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 p110alpha, a ubiquitously expressed PI(3)K involved in tyrosine kinase and Ras signalling, here we generated mice carrying a knockin mutation (D933A) that abrogates p110alpha kinase activity. Homozygosity for this kinase-dead p110alpha 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 p110alpha derives from its highly selective recruitment and activation to IRS signalling complexes compared to p110beta, the other broadly expressed PI(3)K isoform, which did not contribute to IRS-associated PI(3)K activity. p110alpha was the principal IRS-associated PI(3)K in cancer cell lines. These findings demonstrate a critical role for p110alpha in growth factor and metabolic signalling and also suggest an explanation for selective mutation or overexpression of p110alpha in a variety of cancers.

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.

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We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC 50 ‫؍‬ 75 M for p110␦, 400 M for p110␣ and p110␤, and 1 mM for p110␥), and theophylline has similar effects (IC 50 ‫؍‬ 75 M for p110␦, 300 M for p110␣, and 800 M for p110␤ and p110␥) and also inhibits the ␣ isoform of class II PI3K (PI3K-C2␣) (IC 50 Ϸ 400 M). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)-triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110␦ (IC 50 < 10 M). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC 50 Ϸ 10 mM for p110␣ , 3 mM for p110␤, and 10 mM for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC 50 values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mM. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110␦ lipid kinase. Finally, we conclude that inhibition of PI3K (p110␦ in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.Caffeine and theophylline are naturally occurring methylxanthine compounds that can be found in micromolar concentrations in human circulation as a result of dietary intake or pharmacological use. These compounds have been the subject of intense study to determine how they act at physiological concentrations, and a number of effects have been ascribed to these compounds at such concentrations including stimulation of muscle contraction levels (1), anti-inflammatory and immunomodulatory effects (2), alterations in glucose metabolism (3-8), attenuation of the antilipolytic effect of insulin (3), and induction of apoptosis (9, 10). Several mechanisms of action have been identified for these methylxanthines, and these can explain some of the pleiotropic effects these compounds have on cells at their physiologically achievable concentrations. These include their abilities to directly inhibit phosphodiesterases and thus increase cellular cAMP levels, to directly antagonize adenosine receptors, and to cause increases in cytosolic C...

Small molecule inhibitors of PI3K for oncology mainly target the class I PI3Ks, comprising the p110α, β, γ, and δ isoforms, of which only p110α is mutated in cancer. To assess the roles of class I PI3K isoforms in cell proliferation and survival, we generated immortalized mouse leukocyte and fibroblast models in which class I PI3Ks were inactivated by genetic and pharmacological approaches. In IL3-dependent hemopoietic progenitor cells (which express all four class I PI3K isoforms), genetic inactivation of either p110α or p110δ did not affect cell proliferation or survival or sensitize to p110β or p110γ inactivation. Upon compound inactivation of p110α and p110δ, which removed >90% of p85-associated PI3K activity, remarkably, cells continued to proliferate effectively, with p110β assuming an essential role in signaling and cell survival. Furthermore, under these conditions of diminished class I PI3K activity, input from the ERK pathway became important for cell survival. Similar observations were made in mouse embryonic fibroblasts (which mainly express p110α and p110β) in which p110α or p110β could sustain cell proliferation as a single isoform. Taken together, these data demonstrate that a small fraction of total class I PI3K activity is sufficient to sustain cell survival and proliferation. Persistent inhibition of selected PI3K isoforms can allow the remaining isoform(s) to couple to upstream signaling pathways in which they are not normally engaged. Such functional redundancy of class IA PI3K isoforms upon sustained PI3K inhibition has implications for the development and use of PI3K inhibitors in cancer.apoptosis | cancer therapeutics | inhibitors | combination therapy | MAP kinase T he class I subset of PI3K comprises the p110 catalytic subunits, which have been further subdivided into class IA and IB, depending on whether they occur in complex with a p85-(in the case of p110α, p110β, and p110δ) or p87/p101-type regulatory subunit (in the case of p110γ). Unlike p87/p101, which have no homology to other proteins, the p85 subunits have SH2 domains, which can link the class IA PI3Ks to tyrosine kinase signaling pathways. In mammals, the four class I PI3K isoforms are present in all cell types, with p110δ and p110γ highly enriched in leukocytes (1). Among the p110s, the most prominent role in cancer has thus far been attributed to p110α, encoded by the PIK3CA gene, found to be amplified and mutated in a wide range of solid tumors (2, 3). Consistent with their roles in signaling downstream of tyrosine kinases and Ras, class IA PI3Ks are currently being pursued as therapeutic targets in oncology (4, 5). However, the relative importance of p110β in tyrosine kinase signaling is not entirely clear, as this isoform has recently been shown to mainly signal downstream of G protein-coupled receptors (GPCRs) (6). A role for the GPCR-coupled p110γ in cancer is less clear, but activity against this isoform is often represented in small PI3K molecule inhibitors under development or in trials. Moreover, overexpression of n...