Bart Vanhaesebroeck

Phosphoinositide 3-kinases: A conserved family of signal transducers

Phosphoinositide 3-kinases (PI3Ks) generate lipids that are implicated in receptor-stimulated signalling and in the regulation of membrane traffic. Several distinct classes of PI3Ks have now been identified that have been conserved throughout eukaryotic evolution. Potential signalling pathways downstream of PI3Ks have been elucidated and PI3K function is now being characterised in several model organisms.

The emerging mechanisms of isoform-specific PI3K signalling

Phosphoinositide 3-kinases (PI3Ks) function early in intracellular signal transduction pathways and affect many biological functions. A further level of complexity derives from the existence of eight PI3K isoforms, which are divided into class I, class II and class III PI3Ks. PI3K signalling has been implicated in metabolic control, immunity, angiogenesis and cardiovascular homeostasis, and is one of the most frequently deregulated pathways in cancer. PI3K inhibitors have recently entered clinical trials in oncology. A better understanding of how the different PI3K isoforms are regulated and control signalling could uncover their roles in pathology and reveal in which disease contexts their blockade could be most beneficial.

PI3K in lymphocyte development, differentiation and activation

Phosphoinositide 3-kinases (PI3Ks) regulate numerous biological processes, including cell growth, differentiation, survival, proliferation, migration and metabolism. In the immune system, impaired PI3K signalling leads to immunodeficiency, whereas unrestrained PI3K signalling contributes to autoimmunity and leukaemia. New insights into the role of PI3Ks in lymphocyte biology have been derived from gene-targeting studies, which have identified the PI3K subunits that are involved in B-cell and T-cell signalling. In particular, the catalytic subunit p110δ seems to be adapted to transmit antigen-receptor signalling in B and T cells. Additional recent work has provided new insights into the molecular interactions that lead to PI3K activation and the signalling pathways that are regulated by PI3K.

Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3‐kinase (PI 3‐kinase) in a GTP‐dependent manner. The affinity of the interaction of Ras‐GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3‐kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP‐dependent interaction of PI 3‐kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3‐kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3‐kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3‐kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras‐GTP, but not Ras‐GDP, with PI 3‐kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3‐kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

PI3K signalling: the path to discovery and understanding

Over the past two decades, our understanding of phospoinositide 3-kinases (PI3Ks) has progressed from the identification of an enzymatic activity associated with growth factors, GPCRs and certain oncogene products to a disease target in cancer and inflammation, with PI3K inhibitors currently in clinical trials. Elucidation of PI3K-dependent networks led to the discovery of the phosphoinositide-binding PH, PX and FYVE domains as conduits of intracellular lipid signalling, the determination of the molecular function of the tumour suppressor PTEN and the identification of AKT and mTOR protein kinases as key regulators of cell growth. Here we look back at the main discoveries that shaped the PI3K field.

Essential role for the p110δ phosphoinositide 3-kinase in the allergic response

Inflammatory substances released by mast cells induce and maintain the allergic response. Mast cell differentiation and activation are regulated, respectively, by stem cell factor (SCF; also known as Kit ligand) and by allergen in complex with allergen-specific immunoglobulin E (IgE). Activated SCF receptors and high-affinity receptors for IgE (FcɛRI) engage phosphoinositide 3-kinases (PI(3)Ks) to generate intracellular lipid second messenger signals. Here, we report that genetic or pharmacological inactivation of the p110δ isoform of PI(3)K in mast cells leads to defective SCF-mediated in vitro proliferation, adhesion and migration, and to impaired allergen–IgE-induced degranulation and cytokine release. Inactivation of p110δ protects mice against anaphylactic allergic responses. These results identify p110δ as a new target for therapeutic intervention in allergy and mast-cell-related pathologies.

Critical role for the p110α phosphoinositide-3-OH kinase in growth and metabolic regulation

The eight catalytic subunits of the mammalian phosphoinositide-3-OH kinase (PI(3)K) family form the backbone of an evolutionarily conserved signalling pathway; however, the roles of most PI(3)K isoforms in organismal physiology and disease are unknown. To delineate the role of p110α, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110α kinase activity. Homozygosity for this kinase-dead p110α led to embryonic lethality. Mice heterozygous for this mutation were viable and fertile, but displayed severely blunted signalling via insulin-receptor substrate (IRS) proteins, key mediators of insulin, insulin-like growth factor-1 and leptin action. Defective responsiveness to these hormones led to reduced somatic growth, hyperinsulinaemia, glucose intolerance, hyperphagia and increased adiposity in mice heterozygous for the D933A mutation. This signalling function of p110α derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110β, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110α was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110α in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110α in a variety of cancers.

Angiogenesis selectively requires the p110α isoform of PI3K to control endothelial cell migration

Phosphoinositide 3-kinases (PI3Ks) signal downstream of multiple cell-surface receptor types. Class IA PI3K isoforms couple to tyrosine kinases and consist of a p110 catalytic subunit (p110α, p110β or p110δ), constitutively bound to one of five distinct p85 regulatory subunits. PI3Ks have been implicated in angiogenesis, but little is known about potential selectivity among the PI3K isoforms and their mechanism of action in endothelial cells during angiogenesis in vivo. Here we show that only p110α activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110α led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110α exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110α activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110β in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1α, whereas p110δ is expressed at low level and contributes only minimally to PI3K activity in endothelial cells. These results provide the first in vivo evidence for p110-isoform selectivity in endothelial PI3K signalling during angiogenesis.

A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system.

Phosphoinositide (PI) 3‐kinases have been characterized as enzymes involved in receptor signal transduction in mammalian cells and in a complex which mediates protein trafficking in yeast. PI 3‐kinases linked to receptors with intrinsic or associated tyrosine kinase activity are heterodimeric proteins, consisting of p85 adaptor and p110 catalytic subunits, which can generate the 3‐phosphorylated forms of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 as potential second messengers. Yeast Vps34p kinase, however, has a substrate specificity restricted to PtdIns and is a PtdIns 3‐kinase. Here the molecular characterization of a new human PtdIns 3‐kinase with extensive sequence homology to Vps34p is described. PtdIns 3‐kinase does not associate with p85 and phosphorylates PtdIns, but not PtdIns4P or PtdIns(4,5)P2. In vivo PtdIns 3‐kinase is in a complex with a cellular protein of 150 kDa, as detected by immunoprecipitation from human cells. Protein sequence analysis and cDNA cloning show that this 150 kDa protein is highly homologous to Vps15p, a 160 kDa protein serine/threonine kinase associated with yeast Vps34p. These results suggest that the major components of the yeast Vps intracellular trafficking complex are conserved in humans.

The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110α, p110β, and p110δ) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110α and p110δ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110γ class IB PI3K lack SH2 domains and instead couple p110γ to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110β and cells derived from a p110β-deficient mouse line, that p110β is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110β and p110γ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110β but not p110γ, p110β mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110γ in these cells reduced the contribution of p110β to GPCR signaling. Taken together, these data show that p110β and p110γ can couple redundantly to the same GPCR agonists. p110β, which shows a much broader tissue distribution than the leukocyte-restricted p110γ, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110γ expression is low or absent.