α was observed, suggesting a specific mechanism of regulation
of the β isoform.
CELLULAR FUNCTIONS
PI3K-C2α
In the last few years, several studies have investigated the
physiological roles of PI3K-C2α [48]. A summary of the cellular
functions regulated by this enzyme is given in Table 1.
Glucose transport
We have demonstrated that PI3K-C2α is required for full
translocation of GLUT (glucose transporter) 4 to the plasma
membrane of muscle cells [20], a key process for regulation of
glucose removal from the bloodstream. Indeed down-regulation
of the enzyme in muscle cells reduces both GLUT4 translocation
and glucose transport [20]. Consistent with this, several results in
the literature support a critical role for PtdIns3P in the regulation
of GLUT4 translocation and glucose transport [19,36,49–53].
How PI3K-C2α and PtdIns3P can control these processes
is still not understood. Since addition of exogenous PtdIns3P is
able to induce GLUT4 translocation, but is not sufficient to
induce glucose uptake [19], we originally proposed that the
enzyme might be involved in movement of the GLUT4-containing
vesicles to the plasma membrane [20]. New evidence arising
from exocytosis processes has recently suggested alternative
interesting possibilities [48]. Therefore the similarities between
these processes led us to hypothesize that PI3K-C2α may control
steps which are common to these processes, as discussed below.
Neurosecretory granule release
The observation that treatment of adrenal chromaffin cells
with anti-PI3K-C2α antibody inhibited carbachol-induced
catecholamine release [21] suggested that this enzyme could
be involved in neurosecretory granule exocytosis (Figure 3B).
This was supported further by the observation that release of
hGH (human growth hormone) by PC12 cells upon treatment
with high K + was enhanced by overexpression of wild-type
PI3K-C2α and inhibited by overexpression of a kinase-dead
PI3K-C2α mutant. Specifically, it was reported that PI3K-C2α
regulates the ATP-dependent priming of neurosecretory granules
[21]. The observation that overexpression of the binding probe
GFP–2×FYVE (but not the mutant GFP–2×FYVEC215S, which
is unable to bind PtdIns3P) was also able to inhibit the release of
Class II phosphoinositide 3-kinases
hGH in PC12 and of catecholamine in chromaffin cells supported
the hypothesis of a specific role for PtdIns3P in this process,
which was confirmed by subsequent studies [21,22].
Insulin secretion
A role for PI3K-C2α in regulation of different steps during insulin
secretion has been recently demonstrated (Figure 3C). Downregulation of PI3K-C2α in MIN6 pancreatic β-cells reduces the
glucose-induced stimulation of insulin secretion. This seems to
occur in a mechanism involving an insulin-dependent PI3K-C2αmediated activation of Akt1, partially through up-regulation of
the gene encoding β-GK (β-cell glucokinase) [24]. This would be
consistent with a previous study reporting that the insulin-induced
transcription of β-GK was not inhibited by high concentrations
of PI3K inhibitors or by a dominant-negative mutant of the
class IA regulatory subunit p85 [54]. Evidence suggests that
PI3K-C2α may have additional roles in insulin secretion. Indeed
we have also recently found that down-regulation of PI3K-C2α
in INS1 rat insulinoma cells inhibits insulin secretion induced
by membrane depolarization [45], possibly by regulating the
fusion of the insulin granules to the plasma membrane. Downregulation of the enzyme did not affect the total insulin content,
the increase in intracellular Ca2 + , the number of insulin granules
proximal to the plasma membrane in resting cells or the expression
levels of proteins important for exocytosis [45]. On the other hand,
proteolysis of SNAP-25 (25 kDa synaptosome-associated protein)
was strongly reduced in cells lacking PI3K-C2α [45].
Endocytosis
Several lines of evidence suggest that PI3K-C2α has a
role in different processes of endocytosis. For instance,
cells overexpressing PI3K-C2α showed reduced endocytosis
of transferrin, reduced displacement of mannose 6-phosphate
receptors from the trans-Golgi network and reduced localization
of Lamp (lysosome-associated membrane protein) 1 and
Lamp2 in lysosomes, suggesting that this enzyme is involved in
regulation of clathrin-dependent endocytosis [18]. More recently,
a reduced internalization of diphtheria toxin, but not of transferrin
receptor, has been observed in HeLa cells upon down-regulation
of PI3K-C2α, suggesting an additional role for PI3K-C2α in
dynamin-independent endocytosis [37]. Interestingly, the process
does not seem to require other PI3Ks, including PI3K-C2β [37].
VSMC (vascular smooth muscle cell) contraction
Several lines of evidence indicate that PI3K-C2α is involved
in regulation of VSMC contraction (Figure 3B). Indeed, it
has been reported that specific down-regulation of this enzyme
inhibits contraction of VSMCs induced by noradrenaline [43] and
ionomycin [55]. This appears to occur through regulation of key
events for muscle contraction, including Rho GTP-loading, and
phosphorylation of 20 kDa myosin light chain [43,55]. Activation
of PI3K-C2α has also been recently detected in aorta and
mesenteric arteries stimulated with KCl, with parallel activation
of Rho [56].
Cell growth and survival
It has been reported that down-regulation of PI3K-C2α increases
apoptosis in HeLa cells in a mechanism involving the intrinsic
pathway, but with no effect on Akt phosphorylation at Ser473 and
593
on GSK3β (glycogen synthase kinase 3β) phosphorylation [57].
When tested in a panel of 23 carcinoma cell lines, PI3K-C2α
down-regulation reduced viability in more than half of them [57].
Similarly, reduced cell proliferation and anchorage-independent
growth together with increased apoptosis was observed in the
Mahlavu hepatoma cell line, upon PI3K-C2α down-regulation
[58], and increased apoptosis was reported in CHO-IR cells
expressing antisense sequences targeting PI3K-C2α [59]. More
recently, it has been reported that overexpression of PI3K-C2α
in mesenchymal stem cells increases the survival rate of these
cells under hypoxic conditions [60] and that down-regulation of
this enzyme reduces cell growth in DLD1 CRC cells [46]. In
contrast with these data, we did not detect any effect on cell
proliferation and growth in L6 muscle cells upon stable downregulation of PI3K-C2α [20], consistent with no effect detected
in human BdSMCs (bladder smooth muscle cells) and WI-38
human lung epithelial fibroblast cells [57]. Similarly, no effect on
proliferation was detected in rat insulinoma cells INS1, at least
when analysed in normal growing conditions [45].
Whether PI3K-C2α has a role in regulation of cell proliferation
and/or survival specifically in some cancer cells is an interesting
hypothesis that needs to be investigated further. Moreover, the
specific signalling pathways regulated by PI3K-C2α in this
process remain to be defined.
PI3K-C2β
A summary of the cellular functions regulated by this enzyme is
given in Table 1.
Cell migration
A specific role for PI3K-C2β in control of cell migration has
been supported by our study demonstrating that down-regulation
of this enzyme inhibits the LPA-dependent migration of ovarian
and cervical cancer cell lines [25] (Figure 4A). Under the same
experimental conditions, down-regulation of PI3K-C2α did not
affect the process, suggesting a specific role of the β isoform
in cell migration in these cells. This conclusion was supported
further by a study investigating the role of PI3K-C2β in HEK-293
cells [27]. Overexpression of PI3K-C2β increased cell motility,
decreased cell adhesion and stimulated the formation of actinrich lamellipodia and filopodia. Both studies demonstrated a key
role for PtdIns3P in the PI3K-C2β-dependent regulation of cell
migration. It was later reported that overexpression of wildtype PI3K-C2β in A-431 epidermoid carcinoma cells induced
an increase in F-actin and E-cadherin at cell–cell junctions and
lamellipodia formation, whereas overexpression of a kinasedead PI3K-C2β isoform resulted in an increase in perinuclear
F-actin [38]. Moreover, overexpression of wild-type PI3K-C2β
was associated with increased membrane ruffling and migration
speed in A-431 cells either in the presence or in the absence
of EGF stimulation [38]. Consistent with this, overexpression of
kinase-dead PI3K-C2β reduced cell speed and migration. On the
other hand, down-regulation of PI3K-C2β in freshly isolated Tlymphocytes, followed by 3 days of ex vivo culture, did not appear
to affect CXCL12-induced cell migration [61], again supporting
the hypothesis that PI3K-C2β may regulate migration in specific
cancer cells.
