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Dipartimento di Genetica, Biologia e Biochimica, Molecular Biotechnology Center, University of Torino, Via Nizza 52, Torino 10126, Italy

(Requests for offprints should be addressed to E Hirsch; Email: emilio.hirsch{at}unito.it)

	Abstract

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

 
In multicellular organisms, concerted actions of different tissues are regulated inside single cells by signal transduction mechanisms that, subsequently to hormones sensing, trigger intracellular responses. In recent years, increasing evidence indicates phosphoinositide 3-kinases (PI3K) as crucial signal transducing elements that regulate communication across the plasma membrane. PI3K generate lipid secondary messengers that trigger a plethora of intracellular responses ranging from metabolic regulation to cell proliferation, survival, and migration. The growing number of hormones that relay signals by activating PI3K suggests not only multiple roles of these enzymes in the regulation of different physiological responses but also a way by which common reactions can be stimulated by different inputs. This review will thus focus on the different pathways that converge on PI3K activation, with particular attention to the paradigmatic PI3K involvement in insulin signaling.

	Signal transduction: a matter of amplifiers

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

 
Homeostasis of multicellular organisms requires precise communication between different cells. The paradigm of hormonal function defines a cellular source of signals and a definite target able to interpret and respond to the stimulation. Currently, multiple signaling mechanisms are known that, at the target site, detect extracellular cues, provide intracellular amplification, and trigger specific responses. Receptor stimulation triggers the production of second messenger molecules that intensify signal detection. Among these molecules, cyclic AMP, Ca²⁺, and lipid derivatives are able to bind effector proteins and elicit cellular reactions. While cAMP and Ca²⁺ so far appear to bind a limited number of effector proteins (e.g. only protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are direct targets of cAMP), phosphorylated lipids of the phosphatidyl inositol (PtdIns) family are interacting with hundreds of different proteins through common specialized binding domains. This highlights the versatility of phosphoinositide phosphate signaling cascade and supports the view that PtdIns signaling is a crucial ancestral biological mechanism.

	PtdInsPs and PI3K

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

 
Phosphoinositides

Whereas inositol and its phosphorylated derivatives are found in bacteria, it is only in eukaryotes that PtdIns and its phosphorylated products (phosphoinositides) appear as lipid components of cell membranes involved in signaling (Michell 2007). Phosphoinositides are present in yeast and they are likely evolved as a means to mark membranes and regulate organelle identity as well as vesicular trafficking (Di Paolo & De Camilli 2006). In contrast to other types of signaling molecules, PtdIns phosphates label the membrane–cytosol interface without diffusion from one isolated membrane to another, thus stably marking, for example, vesicles and organelles. Enzymes that produce or degrade phosphoinositides probably evolved as a spatially restricted machinery that specifically scores membranes, promoting segregation of different compartments and providing a code to identify the fate of vesicles in their intracellular traffic. Similarly, protein binding to phosphoinositides could have appeared to ensure detection of such code as well as achievement of the targeted membrane fusion necessary for vectorial vesicular traffic. A further wave of diversification, dated at least one billion years ago, probably led to the appearance of the signaling properties of phosphoinositides and their derivatives. It is likely that the first signaling mechanisms involving PtdIns to appear in eukaryotes was the metabolism of PtdIns(4,5)P₂ (also known as PIP₂). PIP₂ is diffusely present in the plasma membrane in resting conditions but is hydrolyzed to diacylglycerol (DAG) and inositol(1,4,5)trisphosphate (also known as IP3) upon receptor activation. IP3 and DAG are then able to trigger signal cascades involving Ca²⁺ release from intracellular stores and protein kinase C (PKC) activation respectively (Berridge 2005).

Though the effects of constitutive PtdIns production (Di Paolo & De Camilli 2006) and PIP₂ metabolism (Berridge 2005) are of well-established importance, this review will focus on the mechanisms of production and on the biological effects of PtdIns phosphorylated at the D3 hydroxyl of the inositol ring, where the D prefix determines the direction of the phosphate away from the viewer (i.e. when the rings drawn in Fig. 1 are viewed from above; Hinchliffe & Irvine 1997). Cells contain at least four species of PtdIns phosphorylated in the D3 position (Fig. 1): PtdIns3P, PtdIns(3,4)P₂, PtdIns(3,5)P₂, and PtdIns(3,4,5)P₃ (also known as phosphatidyl inositol phosphate₃ (PIP₃)). While PtdIns3P can be found in lower eukaryotes like yeasts, PtdIns(3,4)P₂ and PIP₃ appear in the phylogenetic tree with ameboid organisms and are detectable in nearly all higher eukaryotic cells, with the exclusion of plants that so far have never been described to produce PIP₃ (Vanhaesebroeck et al. 2001). In a resting cell, only PtdIns3P can be detected and, though the other D3-phosphorylated molecular forms are virtually absent, they appear as pulses with different kinetics after receptor stimulation.

