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

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 2-position, but they are differentially phosphorylated at the 3-, 4- and 5-hydroxy groups within the myo-inositol ring (Figure 1A).

Structure of PtdIns and lipid products of PI3Ks

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)P2, derived from phosphorylation of PtdIns4P; and PtdIns(3,4,5)P3, derived from phosphorylation of PtdIns(4,5)P2.

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)P2, derived from phosphorylation of PtdIns4P; and PtdIns(3,4,5)P3, derived from phosphorylation of PtdIns(4,5)P2.

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 [13]. Deregulation of PI3K-dependent cellular pathways is associated with several diseases, including cancer and diabetes [1,2,46].

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 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-protein-coupled 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.

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 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.

Structure of class II PI3K isoforms

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.

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.

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 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

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 Mg-ATP 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 PI3K-C2α 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)P2in 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

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 (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 insulin-induced 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 down-regulation 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 PI3K-C2α 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 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 N-terminal 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 clathrin-binding site within PI3K-C2α in regulation of the activity of the enzyme. Interestingly, although lacking the specific clathrin-binding 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 PI3K-C2β 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 N-terminal 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 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 insulin-dependent 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 PI3K-C2α 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 insulin-stimulated 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 co-immunoprecipitation 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].

Mechanisms of activation and action of PI3K-C2α

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)P2 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).

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)P2 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).

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 co-localizes 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 PI3K-C2β 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].

Mechanisms of activation and action of PI3K-C2β

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.

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.

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 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.

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 GAPEX-5 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.

Other protein associations

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 C-terminal 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 PI3K-C2β 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 post-translational 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.

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, PI3K-C2β 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].

Calcium

Several lines of evidence indicate that the activity of PI3K-C2α 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 PI3K-C2α 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 PI3K-C2α 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 PI3K-C2α 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 PI3K-C2α, 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 co-expression 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 and GFP–PI3K-C2α was also detected, no ubiquitination of PI3K-C2α 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.

Table 1
Intracellular functions regulated by class II PI3Ks assessed by in vitro studies
Enzyme Cellular function Reference(s) 
PI3K-C2α Glucose transport [20
 Neurosecretory granule release [21,22
 Insulin secretion [24,45
 Endocytosis [18,37
 VSMC contraction [43,55,56
 Cell growth and survival [46,57,58,59,60
PI3K-C2β Cell migration [25,27,38
 K+ channel activation [28,47
 Cell growth and survival [38,65
 Cell-cycle progression [26
PI3K-C2γ Homing of leukaemic cells [66
Enzyme Cellular function Reference(s) 
PI3K-C2α Glucose transport [20
 Neurosecretory granule release [21,22
 Insulin secretion [24,45
 Endocytosis [18,37
 VSMC contraction [43,55,56
 Cell growth and survival [46,57,58,59,60
PI3K-C2β Cell migration [25,27,38
 K+ channel activation [28,47
 Cell growth and survival [38,65
 Cell-cycle progression [26
PI3K-C2γ Homing of leukaemic cells [66

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 [19,36,4953]. 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 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). Down-regulation 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-C2α-mediated 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 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 down-regulation 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 actin-rich 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 wild-type 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 kinase-dead 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 T-lymphocytes, 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 K+ channel KCa3.1 [28]. Specifically, down-regulation of PI3K-C2β 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 [6264]. As discussed above, a role for TRIM27 in regulation of the PI3K-C2β-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 wild-type 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 anchorage-independent 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 PI3K-C2β is the only PI3K involved in this process.

Cell-cycle progression

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.

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 down-regulation 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 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 PI3K-C2β 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.

DOWNSTREAM EFFECTORS

A downstream effector of class II PI3Ks regulating cell survival: the usual Akt?

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 (extracellular-signal-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 insulin-induced Akt1 activation was reported in these cells upon down-regulation 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 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 down-regulation may result in accumulation of this phosphoinositide. Co-silencing of PI3K-C2β but not PI3K-C2α restores the EGF-dependent 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.

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 (vesicle-associated 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 PI3K-C2α may have a role in regulation of calpain 10, a protease involved in GLUT4 translocation [7375] 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 dominant-negative 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 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 PtdIns3P-binding 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

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.

Table 2
Potential connections of class II PI3Ks and human diseases
Class II isoform Human disease Evidence Reference(s) 
PI3K-C2α Diabetes In vitro studies [20,24,45
  mRNA down-regulation in islets from Type 2 diabetic patients [45
 Cancer Decrease in DNA copy number/increase in mRNA levels in hepatocellular carcinoma [58
  In vitro studies [58,46,79
 Cardiovascular diseases In vitro studies [43,44,55
  In vivo study [56
PI3K-C2β Cancer Single-nucleotide polymorphism associated with prostate cancer risk [81
  Amplification of PIK3C2B found in glioblastoma [8284
  DNA copy number gain in ovarian cancer [85
  Overexpression in ESCC tissues [88
  High levels in neuroblastoma cell lines and primary tumours [65
  In vitro studies [86,87
PI3K-C2γ Diabetes Polymorphisms associated with Type 2 diabetes [78
Class II isoform Human disease Evidence Reference(s) 
PI3K-C2α Diabetes In vitro studies [20,24,45
  mRNA down-regulation in islets from Type 2 diabetic patients [45
 Cancer Decrease in DNA copy number/increase in mRNA levels in hepatocellular carcinoma [58
  In vitro studies [58,46,79
 Cardiovascular diseases In vitro studies [43,44,55
  In vivo study [56
PI3K-C2β Cancer Single-nucleotide polymorphism associated with prostate cancer risk [81
  Amplification of PIK3C2B found in glioblastoma [8284
  DNA copy number gain in ovarian cancer [85
  Overexpression in ESCC tissues [88
  High levels in neuroblastoma cell lines and primary tumours [65
  In vitro studies [86,87
PI3K-C2γ Diabetes Polymorphisms associated with Type 2 diabetes [78

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].

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 PI3K-C2α 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 miR-30e-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.

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 whole-genome 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 down-regulation 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 PI3K-C2β may actually promote resistance of leukaemia cells to specific chemotherapeutics [89]. The authors showed that down-regulation 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 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 PI3K-C2β 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].

Cardiovascular diseases

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 PI3K-C2α may have a role in hypertension.

Neuromuscular diseases

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 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?

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 co-operative 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.

Are class II and class III PI3Ks redundant?

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 PI3K-dependent 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 (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 co-operate to control these processes cannot be completely ruled out. Similarly, evidence suggesting the possibility that PI3K-C2α 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 down-regulation 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 down-regulation 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 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.

Abbreviations

     
  • 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

FUNDING

We thank the Pancreatic Cancer Research Fund, the British Heart Foundation and Diabetes UK [grant number BDA:09/0003971] for their support.

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