K + channel activation
It has been demonstrated that PI3K-C2β can be activated by
the TCR (T-cell receptor) and that in turn it can activate the
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M. Falasca and T. Maffucci
K + channel KCa3.1 [28]. Specifically, down-regulation of PI3KC2β by siRNA (small interfering RNA) inhibits KCa3.1 channel
activity in human CD4 + T-cells and in Jurkat T-cells transfected
with KCa3.1, whereas overexpression of the enzyme in these
latter cells increased TCR-stimulated activation of KCa3.1. A
specific role for PtdIns3P in this process has been demonstrated
[28], consistent with several other lines of evidence supporting
the role of this specific phosphoinositide in this process [62–64].
As discussed above, a role for TRIM27 in regulation of the PI3KC2β-dependent KCa3.1 activation has been reported recently [47]
(Figure 4C).
Cell growth and survival
Increased proliferation and protection from anoikis induced by
cell detachment was detected in A-431 cells overexpressing wildtype PI3K-C2β compared with parental cells [38]. Interestingly,
both processes appear to be independent of Akt activation
[38]. Overexpression of PI3K-C2β inhibits apoptosis induced
by down-regulation of intersectin in N1E-115 cells during
differentiation, suggesting that this enzyme may be involved in
survival of neuronal cells [41]. Consistent with this, it has been
reported recently that overexpression of PI3K-C2β counteracts
the inhibitory effect of intersectin down-regulation on anchorageindependent growth of neuroblastoma cell line IMR-5 [65]. No
effect on growth of adherent cells was detected upon PI3K-C2β
overexpression [65]. The effect of PI3K-C2β down-regulation
was not determined in these studies neither was whether PI3KC2β is the only PI3K involved in this process.
podocyte morphology and function. It must be mentioned that,
although the PI3K activity in brain and liver was almost blunted
in PI3K-C2α − / − mice, a truncated form of the enzyme was still
detected, albeit with a lower expression.
PI3K-C2β
A knockout model for PI3K-C2β was generated few years ago
using targeted gene deletion [68]. No major phenotype was
detected, with mice appearing to be viable and fertile. This
particular study focused on investigation of a potential role for
PI3K-C2β in epidermal differentiation and it reported that these
mice displayed normal epidermal growth, differentiation and
normal barrier function and wound healing, suggesting that PI3KC2β does not have a major role in epidermal differentiation. No
further investigation of the potential effects of PI3K-C2β deletion
on other cellular functions or in relation to human diseases has
been reported so far.
Whether other phenotypes will be revealed by generation of
distinct knockout and/or knockin mouse models for PI3K-C2α
and PI3K-C2β remain to be defined.
PI3K-C2γ
No report of a knockout or knockin mouse model for PI3K-C2γ
has been published so far, confirming that, of the three class II
PI3K isoforms, this enzyme is definitely the least investigated and
characterized.
Cell-cycle progression
DOWNSTREAM EFFECTORS
The demonstration that the activity of nuclear PI3K-C2β appears
to increase in the nuclei and nuclear envelopes when HL-60 cells
progress to the G2 /M-phase [26] suggests that this enzyme may
have a role in the control of cell cycle. A clear demonstration that
the enzyme is indeed involved in this process and its potential
specific function is, however, missing.
A downstream effector of class II PI3Ks regulating cell survival: the
usual Akt?
PI3K-C2γ
As far as we are aware, there is no evidence in the literature
of an intracellular function specifically regulated by PI3K-C2γ .
Recently, a paper has reported a specific down-regulation of
this isoform in Ba/F3 cells transformed by p185Bcr-Abl compared
with parental cells [66]. The authors have proposed that downregulation of PI3K-C2γ may represent a mechanism by which
Bcr-Abl inhibits chemotaxis and induces abnormal homing of
leukaemic cells. However, no demonstration that PI3K-C2γ can
indeed regulate these processes directly was provided.
MOUSE MODELS
PI3K-C2α
A recent knockout model for PI3K-C2α showed that PI3K-C2α
was essential for normal postnatal development, as judged by
the survival curve [67]. PI3K-C2α − / − mice presented reduced
body fat and lean body mass compared with wild-type mice at 4–
6 weeks of age. The authors reported further that approximately
30 % of PI3K-C2α − / − mice died by 6 months of age (compared
with only 5 % of wild-type mice) and they presented all features
of chronic renal failure. Histological analysis revealed a severe
glomerulonephropathy with defects specifically at the level of
c The Authors Journal compilation c 2012 Biochemical Society
On the basis of the possibility that class II PI3Ks could generate
PtdIns(3,4)P2 (at least in vitro), a few studies have investigated
whether these enzymes might also activate Akt. A careful
analysis of the data presented in the literature shows that there
is contrasting evidence on this issue.
We reported that phosphorylation of Akt at Ser473 and of its
downstream target GSK3β upon a short stimulation with insulin
or PDGF was not affected by down-regulation of PI3K-C2α in L6
cells, consistent with the fact that the insulin-induced synthesis of
PtdIns(3,4)P2 and PtdIns(3,4,5)P3 was not inhibited in these cells
[20]. Recently, it has been demonstrated that ERK (extracellularsignal-regulated kinase), but not Akt Ser473 , phosphorylation
was inhibited in CHO-IR and HepG2 cells expressing antisense
sequences targeting PI3K-C2α and stimulated with insulin for
10 min [69]. Furthermore, down-regulation of PI3K-C2α did not
affect Akt (Ser473 ) and GSK3β phosphorylation in HeLa cells
in serum [57]. On the other hand, an increase in Akt1 activity
was detected in MIN6 cells overexpressing wild-type, but not
catalytically inactive PI3K-C2α, and inhibition of the insulininduced Akt1 activation was reported in these cells upon downregulation of PI3K-C2α [24].
Similarly, contrasting data are present in the literature on
the role of PI3K-C2β in Akt activation. Overexpression of a
dominant-negative PI3K-C2β mutant inhibited the EGF-induced
Akt phosphorylation in HEK-293 and COS7 cells [32]. Similar
results were obtained in HEK-293 cells expressing an antisense
construct targeting PI3K-C2β. Furthermore, overexpression of
a potentially active PI3K-C2β (a mutant lacking the C2
domain) isoform increased Akt phosphorylation. A potential
PI3K-C2β/Akt pathway was also proposed to be involved
Class II phosphoinositide 3-kinases
in the intersectin-dependent regulation of neuronal survival
[41]. However, no direct demonstration that Akt was indeed
phosphorylated downstream of PI3K-C2β was provided in that
study.
Although these data suggested a positive role for PI3K-C2β
in Akt regulation, no difference in Akt phosphorylation at Ser473
was detected in A-431 cells overexpressing PI3K-C2β compared
with parental cells, and Akt did not seem to regulate the increase
in proliferation and resistance to anoikis detected in these cells
[38]. An interesting novel pathway has been proposed recently
which would indicate a specific role for PI3K-C2β in a negative
regulation of Akt phosphorylation [70]. This study showed that
down-regulation of the phosphatase MTM (myotubularin) 1
inhibits the EGF-induced Akt phosphorylation in HeLa cells [70].
Notably, MTM1 dephosphorylates PtdIns3P, therefore its downregulation may result in accumulation of this phosphoinositide.
Co-silencing of PI3K-C2β but not PI3K-C2α restores the EGFdependent Akt phosphorylation in MTM1-down-regulated cells,
suggesting that a pool of PtdIns3P specifically generated by
PI3K-C2β inhibits phosphorylation of Akt upon EGF stimulation.
Consistent with this hypothesis, blockade of PtdIns3P through
overexpression of the binding domain 2×FYVE counteracts the
effect of MTM1 down-regulation on Akt.
One interesting hypothesis, which could help explain
the contrasting evidence, is that class II PI3Ks do not
regulate Akt activation directly through synthesis of a specific
phosphoinositide but indirectly through cross-talk with other
signalling molecules. More studies are required to determine
definitively whether class II PI3Ks are indeed involved in
regulation of Akt activation either directly or indirectly.
595
effectors of PI3K-C2β in control of cell migration. Similarly, Rho
activation seems to be crucial for PI3K-C2α-dependent regulation
of VSMC contraction [43,55,56], as discussed above (Figure 3B).
How exactly class II PI3Ks can control small GTPases remains
to be defined. An intriguing possibility is that interaction of the
Rac-specific guanine-nucleotide-exchange factor Tiam1 (T-cell
lymphoma invasion and metastasis 1) with PtdIns3P is involved
in the PI3K-C2β-dependent regulation of Rac (Figure 4A), but
this hypothesis needs to be tested.
New downstream effectors?
