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.

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 (p110alpha, p110beta or p110delta), 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 p110alpha activity is essential for vascular development. Ubiquitous or endothelial cell-specific inactivation of p110alpha led to embryonic lethality at mid-gestation because of severe defects in angiogenic sprouting and vascular remodelling. p110alpha exerts this critical endothelial cell-autonomous function by regulating endothelial cell migration through the small GTPase RhoA. p110alpha activity is particularly high in endothelial cells and preferentially induced by tyrosine kinase ligands (such as vascular endothelial growth factor (VEGF)-A). In contrast, p110beta in endothelial cells signals downstream of G-protein-coupled receptor (GPCR) ligands such as SDF-1alpha, whereas p110delta 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.

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110␣, p110␤, and p110␦) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110␣ and p110␦ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110␥ class IB PI3K lack SH2 domains and instead couple p110␥ to G protein-coupled receptors (GPCRs). Here, we show, using smallmolecule inhibitors with selectivity for p110␤ and cells derived from a p110␤-deficient mouse line, that p110␤ is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110␤ and p110␥ 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 p110␤ but not p110␥, p110␤ 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 p110␥ in these cells reduced the contribution of p110␤ to GPCR signaling. Taken together, these data show that p110␤ and p110␥ can couple redundantly to the same GPCR agonists. p110␤, which shows a much broader tissue distribution than the leukocyterestricted p110␥, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110␥ expression is low or absent.gene targeting ͉ signaling ͉ tyrosine kinase ͉ Akt ͉ insulin T he lipid second messengers generated by phosphoinositide 3-kinases (PI3Ks) regulate a wide variety of cellular functions such as cell growth, proliferation, differentiation, and survival and have been implicated in cancer, inflammation, and diabetes. Mammals have eight isoforms of PI3K, which have been divided in three classes (1). Thus far, attention has focused mainly on the class I PI3Ks that are acutely activated by extracellular ligands. These heterodimers consist of a p110 catalytic subunit in complex with a regulatory subunit and have further been subdivided into class IA and IB PI3Ks. The class IA catalytic subunits (p110␣, p110␤, and p110␦) are in complex with an Src homology 2 (SH2) domain-containing regulatory subunit (of which there are five species, often referred to as p85s) that binds phosphoTyr in intracellular proteins, allowing recruitment of p85/p110 complexes to the membrane. The class IB regulatory subunits (p84, p101) do not have SH2 domains and link the single class IB PI3K catalytic subunit (p110␥) to G protein-coupled receptors (GPCRs).Over the last few years, the cellular signaling contexts and physiological roles of p110␣, p110␦, and p110␥ have become clearer, because of the generation of gene-targeted mice and small-molecule inhibitors for these PI3K isoforms. In contrast, very little is known about p110␤, both at the cellular and organismal level. One confounding factor has been the very early embryonic lethality of the p110␤ KO ...

SummaryPI3K signaling is thought to mediate leptin and insulin action in hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, through largely unknown mechanisms. We inactivated either p110α or p110β PI3K catalytic subunits in these neurons and demonstrate a dominant role for the latter in energy homeostasis regulation. In POMC neurons, p110β inactivation prevented insulin- and leptin-stimulated electrophysiological responses. POMCp110β null mice exhibited central leptin resistance, increased adiposity, and diet-induced obesity. In contrast, the response to leptin was not blocked in p110α-deficient POMC neurons. Accordingly, POMCp110α null mice displayed minimal energy homeostasis abnormalities. Similarly, in AgRP neurons, p110β had a more important role than p110α. AgRPp110α null mice displayed normal energy homeostasis regulation, whereas AgRPp110β null mice were lean, with increased leptin sensitivity and resistance to diet-induced obesity. These results demonstrate distinct metabolic roles for the p110α and p110β isoforms of PI3K in hypothalamic energy regulation.

During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110␤ null mouse model and demonstrated a critical role of PI3K␤ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glycoprotein VI-Fc receptor ␥-chain complex, and its contribution in response to Gprotein-coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110␤ null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein-coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3K␤ is also acting downstream of ␣ IIb ␤ 3 . In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl 3 injury of the carotid, suggesting that PI3K␤ is a potential target for antithrombotic drugs. IntroductionPlatelet activation is a highly regulated process involving various signaling pathways initiated by specific receptors coupled to heterotrimeric G proteins (GPCRs), integrins, or immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins. In all cases, key signaling enzymes, such as phospholipases C (PLC) and phosphoinositide 3-kinase (PI3K) isoforms, are activated. If the situation is clear for PLC (ie, the PLC␤ isoforms are activated by heterotrimeric Gq proteins, whereas the ␥ isoforms are stimulated via tyrosine phosphorylation and ITAM signaling), the implication of the different PI3K isoforms downstream of the major platelet receptors is still poorly known. Class Ia PI3Ks (␣,␤,␦), composed of a catalytic subunit (p110) and a regulatory subunit, are classically activated by their association with phosphotyrosine residues containing sequences via the SH2 domains of their regulatory subunit. 1 However, the p110␤ isoform may not follow this rule because its activation has been proposed to involve both G␤␥ and phosphotyrosyl peptides. 2-5 Using a selective inhibitor of p110␤, Jackson et al 6 have proposed a role of p110␤ in the regulation of ␣ IIb ␤ 3 integrin in a shear-dependent manner. Interestingly, this inhibitor prevented the formation of an occlusive thrombus generated in vivo. To firmly establish the role of the PI3K␤ in platelet activation and evaluate its impact on hemostasis in vivo, we created a mouse line in which this isoform has been inactivated by gene targeting selectively in the megakaryocyte lineage. Methods MaterialsCollagen was from Nycomed, U46619 from QBiogen Inc; integrilin from Glaxo Group Ltd; p110␣, ␤, ␥, and ␦ antibodies from Santa Cruz Biotechnology; p85 antibody from Upstate Biotechnology; TGX-221 from Cayman Chemical; and other reagents from Sigma-Aldrich. An...