View larger version (21K): [in this window] [in a new window]   Figure 1 Structure of selected phosphoinositides. A schematic of PtdIns and some of its phosphorylated derivatives. Black arrows indicate that a kinase is involved; conversely, a white arrow indicates that phosphatase is necessary for the reaction. CI, II, III PI3K, class I, II, and III PI3K. Refer to text for explanation of acronyms.

Turnover of phosphoinositides is assured by the concerted action of kinases and phosphatases, and phosphoinositide 3-kinases (PI3K) are responsible for the phosphorylation of the D3 position of PtdIns. The number of PI3K present in different organisms varies throughout the phylogenetic tree: whereas yeast possesses only one PI3K gene, mammalian cells carry at least eight different genes with significant degrees of homology (Engelman et al. 2006). Some of these mammalian proteins are not only lipid kinases but also protein kinases, suggesting a possible origin from a class of distantly related protein kinases including mTOR (mammalian target of rapamycin) and DNA-PK. In the recent past, our notions on how PI3K works has tremendously increased and presently these kinases can be divided into three classes depending on their substrate specificity and ways of activation (Hawkins et al. 2006).

It is now clear, for example, that class I PI3K phosphorylate in vivo PIP₂ to produce PIP₃ and that class III enzymes produce PtdIns3P from PtdIns (Engelman et al. 2006). Nonetheless, the activity of class II PI3K is still debated and probably they are involved in the production of both PIP₃ and PtdIns3P (Engelman et al. 2006). So far, though mounting evidences indicate that classes II and III PI3K might be involved in vesicular trafficking (Maffucci et al. 2003, Lindmo & Stenmark 2006), class I PI3K represent the best characterized type. Class I PI3K are usually heterodimers consisting of a catalytic subunit and an adaptor/regulator subunit. All class I catalytic subunits share substantial homology, show a molecular mass of ~110 kDa, and are collectively referred to as p110 subunits. Mammals possess four class I PI3K p110 genes named Pik3ca, Pik3cb, Pik3cg, and Pik3cd that are, however, usually termed PI3K, ß, , and . While Pik3ca and Pik3cb are ubiquitously expressed, Pik3cg and Pik3cd are preferentially found in leukocytes, with the exception of Pik3cg being recently detected in the cardiovascular system (Hirsch et al. 2006). Proteins encoded by these genes share four regions of highest homology that comprises, from the N-terminal end, the domain for binding to the small GTPase Ras, the lipid-binding C2 domain, the PIK domain (a helical motif common to all PI3K), and the catalytic domain. Of these four enzymes, only the structure of PI3K has been resolved (Walker et al. 1999). The catalytic pocket of PI3K shows a two-lobe organization, where the first is involved in ATP binding (through a fundamental lysine residue found in all PI3K) and the second is more involved in substrate specificity (Walker et al. 1999). This catalytic cleft is able to specifically accommodate small molecule inhibitors that like wortmannin and LY294002 effectively block enzymatic activity of all PI3K (Walker et al. 2000). Nonetheless, the presence in the pocket of residues specific to the PI3K isoform allows the generation of selective inhibitors able to act more specifically on PI3K (Camps et al. 2005). While binding of PI3K to Ras has been described at atomic resolution (Pacold et al. 2000), an in-depth description of the structural details leading to the interaction of p110s with their adaptor/regulator subunit is still missing.

Two class I PI3K types

The presence of different families of adaptor/regulator subunits defines two subgroups in the family of class I PI3K. PI3K, ß, and enzymes bind to adaptor/regulators of the so-called p85 family and embody the IA subgroup (Fig. 2). Of these p85 adaptor/regulators, three genes named Pik3r1, Pik3r2, and Pik3r3 are found in mammals. Pik3r1 can be expressed in three splice variants that encode p85, p55, and p50 respectively; on the other hand, Pik3r2 and Pik3r3 are known to present only one transcript and encode p85ß and p55 respectively. While p85 and p85ß are ubiquitously expressed (Gout et al. 1992), p50 and p55 are present in fat, muscle, liver, and brain (Antonetti et al. 1996, Inukai et al. 1996), and p55 is mainly expressed in the brain (Pons et al. 1995). All members of the p85 family contain a p110-binding region tethering them to a specific domain located at the N-terminal end of class IA p110s. In p85 proteins, the p110-binding region is sandwiched between two SH2 domains and these two regions bind to phosphorylated tyrosine residues in the YXXM motif of tyrosine-phosphorylated receptors or receptor-associated adaptor signaling molecules. This property defines a way of activation peculiar to class IA PI3K that generally act downstream tyrosine kinase receptors. In the currently accepted model of class IA activation, p110 subunits are constitutively bound to p85 and kept inactive in the cytosol. Upon receptor activation, p85 is recruited to tyrosine-phosphorylated motifs and this event not only liberates the p85-mediated constraint on PI3K catalytic activity (Fu et al. 2003) but also localizes the enzyme next to the plasma membrane in the proximity of its lipid substrate (Yu et al. 1998). In addition to controlling activity and localization of PI3K, p85 family members possess specific activities: for example, p85 can control cell cycle in a p110-independent way (Garcia et al. 2006). In addition, p85 and ß contain an SH3 domain that ensures interaction with intracellular protoncogenes and oncogenes like Cbl or breakpoint cluster region (BCR)/Abl (Ren et al. 2005).