The main downstream effectors of class II PI3Ks, namely
the proteins activated by the class II PI3K-dependent pool
of PtdIns3P, are still unknown. Proteins possessing PtdIns3Pbinding domains, such as PX, FYVE and PH domains, are the
most likely candidates, but they are still undefined.
The possibility that the PtdIns3P generated by class II PI3Ks
acts as a precursor for the synthesis of PtdIns(3,5)P2 by PIKFyve
and that it is actually the bisphosphate which may ultimately
regulate some cellular functions cannot be excluded (Figure 3A).
In this respect, it is important to mention that several reports
have demonstrated that PtdIns(3,5)P2 is involved in regulation
of glucose transport [77]. Whether PtdIns3P and PtdIns(3,5)P2
act independently in this process remain to be addressed. On the
other hand, it has been reported that PIKFyve negatively regulates
neurosecretory granules exocytosis [23] (Figure 3B).
CLASS II PI3Ks AND HUMAN DISEASES
A common downstream effector in PI3K-C2α-mediated exocytosis?
As discussed above, PI3K-C2α is involved in regulation of
neurosecretory granules and insulin granules exocytosis, as
well as GLUT4 translocation that, although not a proper
exocytotic process, still requires similar processing steps. We have
proposed previously that the enzyme may control steps which are
common between these processes, for instance by facilitating the
interaction between granules/vesicles and plasma membrane [48].
We suggested a potential role for the protein VAMP8 (vesicleassociated membrane protein 8), which has been shown to bind
PtdIns3P [71], and it is associated with the insulin secretory
granules in INS-1E cells [72]. We have also suggested that PI3KC2α may have a role in regulation of calpain 10, a protease
involved in GLUT4 translocation [73–75] and in insulin secretion
[76]. Alternatively, PtdIns3P may be directly involved in the
fusion event during exocytosis [48]. These hypotheses need to
be tested.
Small GTP-binding proteins
Evidence suggests that small GTPases can act as downstream
effectors of class II PI3Ks. For instance, overexpression
of PI3K-C2β appears to be able to stimulate Rac activity
in A-431 epidermoid carcinoma cells under both basal
and EGF-induced conditions (Figure 4B), and this is
associated with enhanced membrane ruffling and migration
speed of the cells [38]. Consistently, expression of dominantnegative PI3K-C2β reduces Rac activity as well as membrane
ruffling and migration speed of the cells [38]. Another study has
shown that overexpression of PI3K-C2β increases GTP loading
of Cdc42 in HEK-293 cells [27]. Taken together, these data
suggest that small GTP-binding proteins can act as downstream
Although it is well established that deregulation of class I
PI3K-dependent pathways is associated with several diseases,
including diabetes and cancer, there is currently no clear and direct
demonstration of a role for class II PI3Ks in human diseases. Data
are now emerging suggesting that this may be the case. A list of
potential connections between class II PI3Ks and human diseases
is given in Table 2.
Diabetes
PI3K-C2α
PI3K-C2α is involved in both glucose disposal into muscle cells
[20] and insulin secretion [24,45]. It is therefore tempting to
speculate that deregulation of signals controlled by this enzyme
may result in inhibition of glucose disposal, a hallmark of
insulin resistance, and in reduced insulin secretion, indicative
of pancreatic β-cell dysfunction, and it may ultimately have a
role in Type 2 diabetes. Although this hypothesis needs to be
formally tested, we have reported recently that PI3K-C2α mRNA
is down-regulated in islets from Type 2 diabetic compared with
non-diabetic individuals [45]. No difference in the mRNA for
PI3K-C2β and PI3K-C2γ was observed. Whether this is an early
event in the disease progression or a secondary effect and whether
this contributes to pancreatic β-cell loss of function remains to be
determined.
PI3K-C2γ
Polymorphisms in the gene encoding PI3K-C2γ have been
detected in a Japanese population and found to be associated
with Type 2 diabetes [78].
c The Authors Journal compilation c 2012 Biochemical Society
596
Table 2
M. Falasca and T. Maffucci
Potential connections of class II PI3Ks and human diseases
Class II isoform
Human disease
Evidence
Reference(s)
PI3K-C2α
Diabetes
In vitro studies
mRNA down-regulation in islets from Type 2 diabetic patients
Decrease in DNA copy number/increase in mRNA levels in hepatocellular carcinoma
In vitro studies
In vitro studies
In vivo study
Single-nucleotide polymorphism associated with prostate cancer risk
Amplification of PIK3C2B found in glioblastoma
DNA copy number gain in ovarian cancer
Overexpression in ESCC tissues
High levels in neuroblastoma cell lines and primary tumours
In vitro studies
Polymorphisms associated with Type 2 diabetes
[20,24,45]
[45]
[58]
[58,46,79]
[43,44,55]
[56]
[81]
[82–84]
[85]
[88]
[65]
[86,87]
[78]
Cancer
Cardiovascular diseases
PI3K-C2β
Cancer
PI3K-C2γ
Diabetes
Cancer
PI3K-C2α
Few data have appeared supporting the hypothesis that PI3K-C2α
may have a role in cancer. A slight decrease in the DNA copy
number, but a slight increase in mRNA levels, was reported in
19 hepatitis B-positive hepatocellular carcinoma compared with
matched non-tumour counterparts [58]. We also detected PI3KC2α in a subset of pancreatic ductal adenocarcinoma specimens
and normal tissues, and we observed that the enzyme localized
in acini and ducts both in cancer and normal tissue. A higher
expression was detected in acini with high cellular atypia and in
dysplastic ducts. In contrast, PI3K-C2β was only visibly stained
in a few samples [79].
PIK3C2A, the gene encoding PI3K-C2α, was one of the genes
expressed at higher levels in a cell population of MCF7 breast
cancer cells enriched in cancer stem-like cells with increased
tumorigenicity in vivo compared with the normal population [80].
Recently, it has been demonstrated that PI3K-C2α is repressed
by miR-30e-3p, mostly at translational levels in DLD1 CRC cells
[46]. Interestingly, the authors showed that miR-30e-3p is one
of the miRNAs (microRNAs) which appear to be significantly
down-regulated in CRC tissue compared with healthy mucosa,
possibly as an early event in CRC carcinogenesis. Although
not investigated directly in this study, this evidence suggests
the hypothesis that PI3K-C2α may be up-regulated in CRC as
a consequence of miR-30e-3p down-regulation and it may be
involved in promoting cell growth. More intriguingly, inhibition
of the Wnt signalling pathway in DLD1 cells up-regulates miR30e-3p. The interesting hypothesis that PI3K-C2α expression can
be regulated by a Wnt/miRNA pathway needs to be urgently
tested.
PI3K-C2β
There are few lines of evidence in literature suggesting a role for
this specific isoform in cancer, although most of the data are still
at the genetic level.
For instance, one single-nucleotide polymorphism in PIK3C2B,
the gene encoding PIK3-C2β, has been reported recently to be
significantly associated with prostate cancer risk [81]. Association
was stronger for men who were diagnosed before the age of
65 years or had a family history and among men in the top tertile
of circulating IGF-1 levels.
c The Authors Journal compilation c 2012 Biochemical Society
Amplification of PIK3C2B was detected in six tumours and
mRNA overexpression in four cases in an analysis of 103
glioblastomas [82], and amplification of PIK3C2B/MDM4 was
reported in an analysis of genome-wide copy number alterations
in glioblastoma multiforme [83]. Gain at 1q32.1 (PIK3C2B and
MDM4) was also reported in a recent study performing wholegenome amplification to amplify DNA from histological sections
of glioblastomas [84].
PIK3C2B displayed significant DNA copy number gain in
ovarian cancer as assessed by a high-resolution array comparative
genomic hybridization in 89 specimens [85]. The increase
in PIK3-C2β was higher than the increase in other class I
PI3K catalytic and regulatory subunits analysed, although only
the mRNA for PIK3R3 (PI3K regulatory subunit 3) appeared
significantly up-regulated in ovarian cancer compared with
normal ovary.
