P.T. Hawkins

Signalling through Class I PI3Ks in mammalian cells

It is now accepted that activation of Class I PI3Ks (phosphoinositide 3-kinases) is one of the most important signal transduction pathways used by cell-surface receptors to control intracellular events. The receptors which access this pathway include those that recognize growth factors, hormones, antigens and inflammatory stimuli, and the cellular events known to be regulated include cell growth, survival, proliferation and movement. We have learnt a great deal about the family of Class I PI3K enzymes themselves and the structural adaptations which allow a variety of cell-surface receptors to regulate their activity. Class I PI3Ks synthesize the phospholipid PtdIns(3,4,5)P3 in the membranes in which they are activated, and it is now accepted that PtdIns(3,4,5)P3 and its dephosphorylation product PtdIns(3,4)P2 are messenger molecules which regulate the localization and function of multiple effectors by binding to their specific PH (pleckstrin homology) domains. The number of direct PtdIns(3,4,5)P3/PtdIns(3,4)P2 effectors which exist, even within a single cell, creates an extremely complex signalling web downstream of PI3K activation. Some key players are beginning to emerge, however, linking PI3K activity to specific cellular responses. These include small GTPases for the Rho and Arf families which regulate the cytoskeletal and membrane rearrangements required for cell movement, and PKB (protein kinase B), which has important regulatory inputs into the regulation of cell-cycle progression and survival. The importance of the PI3K signalling pathway in regulating the balance of decisions in cell growth, proliferation and survival is clear from the prevalence of oncogenes (e.g. PI3Kalpha) and tumour suppressors [e.g. the PtdIns(3,4,5)P3 3-phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)] found in this pathway. The recent availability of transgenic mouse models with engineered defects in Class I PI3K signalling pathways, and the development of PI3K isoform-selective inhibitors by both academic and pharmaceutical research has highlighted the importance of specific isoforms of PI3K in whole-animal physiology and pathology, e.g. PI3Kalpha in growth and metabolic regulation, PI3Kbeta in thrombosis, and PI3Kdelta and PI3Kgamma in inflammation and asthma. Thus the Class I PI3K signalling pathway is emerging as an exciting new area for the development of novel therapeutics.

PtdIns(3)P Regulates the Neutrophil Oxidase Complex by Binding to the PX Domain of p40phox

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b558 (gp91phox and p22phox) and three soluble factors (GTP–Rac, p47phox and p67phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40phox has subsequently been discovered as a binding partner for p67phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40phox to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40phox. These results offer new insights into the roles for PI(3)Ks and p40phox in ROS formation and define a cellular ligand for the orphan PX domain.

PI(3)K|[gamma]| has an important context-dependent role in neutrophil chemokinesis

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P2 and PtdIns(3,4,5)P3 accumulate in a PI(3)Kγ-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kγ activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kγ activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kγ is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.

Phagocytic cells such as neutrophils and macrophages engulf and destroy invading microorganisms. After internalization, material captured within the phagosomal membrane is destroyed by a complex process of coordinated delivery of digestive enzymes and reactive oxygen species. Several endosomal, lysosomal, and oxidase components expected to participate in these events have recently been shown to bind PtdIns3P, suggesting that this lipid may play a role in this process. We used live, digital fluorescence imaging of RAW 264.7 cells stably expressing either a PtdIns3P binding GFP-PX domain or a GFP-FYVE domain to visualize changes in the levels and subcellular localization of PtdIns3P during phagocytic uptake of IgG-opsonized zymosan particles. Very similar results were obtained using both PtdIns3P probes. The basal distribution of each PtdIns3P probe was partially cytosolic and partially localized to EEA-1-positive endosomal structures. Within about 2-3 min of zymosan attachment and concomitant with the closure of the phagosomal membrane, GFP-positive vesicles moved toward and attached to a localized area of the phagosome. A dramatic, transient accumulation of GFP probe around the entire phagosome rapidly ensued, accompanied by a transient drop in cytosolic GFP fluorescence. The magnitude and timing of this rise in PtdIns3P clearly suggest that it is an ideal candidate for controlling the early stages of phagosomal maturation.

Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells

BACKGROUND As phosphoinositides can serve as signalling molecules within cells, the enzymes responsible for their synthesis and cleavage are likely to be involved in the transduction of signals from the cell surface through the cytoplasm. The precise role of the phosphoinositide 3-kinase that has been cloned from mammalian cells is not known, but it has been implicated in receptor-stimulated mitogenesis, glucose uptake and membrane ruffling. The enzyme can use phosphatidylinositol (PtdIns), PtdIns 4-phosphate and PtdIns (4,5)-bisphosphate as substrates in vitro, but it seems to phosphorylate PtdIns (4,5)-bisphosphate preferentially in vivo. The VPS34 gene product of yeast, by contrast, is a phosphoinositide 3-kinase homologue implicated in vacuolar protein sorting that apparently utilizes only PtdIns as a substrate. The significance of this difference in lipid-substrate preference and its relationship to the functions of the two phosphoinositide kinases is unknown. RESULTS We have characterized a distinct PtdIns-specific phosphoinositide 3-kinase activity in mammalian cells. Unlike the previously identified, broad-specificity mammalian phosphoinositide kinase, this enzyme is resistant to the drug wortmannin and uses only PtdIns as a substrate in vitro; it therefore has the capacity to generate PtdIns 3-phosphate specifically. The newly characterized enzyme, which was purified by chromatography from cytosol, has biochemical and pharmacological characteristics distinct from those of the broad-specificity enzyme. CONCLUSIONS The enzyme we have characterized may serve to generate PtdIns 3-phosphate for fundamentally different roles in the cell from those of PtdIns (3,4)-bisphosphate and/or PtdIns (3,4,5)-trisphosphate. Furthermore, the functions of the VSP34 gene product, which may not be relevant to the broad-specificity mammalian phosphoinositide 3-kinase, may be related to those of the enzyme we describe.

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation

BACKGROUND Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

Inactivation of the Class II PI3K-C2β Potentiates Insulin Signaling and Sensitivity

Summary In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2β kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2β inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2β inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2β as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2β as a potential drug target for insulin sensitization.

Signalling via class IA PI3Ks

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that use the g-phosphate of ATP to 3-phosphorylate phosphoinositides on the inositol moiety. Their lipid products, the 3-phosphorylated phosphoinositides, are ubiquitous intracellular messengers that carry signals relevant to a huge range of cell functions in both health and disease. There are three classes of PI3K. The class II PI3Ks are thought to be large, monomeric, multi-domain proteins that primarily use phosphatidylinositol (PtdIns) as a substrate to create PtdIns3P. These are the least well understood of the PI3Ks. The class III PI3Ks also use PtdIns as a substrate and seem to be found in large complexes of proteins comprised of the PI3K catalytic subunit (Vps34) and a number of regulatory subunits, the identity of which depends upon which of the functions that the class III PI3K complex is sub-serving. The class I PI3Ks are the best understood family and use PtdIns(4,5)P2 as a substrate in vivo to yield the intracellular messenger PtdIns(3,4,5)P3. There are four class I PI3Ks in mammalian cells, all are heterodimers comprised of a “p110” catalytic subunits, of which there are four inmammalian cells (a, b, d and g; that give their name to the complex), and one of a range of possible regulatory subunits (p85a, p85b, p55g, p101 and p84 (also called p87(PIKAP))). These have been divided into two sub-groups. Class IA PI3Ks (containing a, b and d subunits) bind SH2 domain-containing regulatory subunits (p85a, p85b and p55g) and are activated by phosphotyrosine-based mechanisms and IB (g) that binds the p101 or p84 regulatory subunits and is primarily regulated by Gbgs. A number of studies have concluded that p110s and p85s can be found outside of the canonical p110/p85 heterodimers in cell extracts, however, the most complete analysis of this question concluded that there was no “free” p85 or p110 in mammalian cell lines (Geering et al., 2007). Many cell types co-express a number of these different class I PI3K components. How these distinct class I PI3K complexes work together to coordinate cell responses remains a huge question in cell biology and its answer is clearly relevant to the development of PI3K inhibitors to treat a wide range of diseases. In this review wewill focus on efforts to understand isoform specific class IA PI3K signalling.

Insulin and ATP stimulate actin polymerization in U937 cells by a wortmannin-sensitive mechanism

ATP and insulin stimulate increases in phosphatidylinositol (3,4,5)‐trisphosphate levels in myeloid‐derived U937 cells. Quantification of FITC‐phalloidin binding by fluorescence‐activated cell sorting reveals that both ATP and insulin stimulate actin polymerization with distinctive kinetics in U937 cells. The response to ATP is rapid and dose‐dependent with an EC50 of 200 nM, and is abolished by pre‐incubation with the Ca2+ chelator BAPTA‐AM. At 800 nM concentration, wortmannin, a potent inhibitor of phosphoinositide 3‐kinase (PI3K), blocks the late, but not the early phase of actin polymerization stimulated by 100 nM ATP. Responses elicited by 10 μg/ml insulin are slower, smaller and more transient than responses to ATP, and are inhibited by preincubation with 100 nM wortmannin. Actin polymerization can also be stimulated by thapsigargin, but not by phorbol ester, providing further evidence for a role for Ca2+ in actin polymerization. These data implicate distinct Ca2+ and PI3K‐mediated pathways in the regulation of actin polymerization.

The roles of PI3Ks in cellular regulation

The term phosphoinositide 30H kinase (PI3K) is given to a family of enzymes which can phosphorylate one or more of the conventional inositol phospholipids found in cells in the 3-position of their inositol headgroup (Fig. 1). It is now clear that these lipids act as regulators of intracellular metabolism and at least one of them, PtdIns(3,4,5)P3, shows all the credentials of being a major ‘second-messenger’ in signalling pathways used by cell-surface receptors for growth factors, inflammatory stimuli and antigens (Stephens et al., 1993; Cantley et al., 1991).