View larger version (32K): [in this window] [in a new window]   Figure 2 Class I PI3K family. Four different class I PI3K are expressed in mammalian cells; depending on their mechanisms of activation, they can be divided into two subtypes: class IA PI3K (PI3K, ß, ) are activated mainly by membrane-bound tyrosine kinase receptor and class IB PI3K is exclusively activated by GPCR generally of the Gi subtype. Class IA PI3K bind to the adaptor subunit p85, which, in turn, is recruited by its phosphotyrosine binding domain to phosphorylated receptors or adaptor proteins like IRS (insulin receptor substrate). Class IB PI3K binds to adaptor/regulators like p101 or p84/87 and to free Gß liberated by active GPCRs. All class I PI3K phosphorylate PIP2 to produce PIP3, a secondary messenger membrane lipid that functions as a docking site to a large number of proteins containing the pleckstrin homology (PH) domain. These effectors in turn control the different cellular responses summarized in Fig. 3.

ß

ß

ß

PIP₃ phosphatases

The product of class I PI3K activity is the substrate for a series of phosphatases that specifically remove phosphate groups from the inositol ring of PIP₃. For example, hydrolysis of the phosphate in the D3 position is achieved by phosphatase and tensin homolog deleted on chromosome ten (PTEN) and is thought to terminate PI3K-mediated signaling (Cully et al. 2006). On the other hand, SH2-containing inositol 5'-phosphatase (SHIP) proteins are able to remove the phosphate in the D5 position, thus indicating that one mechanism of PtdIns(3,4)P₂ appearance is due to PIP₃ degradation (Kalesnikoff et al. 2003).

PI3K downstream effectors

Class I PI3K products exert their large number of biological functions because of their ability to function as docking sites for proteins that contain specific lipid-binding domains such as the pleckstrin homology (PH) domain. Interestingly, PH domain is more abundant in the human proteome, reaching the 11th position of the most common sequences (Lemmon 2007). PH domains are found with different sequence organization and specificities: some, like, for example, that found in Akt, specifically bind PIP₃ but others, like the motif found in phospho lipase C (PLC) 1 (Lemmon et al. 1995), selectively interact with PIP₂. Although not all PH domains containing proteins bind to phosphoinositides with sufficient strength for this to be of functional importance, the high number of proteins with such a domain highlights the complexity of signaling events downstream PI3K. A number of other protein domains are also able to bind to phosphoinositides 3-phosphate. For example, of note is the binding of the phox homology domain (PX; Xu et al. 2001) and domain present in Fab1, YOTB, Vac1 and EEA1 (FYVE) domains to PtdIns3P (Kutateladze 2006). For the sake of clarity, only the best characterized elements binding PIP₃ will be further considered (Fig. 3).

View larger version (45K): [in this window] [in a new window]   Figure 3 Signal transduction pathways downstream PI3K. The product of PI3K activity, PIP3, serves as a docking site for a large number of PH domain (in brown) containing proteins (listed in circle around PIP3). These effector proteins represent the front line of PIP3-dependent signaling. Further signal amplification is achieved by their activation and inhibition of multiple more specialized downstream targets, eventually producing specific biological responses. See text for explanation of the acronyms.

Although PKB/Akt is at the center stage of PI3K signaling, other well-characterized proteins can function as downstream targets of PIP₃ production. It is worth mentioning, for example, that PI3K can trigger the activity of PH domain containing tyrosine kinases Tec and Btk (Lindvall & Islam 2002) or the phospholipase PLC (Maffucci & Falasca 2007). In addition, a PH domain is found in most activators of small GTPases of the Rho and Arf families (Hawkins et al. 2006), thus explaining the role of PI3K in cytoskeletal remodeling, and membrane trafficking respectively. On the other hand, emerging evidence indicates that GAP inhibitors of Rho GTPases can also be regulated by PIP₃ binding and PI3K-mediated signaling (Di Paolo & De Camilli 2006). PI3K activity thus crucially controls the delicate balance between activating and inhibitory stimuli through, for example, the establishment of positive and negative feedback loops that help to transform shallow gradients of extracellular stimuli into robust cellular responses.