The significance and consequences of PI3K-C2β amplification
remain to be demonstrated. A study using complementary RNAs
from cell lines representing sensitive, intermediate and resistant
phenotypes reported that the expression of PIK3-C2β, as well as
of IGF-1, was significantly correlated with resistance to erlotinib
[86], suggesting that this enzyme may be involved in resistance to
chemotherapeutics of glioblastoma multiforme. This possibility
is supported by a study that investigated the effect of downregulation of 779 kinases and related proteins by RNAi (RNA
interference) on viability of MCF7 cells upon treatment with
tamoxifen [87]. The siRNA targeting PIK3C2B was in the list
of the top 20 siRNAs able to sensitize the cells to the effect
of tamoxifen, suggesting that the enzyme may be involved in
resistance to tamoxifen in breast cancer cells [87]. This hypothesis
appears to be supported further by a recent study demonstrating
that overexpression of PI3K-C2β in Eca109, a human ESCC
(oesophageal squamous cell carcinoma) cell line, resulted in
a more than 4-fold reduction in their sensitivity to cisplatin
compared with control cells [88]. The authors showed further
that down-regulation of the enzyme in PI3K-C2β-overexpressing
Eca109 cells by specific siRNA restored their sensitivity to the
drug. These data suggest that PI3K-C2β may have a role in
promoting resistance to chemotherapeutic drugs. Surprisingly,
a more recent study has suggested that deletion of PI3KC2β may actually promote resistance of leukaemia cells to
specific chemotherapeutics [89]. The authors showed that downregulation of PI3K-C2β in CEM human leukaemia cells confers
resistance to treatment with thioguanine and mercaptopurine
without affecting their sensitivity to other drugs. Importantly, the
Class II phosphoinositide 3-kinases
authors showed that down-regulation of PI3K-C2β resulted in
reduction of MSH2 (mutS homologue 2), a key protein for repair
of DNA mismatch, mostly due to increased ubiquitination and
degradation of the protein. Reduction of MSH2 as a consequence
of PI3K-C2β down-regulation resulted in a significant reduction
in the DNA mismatch repair capacity in leukaemia cells.
Recently, a study has evaluated the protein expression
of PI3K-C2β in ESCC tissues from 61 patients using
immunohistochemistry [88]. The authors reported that the enzyme
was not expressed in normal stratified squamous epithelial cells,
whereas 45.9 % of the ESCC cases were positive for PI3K-C2β,
with a diffuse cytoplasmic staining of squamous tumour cells.
No association with tumour stage, differentiation, gender or age
was observed. However a significant association between PI3KC2β protein levels and metastasis was detected, with 68.2 %
of PI3K-C2β-positive cases showing metastases compared with
only 31.8 % of PI3K-C2β-negative cases [88]. These results,
together with data indicating a role for PI3K-C2β in regulation of
cancer cell migration in vitro [20,27,38], including in ESCC [88],
strongly support the hypothesis that PI3K-C2β may be involved
in metastasis by regulating cancer migration and invasion.
High levels of PI3K-C2β protein expression were also
recently reported in neuroblastoma cell lines and primary human
neuroblastoma tumour samples [65].
597
whether mammalian class II PI3Ks (and which specific isoform)
have a similar role and whether deregulation of these enzymes are
associated with these diseases.
OPEN QUESTIONS
Our understanding of class II PI3Ks has greatly improved in
the last few years, but several questions still remain to be
answered.
Are class I and class II PI3Ks redundant?
Hyperactivation of PI3K-C2α, but not p110α, was detected in
aortae from SHRs (spontaneously hypertensive rats) compared
with normotensive rats with concomitant increase of Rho activity
[56]. The Ca2 + channel blocker nicardipine as well as infusion
of high concentrations of wortmannin (which inhibits PI3K-C2α
activity) inhibited all these effects to the values of normotensive
rats, strongly reducing systolic blood pressure in aortae and
mesenteric arteries of SHRs. These data suggested that PI3KC2α may have a role in hypertension.
The strongest data currently available in literature support
the conclusion that class II PI3Ks generate a distinct lipid
product in vivo compared with class I PI3Ks. Since different
phosphoinositides can bind distinct protein domains, it is very
likely that the main downstream effectors of class I and class
II PI3Ks are different. Therefore the most important questions
that need to be answered is whether class II PI3Ks control
the same intracellular processes as those of class I PI3Ks or
whether they regulate different functions or possibly different
steps within the same process. There are several lines of evidence
in the literature already suggesting that class I and II PI3Ks
are not redundant and that they often act together to regulate
cellular processes. For instance, our data suggest that a cooperative action of PI3K-C2α and class I isoforms is necessary
to fully activate GLUT4 translocation and glucose transport [20].
Similarly, insulin secretion appears to require activation of the
class II isoform PI3K-C2α [24,45], as well as members of the class
I subfamily, as demonstrated by data obtained using knockout
mice for p110γ [94,95] and in mice lacking two of the three
genes encoding the class IA regulatory subunits [96].
To definitively answer this question, it will be necessary to
finally determine the selective in vivo lipid product of class II
PI3Ks upon different cellular stimulation and to identify the
specific downstream effectors.
Neuromuscular diseases
Are class II and class III PI3Ks redundant?
Accumulating evidence suggests that class II PI3Ks may be
involved in several human genetic diseases, including myopathy
and neuropathies. Several of these diseases are associated with
mutations in enzymes belonging to the family of MTMs,
phosphatases responsible for dephosphorylation of PtdIns3P and
PtdIns(3,5)P2 . For instance, mutations in MTMs have been
associated with forms of CMT (Charcot–Marie–Tooth) diseases, a
group of neuropathies affecting motor and sensory nerves [90,91].
Mutations all over the gene encoding MTMR (MTM-related
protein) 2 were detected in the autosomal recessive demyelinating
neuropathy CMT4B1 and five distinct mutations in the MTMR13
gene have been reported to cause CMT4B2. Approximately 200
mutations in the gene encoding MTM1 have been found in
patients affected by X-linked centronuclear myopathy [91], a very
severe congenital myopathy. Recent work in D. melanogaster has
highlighted a role for class II PI3K in synthesis of a PtdIns3P pool
specifically regulated by MTMs [92,93]. Indeed, it was reported
that depletion of PI3K68D, the only class II PI3K isoform in
Drosophila, is able to rescue defects in protrusion and distribution
observed in immune cells deficient for the single Drosophila
MTM1/MTMR2 orthologue (mtm) [92]. More recently, it has
been shown that PI3K68D deletion can also rescue the defect
in integrin adhesions detected in mtm-depleted muscle [93],
suggesting that a class II PI3K/MTM pathway may be important
for muscle attachment. It would be very important to determine
As they stand at the moment, results in the literature indicate
that class II enzymes are mainly responsible for synthesis of
PtdIns3P in vivo. Since the class III isoform hVps34 catalyses
the synthesis of the same phosphoinositide, it is important to
determine whether the two classes play redundant functions
intracellularly.
The first studies demonstrating that class II isoforms catalyse
the synthesis of PtdIns3P suggested that the class II PI3Kdependent was distinct from the hVps34-dependent PtdIns3P
[7,19,20,25,49]. Indeed, class II PI3Ks appeared to regulate
a pool of PtdIns3P which was specifically synthesized at the
plasma membrane upon cellular stimulation [49]. In contrast,
it was well established that class III PI3K is responsible for
synthesis of PtdIns3P mainly within the endosomal compartment
[9]. Further data have suggested that class II PI3Ks may also
be involved in the regulation of intracellular pools of PtdIns3P,
including a potential nuclear pool and possibly a Golgi-associated
pool [49], supporting the hypothesis that, although able to
regulate the synthesis of the same phosphoinositide, class II
and class III PI3Ks are not redundant, since they act in distinct
cellular compartments. Consistent with distinct intracellular
compartmentalization, the cellular functions ascribed to class
II and class III PI3Ks are mostly different. hVps34 has a well
established role in endocytic sorting and autophagy, and evidence
suggests that it is also involved in nutrient-dependent mTOR
Cardiovascular diseases
c The Authors Journal compilation c 2012 Biochemical Society
598
M. Falasca and T. Maffucci
(mammalian target of rapamycin) regulation [9]. No study so
far has reported the involvement of class II PI3Ks in these cellular
functions, suggesting that the two classes of PI3K regulate distinct
intracellular functions.
However, it is worth mentioning that a detailed investigation of
the potential involvement of class II PI3Ks in cellular functions
such as autophagy and mTOR regulation has not been performed
yet, therefore the possibility that the two classes of PI3Ks can cooperate to control these processes cannot be completely ruled
out. Similarly, evidence suggesting the possibility that PI3KC2α can also contribute to synthesis of PtdIns3P within the
endosomal compartment has also been reported [49]. It remains to
be established whether hVps34 and PI3K-C2α may act in distinct
subcompartments within the endosomal system to regulate
different functions or whether they co-operate within the same
compartment. Evidence of a co-operative action of class II and
class III PI3Ks has emerged recently from a study demonstrating
that PIKI-1 (the class II PI3K) and Vps34 co-operate to generate
pools of PtdIns3P on phagosomes in Caenorhabditis elegans [97].
