Class II phosphoinositide 3-kinase C2alpha: what we learned so far

More than fifteen years after the first identification of a class II isoform of phosphoinositide 3-kinase (PI3K) in Drosophila melanoǵaster this subfamily remains the most enigmatic among all PI3Ks. What are the functions of these enzymes? What are their mechanisms of activation? Which downstream effectors are specifically regulated by these isoforms? Are class I and class II PI3Ks redundant or do they control different intracellular processes? And, more important, do class II PI3Ks have a role in human diseases? The recent increased interest on class II PI3Ks has started providing some answers to these questions but still a lot needs to be done to completely uncover the contribution of these enzymes to physiological processes and possibly to pathological conditions. Here we will summarise the recent findings on the alpha isoform of mammalian class II PI3Ks (PI3K-C2α ) and we will discuss the potential involvement of this enzyme in human diseases.     

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic β cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in β cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in β cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.     

Phosphatidylinositol-3 kinase acts in parallel to the ERK MAP kinase in the FGF pathway during Xenopus mesoderm induction

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that can phosphorylate phosphaditylinositides leading to the cell type-specific regulation of intracellular protein kinases. PI3Ks are involved in a wide variety of cellular events including mitogenic signalling, regulation of growth and survival, vesicular trafficking, and control of the cytoskeleton. Some of these enzymes also act downstream of receptor tyrosine kinases or G-protein-coupled receptors. Using two strategies to inhibit PI3K signalling in embryos, we have analysed the role of PI3Ks during early Xenopus development. We find that a class 1A PI3K catalytic activity is required for the definition of trunk mesoderm during the blastula stages, but is less important for endoderm and prechordal plate mesoderm induction or for organiser formation. It is required in the FGF signalling pathway downstream of Ras and in parallel to the extracellular signal-regulated kinase (ERK) MAP kinases. In addition, our results show that ERKs and PI3Ks can synergise to convert ectoderm into mesoderm. These data provide the first evidence that class 1 PI3Ks are required for a specific set of patterning events in vertebrate embryos. Furthermore, they bring new insight into the FGF signalling cascade in Xenopus.     

The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism

Phosphatidylinositol 3-kinases (PI3Ks) evolved from a single enzyme that regulates vesicle trafficking in unicellular eukaryotes into a family of enzymes that regulate cellular metabolism and growth in multicellular organisms. In this review, we examine how the PI3K pathway has evolved to control these fundamental processes, and how this pathway is in turn regulated by intricate feedback and crosstalk mechanisms. In light of the recent advances in our understanding of the function of PI3Ks in the pathogenesis of diabetes and cancer, we discuss the exciting therapeutic opportunities for targeting this pathway to treat these diseases.     

Targeted expression of the class II phosphoinositide 3-kinase in Drosophila melanogaster reveals lipid kinase-dependent effects on patterning and interactions with receptor signaling pathways

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.     

Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ^~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.     

Activation loop sequences confer substrate specificity to phosphoinositide 3-kinase alpha (PI3Kalpha ). Functions of lipid kinase-deficient PI3Kalpha in signaling

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein kinases. While the lipid-dependent PI3K downstream signaling is well characterized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes. Here we show that sequences C-terminal to the PI3K ATP-binding site determine the lipid substrate specificity of the class IA PI3Kalpha (p85/p110alpha). Transfer of such activation loop sequences from class II PI3Ks, class III PI3Ks, and a related mammalian target of rapamycin (FRAP) into p110alpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity unaffected. All resulting hybrids lacked the ability to produce phosphatidylinositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Arg) residues in the activation loop are crucial for the functionality of class I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient transfecion of 293 cells, we show that p110alpha hybrids, although unable to support lipid-dependent PI3K signaling, such as activation of protein kinase B/Akt and p70(S6k), retain the capability to associate with and phosphorylate insulin receptor substrate-1, with the same specificity and higher efficacy than wild type PI3Kalpha. Our data lay the basis for the understanding of the class I PI3K substrate selectivity and for the use of PI3Kalpha hybrids to dissect PI3Kalpha function as lipid and protein kinase.     