	PI3K as a common platform for hormonal signal transduction

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

 
PI3K influence a variety of cellular functions, depending on the nature of the target cell and on the multiple signals impinging on a cell at a given time. It has recently become clear that PI3K are implicated in the signal transduction pathways of a wide variety of hormones. Because of the rapid enrichment of the field, this review shall focus on a few paradigmatic cases covering two distinct classes of hormones: peptides and steroids. Most peptide hormones have been reported to trigger PI3K activity, but only for a few like angiotensin II (Ang II), leptin, and insulin, specific knowledge of PI3K-dependent biochemical mechanisms, PI3K isoforms involved, or PI3K-mediated specific biological responses is sufficiently characterized. Similarly, amine and lipid hormones can trigger PI3K activity through their receptors but often with poorly characterized mechanisms. Unexpectedly, however, the PI3K-dependent signaling branch of steroid hormones like estrogen has been extensively analyzed and found to provide new concepts for their function.

PI3K and extranuclear estrogen receptor (ER) signaling

Estrogen actions are mediated by a mixture of both directly controlled gene expression (the so-called ‘genomic or classical’ action) and regulation of cell signaling/phosphorylation cascades, referred to as ‘non-genomic or extranuclear’ action. It is becoming increasingly evident that ER extranuclear signaling involves PI3K and might play central roles in controlling cell proliferation and survival (Fig. 4). Estrogen non-genomic effects on PI3K pathway might indeed be caused by direct interaction of the ER with PI3K (Migliaccio et al. 1996, Castoria et al. 2001), in a process that involves the p85 regulatory subunit binding to ER and a subsequent ER-dependent PKB/Akt activation (Simoncini et al. 2000, Castoria et al. 2001, Sun et al. 2001). An alternative ER-mediated mechanism for PI3K triggering can be found in a restricted number of breast cancer lines and depends on ER-induced ErbB2 activation that subsequently triggers PI3K recruitment/activation (Stoica et al. 2003). Through PI3K, ER also exerts its anti-apoptotic effects: in MCF7 breast cancer cells treated with 17ß-estradiol (E2), modulator of non-genomic action of ER (MNAR) functions as a scaffold for ER, cSrc, and p85 interactions, promoting PI3K activation and cell survival (Greger et al. 2007). Although ER controls PI3K activity, the opposite process has been described as well: in a number of epithelial cells, regulation of ER-dependent transcriptional activity can be a target of PI3K signaling, even in the absence of estrogen, through a mechanism that involves PKBß/Akt2-mediated phosphorylation of ER (Sun et al. 2001).

View larger version (37K): [in this window] [in a new window]   Figure 4 Extranuclear signaling of the estrogen receptor. Upon binding to its ligands, like 17ß-estradiol (E2), the estrogen receptor (ER) not only activates the classic signaling pathway resulting in modulating gene transcription in the nucleus but also triggers PI3K activation in the cytoplasm, by directly binding to the p85 subunit. Listed are some of the biological effects of ER-dependent PI3K activation.

This same signaling cascade is also found to play roles in ER-mediated control of cardiovascular functions (Fig. 4). Estrogen, acting through ERs, plays a critical role in the protection of the cardiovascular system by pleiotropic effects, including vasodilatation, preservation of vascular integrity, and enhancement of cardiomyocyte survival by prevention of apoptosis. Indeed, estrogen-induced direct binding of ER to p85 increases PI3K activity and results in the activation of PKB/Akt, which in turn triggers eNOS-mediated NO production (Simoncini et al. 2000). The activation of NOS indeed reduces inflammatory reactions and protects from the effects of ischemia/reperfusion (Simoncini et al. 2000). Similarly, in an animal model of myocardial infarction, involving permanent coronary occlusion, E2-mediated activation of PI3K and PKB/Akt protects the heart by reducing both infarct size and myocyte apoptosis (Patten et al. 2004). Overall, the activation of the ER/PI3K/Akt/eNOS pathway thus explains, in cultured human endothelial cells as well as in vivo, in intact elastic and muscular arteries, the molecular mechanism underlying the long-known protective effects of estrogen on female cardiovascular system (Haynes et al. 2000, Guo et al. 2005).