Specifically, it has been demonstrated that the two enzymes act
sequentially, with PIKI-1 responsible for the initial synthesis of
PtdIns3P on nascent phagosomes and Vps34 required for the
sustained production of this phosphoinositide [97]. This intriguing
study not only supports the hypothesis that class II and class III
PI3Ks are not redundant, but also represents the first example
of co-operation between these two classes of PI3Ks. It is also
worth mentioning that the authors showed further that degradation
as well as synthesis of PtdIns3P is essential for phagosome
maturation and they identified MTM1 as a key phosphatase in
phagosomal PtdIns3P turnover. These data reveal a complex
mechanism of PtdIns3P regulation, involving the sequential
activation of two distinct PI3K isoforms and the concerted action
of a phosphatase. Interestingly, although MTM-1 antagonizes the
activities of both PIKI-1 and Vps34, results also suggest that
PIKI-1 is the major PI3K that counteracts the activity of MTM1
[97].
The interplay between the two kinases that synthesize PtdIns3P
and the MTMs able to dephosphorylate this lipid appears to be
finely regulated. A question still open is whether the turnover
of class II-dependent and the class III-dependent PtdIns3P
pools is also differentially regulated by the family of MTMs.
It was reported previously that hVps34/hVps15 can associate
with MTM1 or with Rab GTPases in a mutually exclusive way
and that this can represent a mechanism to control endosomal
PtdIns3P turnover [98]. Indeed, down-regulation of MTM1 or
MTMR2 resulted in accumulation of PtdIns3P on early and late
endosomes respectively and in defects in growth factor signalling
[99]. More recent evidence, however, seems to suggest that
MTMs may specifically regulate the class II-mediated PtdIns3P
at least in specific cellular contexts. The first indication that
this may be the case came from a report showing that downregulation of MTM1 and MTM6 in C. elegans was able to rescue
the phenotype of Vps34-null mutant [100]. Interestingly, the
authors showed that the MTM-mediated rescue was reduced
when the C. elegans class II PI3K was also down-regulated.
These data not only indicate that pools of PtdIns3P not generated
by Vps34 exist, but they also suggest that these pools are
specifically regulated by MTMs. It was later reported that
depletion of PI3K68D, but not Vps34, was able to counteract
the effects of mtm depletion in Drosophila [92]. Consistent
with these data, it has been shown recently that knockdown of
PI3K-C2β specifically counteracted the effect of MTM1 downregulation on Akt and pro-apoptotic signalling in HeLa cells
[70]. Importantly, down-regulation of hVps34 or PI3K-C2α did
not rescue Akt phosphorylation or cell survival, suggesting that
c The Authors Journal compilation c 2012 Biochemical Society
MTM1 regulates a pool of PtdIns3P specifically generated by
PI3K-C2β [70]. Whether dephosphorylation and turnover of the
two PtdIns3P pools is distinctly regulated needs to be investigated
further.
CONCLUDING REMARKS
This is a very exciting time for the field of class II PI3K
investigation. Not only is there finally a growing interest in
understanding the physiological roles of these enzymes, but
also evidence suggesting that these enzymes may play a role
in pathological conditions is starting to appear. Importantly,
accumulating data in the literature are unveiling a scenario in
which class II PI3Ks do not simply act redundantly to other PI3K
isoforms, but they possibly regulate distinct processes or different
steps in the same process. This is mainly achieved through
synthesis of a different phosphoinositide compared with class I
PI3Ks or through a distinct intracellular localization compared
with class III PI3Ks. The fact that class II isoforms do not
appear to be redundant is currently giving these enzymes a more
defined identity and it is fuelling a huge interest which is likely
to increase in the near future. However, several questions still
need to be answered. A key step in our future investigation of
these enzymes is the definitive demonstration of their in vivo lipid
product upon different cellular stimulation. This is a very urgent
issue, particularly important considering that too often the product
of class II PI3Ks is simply assumed, but not demonstrated directly.
Reaching a general consensus on this matter will make it easier
to investigate the activation of these enzymes and will represent
an important starting point to identify the specific downstream
effectors of these enzymes. The development of specific class
II PI3Ks inhibitors would also greatly improve our knowledge of
their physiological functions and their contribution to pathological
conditions. For sure, these enzymes will reveal many surprises in
the near future.
FUNDING
We thank the Pancreatic Cancer Research Fund, the British Heart Foundation and Diabetes
UK [grant number BDA:09/0003971] for their support.
REFERENCES
1 Cantley, L. C. (2002) The phosphoinositide 3-kinase pathway. Science 296, 1655–1657
2 Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M. and Bilanges, B. (2010) The
emerging mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 11,
329–341
3 Engelman, J. A., Luo, J. and Cantley, L. C. (2006) The evolution of phosphatidylinositol
3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7, 606–619
4 Wong, K. K., Engelman, J. A. and Cantley, L. C. (2010) Targeting the PI3K signaling
pathway in cancer. Curr. Opin. Genet Dev. 20, 87–90
5 Falasca, M. (2010) PI3K/Akt signalling pathway specific inhibitors: a novel strategy to
sensitize cancer cells to anti-cancer drugs. Curr. Pharm. Des. 16, 1410–1416
6 Foukas, L. C. and Withers, D. J. (2010) Phosphoinositide signalling pathways in
metabolic regulation. Curr. Top. Microbiol. Immunol. 346, 115–141
7 Falasca, M. and Maffucci, T. (2007) Role of class II phosphoinositide 3-kinase in cell
signalling. Biochem. Soc. Trans. 35, 211–214
8 Guillermet-Guibert, J., Bjorklof, K., Salpekar, A., Gonella, C., Ramadani, F., Bilancio, A.,
Meek, S., Smith, A. J., Okkenhaug, K. and Vanhaesebroeck, B. (2008) The p110β
isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled
receptors and is functionally redundant with p110γ . Proc. Natl. Acad. Sci. U.S.A. 105,
8292–8297
9 Backer, J. M. (2008) The regulation and function of Class III PI3Ks: novel roles for
Vps34. Biochem. J. 410, 1–17
Class II phosphoinositide 3-kinases
10 MacDougall, L. K., Domin, J. and Waterfield, M. D. (1995) A family of phosphoinositide
3-kinases in Drosophila identifies a new mediator of signal transduction. Curr. Biol. 5,
1404–1415
11 Domin, J., Pages, F., Volinia, S., Rittenhouse, S. E., Zvelebil, M. J., Stein, R. C. and
Waterfield, M. D. (1997) Cloning of a human phosphoinositide 3-kinase with a C2
domain that displays reduced sensitivity to the inhibitor wortmannin. Biochem. J. 326,
139–147
12 Brown, R. A., Ho, L.K.F., Weber-Hall, S. J., Shipley, J. M. and Fry, M. J. (1997)
Identification and cDNA cloning of a novel mammalian C2 domain-containing
phosphoinositide 3-kinase, HsC2-PI3K. Biochem. Biophys. Res. Comm. 233, 537–544
13 Arcaro, A., Volinia, S., Zvelebil, M. J., Stein, R., Watton, S. J., Layton, M. J., Gout, I,
Ahmadi, K., Downward, J. and Waterfield, M. D. (1998) Human phosphoinositide
3-kinase C2β, the role of calcium and the C2 domain in enzyme activity. J. Biol. Chem.
273, 33082–33090
14 Rozycka, M., Lu, Y. J., Brown, R. A., Lau, M. R., Shipley, J. M. and Fry, M. J. (1998)
cDNA cloning of a third human C2-domain-containing class II phosphoinositide
3-kinase, PI3K-C2γ , and chromosomal assignment of this gene (PIK3C2G ) to 12p12.
Genomics 54, 569–574
15 Virbasius, J. V., Guilherme, A. and Czech, M. P. (1996) Mouse p170 is a novel
phosphatidylinositol 3-kinase containing a C2 domain. J. Biol. Chem. 271,
13304–13307
16 Misawa, H., Ohtsubo, M., Copeland, N. G., Gilbert, D. J., Jenkins, N. A. and Yoshimura,
A. (1998) Cloning and characterization of a novel class II phosphoinositide 3-kinase
containing C2 domain. Biochem. Biophys. Res. Commun. 244, 531–539
17 Arcaro, A., Zvelebil, M. J., Wallasch, C., Ullrich, A., Waterfield, M. D. and Domin, J.
(2000) Class II phosphoinositide 3-kinases are downstream targets of activated
polypeptide growth factor receptors. Mol. Cell. Biol. 20, 3817–3830
18 Gaidarov, I., Smith, M. E., Domin, J. and Keen, J. H. (2001) The class II
phosphoinositide 3-kinase C2α is activated by clathrin and regulates clathrin-mediated
membrane trafficking. Mol. Cell 7, 443–449
19 Maffucci, T., Brancaccio, A., Piccolo, E., Stein, R. C. and Falasca, M. (2003) Insulin
induces phosphatidylinositol-3-phosphate formation through TC10 activation. EMBO J.