Phosphoinositide 3-Kinase C2beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.     

The class II phosphoinositide 3-kinase C2beta is not essential for epidermal differentiation

Phosphoinositide 3-kinases (PI3Ks) regulate an array of cellular processes and are comprised of three classes. Class I PI3Ks include the well-studied agonist-sensitive p110 isoforms; however, the functions of class II and III PI3Ks are less well characterized. Of the three class II PI3Ks, C2alpha and C2beta are widely expressed in many tissues, including the epidermis, while C2gamma is confined to the liver. In contrast to the class I PI3K p110alpha, which is expressed throughout the epidermis, C2beta was found to be localized in suprabasal cells, suggesting a potential role for C2beta in epidermal differentiation. Overexpressing C2beta in epidermal cells in vitro induced differentiation markers. To study a role for C2beta in tissue, we generated transgenic mice overexpressing C2beta in both suprabasal and basal epidermal layers. These mice lacked epidermal abnormalities. Mice deficient in C2beta were then generated by targeted gene deletion. C2beta knockout mice were viable and fertile and displayed normal epidermal growth, differentiation, barrier function, and wound healing. To exclude compensation by C2alpha, RNA interference was then used to knock down both C2alpha and C2beta in epidermal cells simultaneously. Induction of differentiation markers was unaffected in the absence of C2alpha and C2beta. These findings indicate that class II PI3Ks are not essential for epidermal differentiation.     

High glucose promotes cell proliferation and enhances GDNF and RET expression in pancreatic cancer cells

Phosphoinositide 3-kinases (PI3Ks) are key enzymes that activate intracellular signaling molecules when a number of different growth factors bind to cell surface receptors. PI3Ks are divided into three classes (I, II, III), and enzymes of each class have different tissue specificities and physiological functions. The α-isoform (PI3K-C2α) of class II PI3Ks is considered ubiquitous and preferentially activated by insulin. Our previous study showed that suppression of PI3K-C2α leads to apoptotic cell death. The aim of this study is to determine whether depletion of PI3K-C2α affects ERK or PKB/Akt activity following stimulation with serum and insulin growth factors in Chinese hamster ovary cells expressing human insulin receptors (CHO-IR) and human HepG2 liver cells. Different antisense oligonucleotides (ODNs), which were designed based on the sequence of the C2 domain of the human PI3K-C2α gene, were transfected into cells to inhibit PI3K-C2α expression. Insulin- or serum-induced stimulation of ERK was significantly suppressed by depletion of PI3K-C2α, whereas phosphorylation of IRS-1 and the stimulation of PKB/Akt by insulin were not affected. The number of apoptotic cells was also increased by depletion of PI3K-C2α protein levels. Taken together, our data indicate that PI3K-C2α may be a crucial factor in the stimulation of ERK activity in response to serum or insulin, whereas it is less important for the stimulation of PKB/Akt activity in response to insulin.     

Gene-targeting reveals physiological roles and complex regulation of the phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are represented by a family of eight distinct enzymes that can be divided into three classes based on their structure and function. The class I PI3Ks are heterodimeric enzymes that are regulated by recruitment to plasma membrane following receptor activation and which control numerous cellular functions, including growth, differentiation, migration, survival, and metabolism. New light has been shed on the biological role of individual members of the class I PI3Ks and their regulatory subunits through gene-targeting experiments. In addition, these experiments have brought the complexity of how PI3K activation is regulated into focus.     