On the other hand, this signaling pathway might modulate cardiovascular responses to hormones, like Ang II, that might play a role in heart failure and stroke. ER efficiently reduces Ang II- or endothelin 1 (ET1)-induced hypertrophy of individual cardiomyocytes, resulting in a smaller cell surface area and decreased cytoskeletal protein expression. This effect is mediated by the PI3K-dependent activation of MCIP1, a negative regulator of the NFAT transcription factor involved in cardiac hypertrophy (Pedram et al. 2005).

PI3K in Ang II signaling

PI3K might also play a role in the signaling pathways triggered by Ang II itself, the peptide hormone backbone of the rennin–angiotensin system that controls blood pressure homeostasis as well as myocardial cell growth. Ang II and its GPCRs (ATreceptors) play crucial roles by mediating smooth muscle growth, ROS production, and changes in vascular contractility. Indeed, the PI3K branch of Ang II-mediated signaling in vascular smooth muscle cells (VSMCs) is considered of increasing importance. For example, inhibition of PI3K activity with either LY294002 or wortmannin decreases AT1-receptor-stimulated activation of PKB/Akt (Takahashi et al. 1999), mTOR, and p70-S6 kinase as well as VSMC proliferation and gene expression (Saward & Zahradka 1997). Although Ang II regulates cell growth through the independent activation of PI3K and Erk1/2 pathways, it appears that both events are required for Ang II-induced proliferation of rat aortic smooth muscle cells (Dugourd et al. 2003).

In addition, Ang II controls vascular modulation of voltage-gated L-type Ca²⁺ channels by PI3K that thus appear as crucial regulators of Ca²⁺ entry and vascular excitation–contraction coupling. Ang II activates Ca²⁺ entry by stimulating L-type Ca²⁺ channels through G_ß-sensitive PI3K in portal vein myocytes (Le Blanc et al. 2004). Although both PI3K and are expressed in vascular myocytes, Ang II-induced stimulation of vascular L-type Ca²⁺ channel and increase in Ca²⁺ depends only on the PI3K isoform (Macrez et al. 2001, Quignard et al. 2001). Similarly to VSMC, in cardiomyocytes, Ang II-evoked activation of PI3K, but not of PI3K, causes pathological cell growth (Naga Prasad et al. 2000). More recently, Vecchione et al. have demonstrated that PI3K plays a role in Ang II signaling in vivo (Fig. 5). Indeed, mice lacking PI3K are protected from hypertension induced by administration of Ang II. PI3K controls smooth muscle contraction via two distinct pathways: on the one hand, it triggers PKB/Akt activation and the subsequent entry of extracellular Ca²⁺ through L-type Ca²⁺ channels and, on the other hand, PIP₃ produced by PI3K is necessary to trigger the small GTPase Rac possibly through the recruitment of PH domain containing GTP exchange factors (GEF). The activation of Rac, in turn, represents a critical step in the assembly at the plasma membrane and the subsequent activation of the NADPH oxidase complex triggering ROS production. These data indicate that PI3K is a key signaling molecule in the Ang II pathway and suggest that blocking PI3K function might be exploited to improve therapeutic intervention of hypertension (Vecchione et al. 2005).

View larger version (18K): [in this window] [in a new window]   Figure 5 The role of PI3K in Ang II-dependent signaling. Binding of Ang II to its receptors (AT) activates a series of PI3K-dependent effects triggering vascular smooth muscle cell growth and proliferation through activation of PKB/Akt, mTOR, and S6K. At the same time, PKB/Akt phosphorylates the ß-chain of L-type Ca2+ channels, enhancing their plasma membrane exposure and open probability. Furthermore, PI3K-mediated activation of the small GTPase Rac triggers reactive oxygen species (ROS) production, causing vascular contraction and remodeling.