22, 4178–4189
20 Falasca, M., Hughes, W. E., Dominguez, V., Sala, G., Fostira, F., Fang, M. Q., Cazzolli,
R., Shepherd, P. R., James, D. E. and Maffucci, T. (2007) The role of phosphoinositide
3-kinase C2α in insulin signaling. J. Biol. Chem. 282, 28226–28236
21 Meunier, F. A., Osborne, S. L., Hammond, G. R., Cooke, F. T., Parker, P. J., Domin, J. and
Schiavo, G. (2005) Phosphatidylinositol 3-kinase C2α is essential for ATP-dependent
priming of neurosecretory granule exocytosis. Mol. Biol. Cell 16, 4841–4851
22 Wen, P. J., Osborne, S. L., Morrow, I. C., Parton, R. G., Domin, J. and Meunier, F. A.
(2008) Ca2 + -regulated pool of phosphatidylinositol-3-phosphate produced by
phosphatidylinositol 3-kinase C2α on neurosecretory vesicles. Mol. Biol. Cell 19,
5593–5603
23 Osborne, S. L., Wen, P. J., Boucheron, C., Nguyen, H. N., Hayakawa, M., Kaizawa, H.,
Parker, P. J., Vitale, N. and Meunier, F. A. (2008) PIKfyve negatively regulates exocytosis
in neurosecretory cells. J. Biol. Chem. 283, 2804–2813
24 Leibiger, B., Moede, T., Uhles, S., Barker, C. J., Creveaux, M., Domin, J., Berggren, P. O.
and Leibiger, I. B. (2010) Insulin-feedback via PI3K-C2α activated PKBα/Akt1 is
required for glucose-stimulated insulin secretion. FASEB J. 24, 1824–1837
25 Maffucci, T., Cooke, F. T., Foster, F. M., Traer, C. J., Fry, M. J. and Falasca, M. (2005)
Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration.
J. Cell Biol. 169, 789–799
26 Visnjić, D., Curić, J., Crljen, V., Batinić, D., Volinia, S. and Banfić, H. (2003) Nuclear
phosphoinositide 3-kinase C2β activation during G2 /M phase of the cell cycle in HL-60
cells. Biochim. Biophys. Acta 631, 61–71
27 Domin, J., Harper, L., Aubyn, D., Wheeler, M., Florey, O., Haskard, D., Yuan, M. and
Zicha, D. (2005) The class II phosphoinositide 3-kinase PI3K-C2β regulates cell
migration by a PtdIns3P dependent mechanism. J. Cell. Physiol. 205, 452–462
28 Srivastava, S., Di, L., Zhdanova, O., Li, Z., Vardhana, S., Wan, Q., Yan, Y., Varma, R.,
Backer, J., Wulff, H. et al. (2009) The class II phosphatidylinositol 3 kinase C2β is
required for the activation of the K + channel KCa3.1 and CD4 T-cells. Mol. Biol. Cell
20, 3783–3791
29 Brown, R. A., Domin, J., Arcaro, A., Waterfield, M. D. and Shepherd, P. R. (1999) Insulin
activates the α isoform of class II phosphoinositide 3-kinase. J. Biol. Chem. 274,
14529–14532
30 Turner, S. J., Domin, J., Waterfield, M. D., Ward, S. G. and Westwick, J. (1998) The CC
chemokine monocyte chemotactic peptide-1 activates both the class I p85/p110
phosphatidylinositol 3-kinase and the class II PI3K-C2α. J. Biol. Chem. 273,
25987–25995
31 Ktori, C., Shepherd, P. R. and O’Rourke, L. (2003) TNF-α and leptin activate the
α-isoform of class II phosphoinositide 3-kinase. Biochem. Biophys. Res. Commun.
306, 139–143
599
32 Arcaro, A., Khanzada, U. K., Vanhaesebroeck, B., Tetley, T. D., Waterfield, M. D. and
Seckl, M. J. (2002) Two distinct phosphoinositide 3-kinases mediate polypeptide growth
factor-stimulated PKB activation. EMBO J. 21, 5097–5108
33 Wheeler, M. and Domin, J. (2006) The N-terminus of phosphoinositide 3-kinase-C2β
regulates lipid kinase activity and binding to clathrin. J. Cell. Physiol. 206, 586–593
34 Stahelin, R. V., Karathanassis, D., Bruzik, K. S., Waterfield, M. D., Bravo, J., Williams,
R. L. and Cho, W. (2006) Structural and membrane binding analysis of the Phox
homology domain of phosphoinositide 3-kinase-C2α. J. Biol. Chem. 281,
39396–39406
35 Ursø, B., Brown, R. A., O’Rahilly, S., Shepherd, P. R. and Siddle, K. (1999) The
α-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin
receptors than IGF receptors, and activation requires receptor NPEY motifs. FEBS Lett.
460, 423–426
36 Lodhi, I. J., Bridges, D., Chiang, S. H., Zhang, Y., Cheng, A., Geletka, L. M., Weisman,
L. S. and Saltiel, A. R. (2008) Insulin stimulates phosphatidylinositol 3-phosphate
production via the activation of Rab5. Mol. Biol. Cell 19, 2718–2728
37 Krag, C., Malmberg, E. K. and Salcini, A. E. (2010) PI3KC2α, a class II PI3K, is required
for dynamin-independent internalization pathways. J. Cell Sci. 123, 4240–4250
38 Katso, R. M., Pardo, O. E., Palamidessi, A., Franz, C. M., Marinov, M., De Laurentiis, A.,
Downward, J., Scita, G., Ridley, A. J., Waterfield, M. D. and Arcaro, A. (2006)
Phosphoinositide 3-kinase C2β regulates cytoskeletal organization and cell migration
via Rac-dependent mechanisms. Mol. Biol. Cell 17, 3729–3744
39 Didichenko, S. A. and Thelen, M. (2001) Phosphatidylinositol 3-kinase C2α contains a
nuclear localization sequence and associates with nuclear speckles. J. Biol. Chem. 276,
48135–48142
40 Banfic, H., Visnjic, D., Mise, N., Balakrishnan, S., Deplano, S., Korchev, Y. E. and
Domin, J. (2009) Epidermal growth factor stimulates translocation of the class II
phosphoinositide 3-kinase PI3K-C2β to the nucleus. Biochem. J. 422, 53–60
41 Das, M., Scappini, E., Martin, N. P., Wong, K. A., Dunn, S., Chen, Y. J., Miller, S. L.,
Domin, J. and O’Bryan, J. P. (2007) Regulation of neuron survival through an
intersectin–phosphoinositide 3 -kinase C2β–AKT pathway. Mol. Cell. Biol. 27,
7906–7017
42 Chiang, T. M. and Postlethwaite, A. E. (2006) Increase in phosphotidylinositide-3 kinase
activity by nitrotyrosylation of lysates of platelets from patients with systemic sclerosis.
Biochim. Biophys. Acta 1760, 32–37
43 Wang, Y., Yoshioka, K., Azam, M. A., Takuwa, N., Sakurada, S., Kayaba, Y., Sugimoto, N.,
Inoki, I., Kimura, T., Kuwaki, T. and Takuwa, Y. (2006) Class II phosphoinositide 3-kinase
α-isoform regulates Rho, myosin phosphatase and contraction in vascular smooth
muscle. Biochem. J. 394, 581–592
44 Azam, M. A., Yoshioka, K., Ohkura, S., Takuwa, N., Sugimoto, N., Sato, K. and Takuwa, Y.
(2007) Ca2 + -independent, inhibitory effects of cyclic adenosine 5 -monophosphate on
Ca2 + regulation of phosphoinositide 3-kinase C2α, Rho, and myosin phosphatase in
vascular smooth muscle. J. Pharmacol. Exp. Ther. 320, 907–916
45 Dominguez, V., Raimondi, C., Somanath, S., Bugliani, M., Loder, M. K., Edling, C. E.,
Divecha, N., da Silva-Xavier, G., Marselli, L., Persaud, S. J. et al. (2011) Class II
phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic β
cells. J. Biol. Chem. 286, 4216–4225
46 Schepeler, T., Holm, A., Halvey, P., Nordentoft, I., Lamy, P., Riising, E. M., Christensen,
L. L., Thorsen, K., Liebler, D. C., Helin, K. et al. (2011) Attenuation of the
β-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive
microRNAs that target cancer promoting genes. Oncogene, doi:10.1038/onc.2011.453
47 Cai, X., Srivastava, S., Sun, Y., Li, Z., Wu, H., Zuvela-Jelaska, L., Li, J., Salamon, R. S.,
Backer, J. M. and Skolnik, E. Y. (2011) Tripartite motif containing protein 27 negatively
regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2β. Proc. Natl.
