Len R. Stephens

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.

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866, published online 17 October; see Perspective by Conley and Fruman) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders. Gene sequencing of unrelated patients with recurrent airway infections identifies a common underlying mutation. [Also see Perspective by Conley and Fruman] Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P3). PtdIns(3,4,5)P3 regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P3 have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography–mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P3 and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP2 are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P3 in unstimulated mouse and human cells (≥105) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

Moving towards a Better Understanding of Chemotaxis

Eukaryotic cells are thought to move across supporting surfaces through a combination of coordinated processes: polarisation; extension of dynamic protrusions from a leading edge; adhesion-associated stabilisation of some protrusions; centripetal pulling against those leading adhesions; and de-adhesion at the rear. Gradients of extracellular ligands can be detected by cells and then used to guide them either towards the source (in the case of a chemoattractant) or away from the source (in the case of a chemorepellent)--such migration is termed chemotaxis. Recent work suggests that chemotaxis probably emerges from the ability of cells to spatially encode extracellular gradients of ligands, a process for which phosphoinositide 3-kinase (PI3K) signals alone are insufficient, and to use that vectorial information to bias movement by enhancing the survival, and not the formation, of the protrusions that experience the greatest stimulation.

SCFAs Induce Mouse Neutrophil Chemotaxis through the GPR43 Receptor

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43−/− BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

PI3K signalling in inflammation.

PI3Ks regulate several key events in the inflammatory response to damage and infection. There are four Class I PI3K isoforms (PI3Kα,β,γ,δ), three Class II PI3K isoforms (PI3KC2α, C2β, C2γ) and a single Class III PI3K. The four Class I isoforms synthesise the phospholipid PIP3. PIP3 is a second messenger used by many different cell surface receptors to control cell movement, growth, survival and differentiation. These four isoforms have overlapping functions but each is adapted to receive efficient stimulation by particular receptor sub-types. PI3Kγ is highly expressed in leukocytes and plays a particularly important role in chemokine-mediated recruitment and activation of innate immune cells at sites of inflammation. PI3Kδ is also highly expressed in leukocytes and plays a key role in antigen receptor and cytokine-mediated B and T cell development, differentiation and function. Class III PI3K synthesises the phospholipid PI3P, which regulates endosome-lysosome trafficking and the induction of autophagy, pathways involved in pathogen killing, antigen processing and immune cell survival. Much less is known about the function of Class II PI3Ks, but emerging evidence indicates they can synthesise PI3P and PI34P2 and are involved in the regulation of endocytosis. The creation of genetically-modified mice with altered PI3K signalling, together with the development of isoform-selective, small-molecule PI3K inhibitors, has allowed the evaluation of the individual roles of Class I PI3K isoforms in several mouse models of chronic inflammation. Selective inhibition of PI3Kδ, γ or β has each been shown to reduce the severity of inflammation in one or more models of autoimmune disease, respiratory disease or allergic inflammation, with dual γ/δ or β/δ inhibition generally proving more effective. The inhibition of Class I PI3Ks may therefore offer a therapeutic opportunity to treat non-resolving inflammatory pathologies in humans. This article is part of a Special Issue entitled Phosphoinositides.

Structure of Lipid Kinase p110β/p85β Elucidates an Unusual SH2-Domain-Mediated Inhibitory Mechanism

Summary Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTKs pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

PI3Kβ Plays a Critical Role in Neutrophil Activation by Immune Complexes

The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases. The Integrating Isoform The class I phosphoinositide 3-kinases (PI3Ks) are implicated in processes such as growth factor signaling and inflammation. PI3Kγ is activated by G protein–coupled receptors (GPCRs), whereas PI3Kα and PI3Kδ are activated by protein tyrosine kinase–coupled receptors. PI3Kβ is unusual in that it appears to respond to signals from both types of receptors, depending on the cellular context. Kulkarni et al. investigated the responses of mouse neutrophils to immune complexes of antibody and antigen, which trigger chronic inflammation in conditions such as autoimmune arthritis. Genetic and pharmacological evidence suggested that immune complexes stimulated PI3Kβ in a process involving activation of FcγR, a tyrosine kinase–coupled low-affinity antibody receptor, and autocrine signaling by a proinflammatory lipid (LTB4) through its GPCR. Mice deficient in PI3Kβ fared better than did controls in models of arthritis and inflammatory skin disease. These data implicate PI3Kβ in the integration of signals from tyrosine kinase–coupled receptors and GPCRs—and suggest that this isoform may be an effective therapeutic target in inflammatory diseases. Neutrophils are activated by immunoglobulin G (IgG)–containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength–dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase–coupled receptor (FcγR) and a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns(3)P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3)P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns(3)P-regulation of the oxidase must exist.

The cytotoxic T cell proteome and its shaping by the kinase mTOR

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.