Leptin is another peptide hormone that modulates vascular tone as well as cell metabolism by stimulating PI3K. It binds the long form of leptin receptor (LRb) and triggers the tyrosine kinase JAK, eventually controlling STAT transcription factors and gene expression (Munzberg & Myers 2005). The way by which leptin also stimulates PI3K is not yet precisely defined, although different studies suggest JAK2-dependent and -independent mechanisms that trigger p85 binding to tyrosine-phosphorylated insulin receptor substrate (IRS, see below for description) adaptor proteins (Kellerer et al. 1997, Munzberg & Myers 2005, Mansour et al. 2006; Fig. 6). Although many effects of leptin are mediated through the central nervous system (for PI3K-mediated effects of leptin on the brain refer to review; Plum et al. 2005), this peptide can regulate metabolism through a direct action on tissues, such as pancreas and liver. In agreement with this finding, previous studies have shown that leptin activates PI3K in ß-cells and hepatocytes (Wang et al. 1997, Harvey et al. 2000, Szanto & Kahn 2000). Moreover, in primary rat hepatocytes, leptin induces PI3K-dependent activation of phosphodiesterase 3B, a cAMP-degrading enzyme, to suppress glucagon-induced cAMP elevation (Zhao et al. 2000). Leptin function in other tissues appears to similarly rely on PI3K signaling: for example, in VSMCs, leptin stimulates NOS activity and NO release, inhibiting the contractile response induced by Ang II, through mechanisms involving PKB/Akt and JAK2/STAT3 pathways (Rodriguez et al. 2007). On the other hand, it has recently become clear that leptin causes systemic effects on the regulation of immune function and some of these effects are PI3K mediated. Indeed, leptin causes a delayed apoptosis of mature neutrophils in vitro, through a signaling cascade involving PI3K (Bruno et al. 2005). Altogether, these indications suggest that leptin can induce PI3K signaling to influence different responses in distinct cell types.

View larger version (30K): [in this window] [in a new window]   Figure 6 Class IA PI3K function in leptin (right) and insulin (left) induced signaling. Refer to text for details. Binding of free p85 subunits to insulin receptors/IRS complexes might be the cause of the paradoxical effects detected in mice that lack p85 genes. Excess of p85 could thus negatively regulate p110 function, which in the case of p85 reduction can eventually increase. Though a wealth of evidence points to a major role of PI3K in insulin signaling, it is still unclear whether PI3Kß has indeed no importance in the process.

	PI3K signaling in insulin-mediated metabolic control

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

IRS activity downstream the IR is tightly regulated by positive and negative regulation. IRS is activated by tyrosine phosphorylation and negatively regulated by protein tyrosine phosphatases (Hayashi et al. 2004, Gonzalez-Rodriguez et al. 2007). Other inhibitory mechanisms include blockade of their binding to the IR (Ueki et al. 2002a), or serine phosphorylation mediated by a PI3K-mediated negative feedback that involves IRS phosphorylation by S6K (Um et al. 2004).

Upon phosphorylation by active IR, IRS serves as a docking site for SH2 domains of class IA p85 regulatory/adaptor subunits that consequently recruit p110 enzymes next to the activated receptor. PIP₃ production in turn activates downstream effectors that control various metabolic processes such as glucose uptake, lipolysis inhibition, triglyceride formation, and glycogen synthesis. In these processes, the initiating event is the activation of PDK1 that, after PIP₃ binding, activates atypical PKC (aPKC), PKB/Akt, and S6K (Vanhaesebroeck & Alessi 2000). Among these downstream effectors, PKB/Akt stimulates blood glucose disposal, promotes synthesis of glycogen, and inhibits gluconeogenesis. Three different isoforms of PKB/Akt, encoded by different genes, are found in mammals (PKB– or, alternatively, Akt1–3). All isoforms share a common structure including an N-terminal PH domain and a C-terminal catalytic domain. Recent data, obtained using knockout model mice, associate each PKB/Akt isoform with a specific function and indicate that not all are involved in insulin-mediated metabolic control. While PKB/Akt3 appears to be principally implicated in neuronal development (Tschopp et al. 2005), PKB/Akt1 is involved in growth and longevity (Chen et al. 2001, Cho et al. 2001a), but only PKBß/Akt2 is the main regulator in vivo of glucose homeostasis. Indeed, the absence of PKBß/Akt2 causes type 2 diabetes in both mice (Cho et al. 2001b) and humans (George et al. 2004).

Insulin, PI3K, and glucose metabolism

In adipocytes and muscle cells, PI3K-dependent activation of PKBß/Akt2 (and to a lesser extent of PKB/Akt1) controls insulin-mediated glucose uptake by stimulating GLUT4 translocation from an intracellular compartment to the plasma membrane (Katome et al. 2003). GLUT4 trafficking is controlled by PKB/Akt-mediated phosphorylation of the RabGAP (Rab GAP) AS160 (Akt substrate of 160 kDa) that when phosphorylated is inhibited by binding to 14-3-3 protein (Ramm et al. 2006). Other PI3K-dependent mediators of glucose uptake are atypical forms of PKC, including PKC and PKC isoforms. Overexpression of PKC and PKC produces an increase in GLUT4 translocation, which is, conversely, inhibited by a dominant-negative mutant of PKC (Bandyopadhyay et al. 1997).