Acad. Sci. U.S.A. 108, 20072–20077
48 Mazza, S. and Maffucci, T. (2011) Class II phosphoinositide 3-kinase C2α: what we
learned so far. Int. J. Biochem. Mol. Biol. 2, 168–182
49 Falasca, M. and Maffucci, T. (2009) Rethinking phosphatidylinositol 3-monophosphate.
Biochim. Biophys. Acta 1793, 1795–1803
50 Chaussade, C., Pirola, L., Bonnafous, S., Blondeau, F., Brenz-Verca, S., Tronchere, H.,
Portis, F., Rusconi, S., Payrastre, B., Laporte, J. and Van Obberghen, E. (2003)
Expression of myotubularin by an adenoviral vector demonstrates its function as a
phosphatidylinositol 3-phosphate [PtdIns(3)P ] phosphatase in muscle cell lines:
involvement of PtdIns(3)P in insulin-stimulated glucose transport. Mol. Endocrinol. 17,
2448–2460
51 Ishiki, M., Randhawa, V. K., Poon, V., Jebailey, J. and Klip, A. (2005) Insulin regulates
the membrane arrival, fusion, and C-terminal unmasking of glucose transporter-4 via
distinct phosphoinositides. J. Biol. Chem. 280, 28792–28802
52 Kanda, H., Tamori, Y., Shinoda, H., Yoshikawa, M., Sakaue, M., Udagawa, J., Otani, H.,
Tashiro, F., Miyazaki, J. and Kasuga, M. (2005) Adipocytes from Munc18c-null mice
show increased sensitivity to insulin-stimulated GLUT4 externalization. J. Clin. Invest.
115, 291–301
c The Authors Journal compilation c 2012 Biochemical Society
600
M. Falasca and T. Maffucci
53 Kong, A. M., Horan, K. A., Sriratana, A., Bailey, C. G., Collyer, L. J., Nandurkar, H. H.,
Shisheva, A., Layton, M. J., Rasko, J. E., Rowe, T. and Mitchell, C. A. (2006)
Phosphatidylinositol 3-phosphate (PtdIns3P ) is generated at the plasma membrane by
an inositol polyphosphate 5-phosphatase: endogenous PtdIns3P can promote GLUT4
translocation to the plasma membrane. Mol. Cell. Biol. 26, 6065–6081
54 Leibiger, B., Leibiger, I. B., Moede, T., Kemper, S., Kulkarni, R. N., Kahn, C. R., de
Vargas, L. M. and Berggren, P. O. (2001) Selective insulin signaling through A and B
insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic
β cells. Mol. Cell 7, 559–570
55 Yoshioka, K., Sugimoto, N., Takuwa, N. and Takuwa, Y. (2007) Essential role for class II
phosphoinositide 3-kinase α-isoform in Ca2 + -induced, Rho- and Rho kinasedependent regulation of myosin phosphatase and contraction in isolated vascular
smooth muscle cells. Mol. Pharmacol. 71, 912–920
56 Seok, Y. M., Azam, M. A., Okamoto, Y., Sato, A., Yoshioka, K., Maeda, M., Kim, I. and
Takuwa, Y. (2010) Enhanced Ca2 + -dependent activation of phosphoinositide 3-kinase
class IIα isoform–Rho axis in blood vessels of spontaneously hypertensive rats.
Hypertension 56, 934–941
57 Elis, W., Triantafellow, E., Wolters, N. M., Sian, K. R., Caponigro, G., Borawski, J.,
Gaither, L. A., Murphy, L. O., Finan, P. M. and Mackeigan, J. P. (2008) Down-regulation
of class II phosphoinositide 3-kinase α expression below a critical threshold induces
apoptotic cell death. Mol. Cancer Res. 6, 614–623
58 Ng, S. K., Neo, S. Y., Yap, Y. W., Karuturi, R. K., Loh, E. S., Liau, K. H. and Ren, E. C.
(2009) Ablation of phosphoinositide-3-kinase class II α suppresses hepatoma cell
proliferation. Biochem. Biophys. Res. Commun. 387, 310–315
59 Kang, S., Song, J., Kang, J., Kang, H., Lee, D., Lee, Y. and Park, D. (2005) Suppression
of the α-isoform of class II phosphoinositide 3-kinase gene expression leads to
apoptotic cell death. Biochem. Biophys. Res. Commun. 329, 6–10
60 Eun, L. Y., Song, B. W., Cha, M. J., Song, H., Kim, I. K., Choi, E., Chang, W., Lim, S.,
Choi, E. J., Ham, O. et al. (2010) Overexpression of phosphoinositide-3-kinase class II
α enhances mesenchymal stem cell survival in infarcted myocardium. Biochem.
Biophys. Res. Commun. 402, 272–279
61 Smith, L. D., Hickman, E. S., Parry, R. V., Westwick, J. and Ward, S. G. (2007) PI3Kγ is
the dominant isoform involved in migratory responses of human T lymphocytes: effects
of ex vivo maintenance and limitations of non-viral delivery of siRNA. Cell. Signalling
19, 2528–2539
62 Srivastava, S., Li, Z., Lin, L., Liu, G., Ko, K., Coetzee, W. A. and Skolnik, E. Y. (2005) The
phosphatidylinositol 3-phosphate phosphatase myotubularin-related protein 6
(MTMR6) is a negative regulator of the Ca2 + -activated K + channel KCa3.1. Mol. Cell.
Biol. 25, 3630–3638
63 Srivastava, S., Choudhury, P., Li, Z., Liu, G., Nadkarni, V., Ko, K., Coetzee, W. A. and
Skolnik, E. Y. (2006) Phosphatidylinositol 3-phosphate indirectly activates KCa3.1 via
14 amino acids in the carboxy terminus of KCa3.1. Mol. Biol. Cell 17, 146–1454
64 Srivastava, S., Ko, K., Choudhury, P., Li, Z., Johnson, A. K., Nadkarni, V., Unutmaz, D.,
Coetzee, W. A. and Skolnik, E. Y. (2006) Phosphatidylinositol-3 phosphatase
myotubularin-related protein 6 negatively regulates CD4 T cells. Mol. Cell. Biol. 26,
5595–5602
65 Russo, A. and O’Bryan, J. P. (2012) Intersectin 1 is required for neuroblastoma
tumorigenesis. Oncogene, doi:10.1038/onc.2011.643
66 Yu, W., Sun, X., Tang, H., Tao, Y. and Dai, Z. (2010) Inhibition of class II
phosphoinositide 3-kinase γ expression by p185Bcr-Abl contributes to impaired
chemotaxis and aberrant homing of leukemic cells. Leuk. Lymphoma 51, 1098–1107
67 Harris, D. P., Vogel, P., Wims, M., Moberg, K., Humphries, J., Jhaver, K. G., Dacosta,
C. M., Shadoan, M. K., Xu, N., Hansen, G. M. et al. (2011) Requirement for the class II
phosphoinositide 3-kinase C2α in maintenance of glomerular structure and function.
Mol. Cell. Biol. 31, 63–80
68 Harada, K., Truong, A. B., Cai, T. and Khavari, P. A. (2005) The class II phosphoinositide
3-kinase C2β is not essential for epidermal differentiation. Mol. Cell. Biol. 25,
11122–11130
69 Cui, Z. G., Hong, N. Y., Kang, H. K., Lee, D. H., Lee, Y. K. and Park, D. B. (2011) The
α-isoform of class II phosphoinositide 3-kinase is necessary for the activation of ERK
but not Akt/PKB. Mol. Cell. Biochem. 346, 95–101
70 Razidlo, G. L., Katafiasz, D. and Taylor, G. S. (2011) Myotubularin regulates
Akt-dependent survival signaling via phosphatidylinositol 3-phosphate. J. Biol. Chem.