Two distinct phosphoinositide 3-kinases mediate polypeptide growth factor-stimulated PKB activation

Eight human isoforms of phosphoinositide 3-kinases (PI3Ks) exist, but their individual functions remain poorly understood. Here, we show that different human small cell lung carcinoma (SCLC) cell lines overexpress distinct subsets of class I(A) and II PI3Ks, which results in striking differences in the signalling cascades activated by stem cell factor (SCF). Over expression of class I(A) p85/p110alpha in SCLC cells increased SCF-stimulated protein kinase B (PKB) activation and cell growth, but did not affect extracellular signal-regulated kinase (Erk) or glycogen synthase kinase-3 (GSK-3). This effect was selective, since it was not observed in SCLC cell lines overexpressing p85/p110beta or p85/p110delta. The SCF receptor associated with both class I(A) p85 and class II PI3KC2beta, and both enzymes contributed to SCF-stimulated PKB activity. A dominant-negative PI3KC2beta blocked both PKB activation and SCLC cell growth in response to SCF. Together our data provide novel insights into the specificity and functional significance of PI3K signalling in human cancer.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.     

Signaling by distinct classes of phosphoinositide 3-kinases

Many signaling pathways converge on and regulate phosphoinositide 3-kinase (PI3K) enzymes whose inositol lipid products are key mediators of intracellular signaling. Different PI3K isoforms generate specific lipids that bind to FYVE and pleckstrin homology (PH) domains in a variety of proteins, affecting their localization, conformation, and activities. Here we review the activation mechanisms of the different types of PI3Ks and their downstream actions, with focus on the PI3Ks that are acutely triggered by extracellular stimulation.     

IA型磷脂酰肌醇3-激酶与肿瘤的关系及其抑制剂分子机制研究进展

磷脂酰肌醇3-激酶(phosphatidylinositol-3 kinase,PI3K)是细胞内重要的信号分子,它具有调节细胞增殖、分化、代谢、凋亡等功能。PI3K的基因易发生突变和扩增,从而导致PI3K被激活,与肿瘤的形成和发展密切相关。IA型的PI3K及其下游的信号分子组成的通路参与调节肿瘤细胞的增殖、存活、黏附、迁移等活动。综述了IA型PI3K——PI3Kα、PI3Kβ和PI3Kδ与肿瘤发生、发展的关系,列举了20个具有代表性的IA型PI3K抑制剂,并讨论了它们的分子抑制机制。     

Hydrogen peroxide contributes to the manganese superoxide dismutase promotion of migration and invasion in glioma cells

Manganese superoxide dismutase (MnSOD) is over-expressed in most brain tumours, and high MnSOD expression is associated with poor prognosis. The mechanisms still remain largely unknown. In the present study, the elevation of hydrogen peroxide (H(2)O(2)) level and the enhancement of glioma migration/invasion by over-expression of MnSOD were demonstrated. Subsequent studies showed that over-expression of MnSOD significantly increased the activation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinases (PI3Ks), including AKTs, s6-ribosomal protein, ERKs and JNKs. Over-expression of MnSOD was also associated with elevations of matrix metalloproteinases-1(MMP-1) and MMP-9 protein. The promotion of migration/invasion, activation of PI3Ks and MAPKs and up-regulation of MMPs were inhibited by the general reactive oxygen species scavenger N-acetyl-l-cysteine (NAC), over-expression of the H(2)O(2)-detoxifying enzyme mitochondrial catalase (mCat) and specific inhibitors of AKTs or ERKs. Collectively, our study indicated that H(2)O(2) would contribute to the MnSOD-promoted migration/invasion in glioma cells through activation of AKTs and ERKs. This study provided new molecular insights into the understanding of glioma migration and invasion.     

Molecular mechanisms of PI4K regulation and their involvement in viral replication

Lipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4-kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4-phosphate (PI4P)-enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein-binding partners and post-translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection.     

Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products

Morphologic polarity is necessary for the motility of mammalian cells. In leukocytes responding to a chemoattractant, this polarity is regulated by activities of small Rho guanosine triphosphatases (Rho GTPases) and the phosphoinositide 3-kinases (PI3Ks). Moreover, in neutrophils, lipid products of PI3Ks appear to regulate activation of Rho GTPases, are required for cell motility and accumulate asymmetrically to the plasma membrane at the leading edge of polarized cells. By spatially regulating Rho GTPases and organizing the leading edge of the cell, PI3Ks and their lipid products could play pivotal roles not only in establishing leukocyte polarity but also as compass molecules that tell the cell where to crawl.