Together with enzymes of class IA, PI3K of other classes might be involved in insulin-mediated glucose disposal. Recent experimental evidences suggest that insulin stimulation triggers the production of PtdIns3P, thus possibly involving either class II or III PI3K. For example, over-expression of myotubularins, the PtdIns3P phosphatases, reduces glucose uptake (Chaussade et al. 2003). PtdIns3P binds the PX and FYVE domains of proteins often involved in the control of vesicular trafficking. It is thus possible that PI3K other than class IA are involved in processes needed for plasma membrane fusion of cytoplasmic vesicles containing GLUT4 (Shepherd 2005). Interestingly, insulin-mediated PtdIns3P production appears insensitive to the action of wortmannin, thus excluding a role for class III enzymes but indicating a function of the wortmannin-insensitive class II PI3K-C2 isoenzymes (Maffucci et al. 2003). In line with this view, PI3K-C2 is activated in response to insulin-dependent (but PI3K-independent) activation of the small GTPase TC10, thus triggering PtdIns3P elevation necessary for full-scale GLUT4 plasma membrane translocation (Maffucci et al. 2003).

Another target of the class IA PI3K pathway is insulin-mediated inhibition of hepatic gluconeogenesis (Agati et al. 1998, Kotani et al. 1999). De novo synthesis of glucose is indispensable in a starved condition and, on the other hand, is dispensable when external resources are available. In this latter condition, insulin negatively regulates gluconeogenesis by suppressing the expression of key gluconeogenic enzymes through the PKB/Akt-mediated phosphorylation of the transcription factor FoxO1. After phosphorylation, FoxO1 is not able to translocate into the nucleus and consequently fails to activate transcription of various genes necessary for gluconeogenesis, such as phosphoenolpyruvate caboxykinase and glucose-6-phosphatase (Nakae et al. 2001).

Role of the regulatory subunit of PI3K

The crucial role of PI3K in insulin signaling suggests that its dysfunction may have deleterious effects on glucose homeostasis. Established lines of evidence indicate that PI3K signaling could be compromised by reducing expression of class IA PI3K enzymes and adaptors in type 2 diabetic ob/ob mice (Folli et al. 1993, Kerouz et al. 1997) as well as humans (Goodyear et al. 1995, Bjornholm et al. 1997, Rondinone et al. 1997).

Gene deletion studies of p85 regulatory subunits have challenged this view and reported unexpected paradoxical effects of loss of p85 proteins. Mice lacking p85, one of the three splicing variants encoded by the Pik3r1 gene, are viable but are hypoglycemic and show increased insulin sensitivity. However, this probably correlates with the up-regulation of the other Pik3r1 splicing variants, p50 and p55, in fat and muscle. This overexpression produces an elevation of PIP₃ levels in adipocytes, which facilitates the GLUT4 translocation to the plasma membrane (Terauchi et al. 1999). On the contrary, deletion of the Pik3r1 gene, determining the ablation of all its products (p85, p55, and p50), results in perinatal lethality, extensive hepatocyte necrosis, enlarged skeletal muscle fibers, brown fat necrosis, and calcification of cardiac tissue. Loss of Pik3r1 causes a substantial decrease in expression and activity of class IA PI3K catalytic subunits. Nonetheless, Pik3r-deficient mice are hypoglycemic and more insulin sensitive than controls because of a more active glucose transport in insulin-responsive tissues like fat and muscle (Fruman et al. 2000). Furthermore, hepatic deletion of Pik3r1 produces similar paradoxical effects on PI3K activation/function. If, on the one hand, hepatic loss of Pik3r1 improves insulin sensitivity in liver, muscle, and fat, on the other hand, it leads to severe reduction in PI3K activation, with a 60% decrease in total hepatic PI3K activity (Taniguchi et al. 2006). Nonetheless, the activation of PKB/Akt downstream PI3K is enhanced as a consequence of reduced activity of the phosphatase PTEN, thus suggesting a role of p85 in PTEN regulation (Taniguchi et al. 2006). Indeed, PTEN is a potent negative regulator of the insulin signaling and loss of PTEN in adipose tissue results in an increased insulin sensitivity and GLUT4 recruitment (Kurlawalla-Martinez et al. 2005).

The paradoxical enhancement of insulin signaling is also detected in mice lacking either p50/p55 or p85ß. Although p50 and p55 are not required for survival, their loss produces a reduction in the insulin levels in the fasting state and an increase in glucose transport in isolated extensor digitorum longus (EDL) muscle and adipocytes (Chen et al. 2004). Similarly, mice lacking p85ß are viable and show hypoinsulinemia, hypoglycemia, and improved insulin sensitivity (Ueki et al. 2002b, 2003).