286, 20005–20019
71 Dai, S., Zhang, Y., Weimbs, T., Yaffe, M. B. and Zhou, D. (2007) Bacteria-generated
PtdIns(3)P recruits VAMP8 to facilitate phagocytosis. Traffic 8, 1365–1374
72 Brunner, Y., Couté, Y., Iezzi, M., Foti, M., Fukuda, M., Hochstrasser, D. F., Wollheim,
C. B. and Sanchez, J. C. (2007) Proteomics analysis of insulin secretory granules. Mol.
Cell. Proteomics 6, 1007–1017
73 Paul, D. S., Harmon, A. W., Winston, C. P. and Patel, Y. M. (2003) Calpain facilitates
GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes.
Biochem. J. 376, 625–632
c The Authors Journal compilation c 2012 Biochemical Society
74 Logie, L. J., Brown, A. E., Yeaman, S. J. and Walker, M. (2005) Calpain inhibition and
insulin action in cultured human muscle cells. Mol. Genet. Metab. 85, 54–60
75 Brown, A. E., Yeaman, S. J. and Walker, M. (2007) Targeted suppression of calpain-10
expression impairs insulin-stimulated glucose uptake in cultured primary human
skeletal muscle cells. Mol. Genet. Metab. 91, 318–324
76 Marshall, C., Hitman, G. A., Partridge, C. J., Clark, A., Ma, H., Shearer, T. R. and Turner,
M. D. (2005) Evidence that an isoform of calpain-10 is a regulator of exocytosis in
pancreatic β-cells. Mol. Endocrinol. 19, 213–224
77 Shisheva, A. (2008) Phosphoinositides in insulin action on GLUT4 dynamics: not just
PtdIns(3,4,5)P 3 . Am. J. Physiol. Endocrinol. Metab. 295, E536–E544
78 Daimon, M., Sato, H., Oizumi, T., Toriyama, S., Saito, T., Karasawa, S., Jimbu, Y., Wada,
K., Kameda, W., Susa, S. et al. (2008) Association of the PIK3C2G gene polymorphisms
with type 2 DM in a Japanese population. Biochem. Biophys. Res. Commun. 365,
466–471
79 Edling, C. E., Selvaggi, F., Buus, R., Maffucci, T., Di Sebastiano, P., Friess, H., Innocenti,
P., Kocher, H. M. and Falasca, M. (2010) Key role of phosphoinositide 3-kinase class IB
in pancreatic cancer. Clin. Cancer Res. 16, 4928–4937
80 Zhou, J., Wulfkuhle, J., Zhang, H., Gu, P., Yang, Y., Deng, J., Margolick, J. B., Liotta,
L. A., Petricoin, 3rd, E. and Zhang, Y. (2007) Activation of the PTEN/mTOR/STAT3
pathway in breast cancer stem-like cells is required for viability and maintenance. Proc.
Natl. Acad. Sci. U.S.A. 104, 16158–16163
81 Koutros, S., Schumacher, F. R., Hayes, R. B., Ma, J., Huang, W. Y., Albanes, D., Canzian,
F., Chanock, S. J., Crawford, E. D., Diver, W. R. et al. (2010) Pooled analysis of
phosphatidylinositol 3-kinase pathway variants and risk of prostate cancer. Cancer Res.
70, 2389–2396
82 Knobbe, C. B. and Reifenberger, G. (2003) Genetic alterations and aberrant expression of
genes related to the phosphatidyl-inositol-3 -kinase/protein kinase B (Akt) signal
transduction pathway in glioblastomas. Brain Pathol. 13, 507–518
83 Rao, S. K., Edwards, J., Joshi, A. D., Siu, I. M. and Riggins, G. J. (2010) A survey of
glioblastoma genomic amplifications and deletions. J. Neurooncol. 96, 169–179
84 Nobusawa, S., Lachuer, J., Wierinckx, A., Kim, Y. H., Huang, J., Legras, C., Kleihues, P.
and Ohgaki, H. (2010) Intratumoral patterns of genomic imbalance in glioblastomas.
Brain Pathol. 20, 936–944
85 Zhang, L., Huang, J., Yang, N., Greshock, J., Liang, S., Hasegawa, K., Giannakakis, A.,
Poulos, N., O’Brien-Jenkins, A., Katsaros, D. et al. (2007) Integrative genomic analysis
of phosphatidylinositol 3 -kinase family identifies PIK3R3 as a potential therapeutic
target in epithelial ovarian cancer. Clin. Cancer Res. 13, 5314–5321
86 Löw, S., Vougioukas, V. I., Hielscher, T., Schmidt, U., Unterberg, A. and Halatsch, M. E.
(2008) Pathogenetic pathways leading to glioblastoma multiforme: association between
gene expressions and resistance to erlotinib. Anticancer Res. 28, 3729–3732
87 Iorns, E., Lord, C. J. and Ashworth, A. (2009) Parallel RNAi and compound screens
identify the PDK1 pathway as a target for tamoxifen sensitization. Biochem. J. 417,
361–370
88 Liu, Z., Sun, C., Zhang, Y., Ji, Z. and Yang, G. (2011) Phosphatidylinositol
3-kinase-C2β inhibits cisplatin-mediated apoptosis via the Akt pathway in oesophageal
squamous cell carcinoma. J. Int. Med. Res. 39, 1319–1332
89 Diouf, B., Cheng, Q., Krynetskaia, N. F., Yang, W., Cheok, M., Pei, D., Fan, Y., Cheng, C.,
Krynetskiy, E. Y., Geng, H. et al. (2011) Somatic deletions of genes regulating MSH2
protein stability cause DNA mismatch repair deficiency and drug resistance in human
leukemia cells. Nat. Med. 17, 1298–12303
90 McCrea, H. J. and De Camilli, P. (2009) Mutations in phosphoinositide metabolizing
enzymes and human disease. Physiology 24, 8–16
91 Nicot, A. S. and Laporte, J. (2008) Endosomal phosphoinositides and human diseases.
Traffic 9, 1240–1249
92 Velichkova, M., Juan, J., Kadandale, P., Jean, S., Ribeiro, I., Raman, V., Stefan, C. and
Kiger, A. A. (2010) Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with
cortical and endolysosomal functions. J. Cell Biol. 190, 407–425
93 Ribeiro, I., Yuan, L., Tanentzapf, G., Dowling, J. J. and Kiger, A. (2011) Phosphoinositide
regulation of integrin trafficking required for muscle attachment and maintenance. PLoS
Genet. 7, e1001295
94 MacDonald, P. E., Joseph, J. W., Yau, D., Diao, J., Asghar, Z., Dai, F., Oudit, G. Y., Patel,
M. M., Backx, P. H. and Wheeler, M. B. (2004) Impaired glucose-stimulated insulin
secretion, enhanced intraperitoneal insulin tolerance, and increased β-cell mass in mice
lacking the p110γ isoform of phosphoinositide 3-kinase. Endocrinology 145,
4078–4083
95 Pigeau, G. M., Kolic, J., Ball, B. J., Hoppa, M. B., Wang, Y. W., Rückle, T., Woo, M.,
Manning Fox, J. E. and MacDonald, P. E. (2009) Insulin granule recruitment and
exocytosis is dependent on p110γ in insulinoma and human β-cells. Diabetes 58,
2084–2092
96 Kaneko, K., Ueki, K., Takahashi, N., Hashimoto, S., Okamoto, M., Awazawa, M., Okazaki,
Y., Ohsugi, M., Inabe, K., Umehara, T. et al. (2010) Class IA phosphatidylinositol
3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms. Cell
Metab. 12, 619–632
Class II phosphoinositide 3-kinases
97 Lu, N., Shen, Q., Mahoney, T. R., Neukomm, L. J., Wang, Y. and Zhou, Z. (2012) Two PI
3-kinases and one PI 3-phosphatase together establish the cyclic waves of phagosomal
PtdIns(3)P critical for the degradation of apoptotic cells. PLoS Biol. 10, e1001245
98 Cao, C., Laporte, J., Backer, J. M., Wandinger-Ness, A. and Stein, M. P. (2007)
Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on
endosomes. Traffic 8, 1052–1067
601
99 Cao, C., Backer, J. M., Laporte, J., Bedrick, E. J. and Wandinger-Ness, A. (2008)
Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and
growth factor receptor trafficking. Mol. Biol. Cell 19, 3334–3346
100 Xue, Y., Fares, H., Grant, B., Li, Z., Rose, A. M., Clark, S. G. and Skolnik, E. Y. (2003)
Genetic analysis of the myotubularin family of phosphatases in Caenorhabditis elegans .
J. Biol. Chem. 278, 34380–34386
Received 9 January 2012/29 February 2012; accepted 1 March 2012
Published on the Internet 16 April 2012, doi:10.1042/BJ20120008
c The Authors Journal compilation c 2012 Biochemical Society