Taken together, these findings assign a positive and negative role to p85 regulatory subunits in the insulin signaling. In fact, p85 proteins are required for p110 stabilization but conversely free p85s exert negative effects on PI3K signaling. Therefore, a critical molecular balance between regulatory and catalytic subunits determines the optimal response of the PI3K pathway to insulin signaling (Ueki et al. 2002a). In physiological conditions, p85 is more abundant than the catalytic p110 subunit, producing a competition between p85 monomers and p85–p110 dimers. Monomers of p85 can thus inhibit PIP₃ production either by binding to phosphorylated IRS proteins (Ueki et al. 2002a) or by altering subcellular localization of p110/p85 dimers (Inoue et al. 1998). Unbalancing p85 levels might thus compromise this regulatory mechanisms and lead to a paradoxical increase in PI3K activity. Finding of increased p85/55/50 protein expression, in mouse models of obesity as well as in type 2 diabetic patients, further confirms this view (Bandyopadhyay et al. 2005). Altogether, these findings suggest that p85 family members have a complex role in regulating PI3K-mediated insulin signaling that probably involves p110-independent activities yet to be fully understood.

Role of the catalytic subunit of PI3K

The use of no isoform-selective PI3K inhibitors has for long hindered the definition of the nature of the p110 catalytic subunits involved in insulin signaling. Nonetheless, recent reports using mutant mice as well as novel selective inhibitors suggest that different p110 subunits might play distinct roles. Although the lethal phenotype of mice lacking either p110 (p110^–/–) or p110ß (p110ß^–/–) hampered the study of their relative role in the insulin signaling, analysis of insulin-mediated responses in heterozygous animals suggests that both proteins might be involved. Heterozygous mice lacking either PI3K or ß show normal responses to insulin; however, p110^+/–/p110ß^+/– compound heterozygotes display decreased insulin sensitivity (Brachmann et al. 2005). Nonetheless, heterozygous mice expressing a catalytically inactive PI3K become insulin resistant with age. While young animals are normal, mice older than 6 months display glucose intolerance, hyperlipidemia, adiposity, as well as hyperglycemia and deregulate hepatic gluconeogenesis, thus assigning a fundamental role of PI3K in insulin-dependent signaling (Foukas et al. 2006). Biochemical analysis further shows that only PI3K is selectively recruited and activated by IRS proteins (Foukas et al. 2006). In agreement with this finding, the use of PI3K inhibitors with significant isoform selectivity shows that PI3K is the major PI3K effector downstream the IR and that, in contrast, PI3Kß maintains a marginal role (Knight et al. 2006). Nonetheless, inhibitor studies indicate that PI3Kß influences PI3K function providing a basal threshold of PIP₃ production that potentiates PI3K activity, thus suggesting that PI3Kß function might be still necessary for achieving full scale signaling responses (Knight et al. 2006). Further studies with mice possibly expressing a catalytically inactive PI3Kß might help to address this issue.

	How can different hormones activate PI3K but exert different biological responses?

Top Abstract Signal transduction: a matter... PtdInsPs and PI3K PI3K as a common... PI3K signaling in insulin... How can different hormones... References

 
Exploration of signaling mechanisms of most hormonal receptor agonists has led to the identification of PI3K activation with such a high frequency that it becomes legitimate to ask oneself if, in higher eukaryotes, receptor agonist that works independently of this pathway indeed exist. Furthermore, the fact that a multitude of extracellular stimuli relay PI3K activation poses important interrogatives concerning the mechanisms by which selective extracellular cues trigger specific responses using the same signaling components. For example, although most hormones signal through PI3K to generally promote cell survival and proliferation, only insulin requires PI3K for the induction of glucose uptake. How this specificity is achieved is not yet understood. Nonetheless, a few speculations can be made: for example, different PI3K isoforms can be involved in determining when and where PIP₃ is produced inside a cell. Despite the wealth of information about the activation mechanisms of distinct PI3K isoforms, little is known about where PIP₃ is indeed located. Indications from studies in leukocytes suggest that subcellular localization of PIP₃ accumulation is fundamental for processes like directional cell migration (Hirsch et al. 2006). Consistent with this view, it is becoming increasingly suspected that compartmentalized pools of PIP₃ exist and that spatially restricted accumulation of second messenger molecules represents the key for solving the paradox of a single pathway eliciting selective distinct cellular responses. The finding that PI3K possess scaffold activity (Patrucco et al. 2004) suggests that signaling complexes involving these enzymes might be more complicated than initially thought and that such multi-molecular interactions might provide the mechanistic basis for localized and selective signaling. The rising expectation that our knowledge on PI3K signal transduction will soon provide new therapeutic tool for a vast number of pathologies (Camps et al. 2005) requires that further studies help to tackle these issues before encountering drawbacks and unwanted side effects.

	Acknowledgements

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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