Benoit Bilanges

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.

Long‐term p110α PI3K inactivation exerts a beneficial effect on metabolism

The insulin/insulin‐like growth factor‐1 signalling (IIS) pathway regulates cellular and organismal metabolism and controls the rate of aging. Gain‐of‐function mutations in p110α, the principal mammalian IIS‐responsive isoform of PI 3‐kinase (PI3K), promote cancer. In contrast, loss‐of‐function mutations in p110α impair insulin signalling and cause insulin resistance, inducing a pre‐diabetic state. It remains unknown if long‐term p110α inactivation induces further metabolic deterioration over time, leading to overt unsustainable pathology. Surprisingly, we find that chronic p110α partial inactivation in mice protects from age‐related reduction in insulin sensitivity, glucose tolerance and fat accumulation, and extends the lifespan of male mice. This beneficial effect of p110α inactivation derives in part from a suppressed down‐regulation of insulin receptor substrate (IRS) protein levels induced by age‐related hyperinsulinemia, and correlates with enhanced insulin‐induced Akt signalling in aged p110α‐deficient mice. This temporal metabolic plasticity upon p110α inactivation indicates that prolonged PI3K inhibition, as intended in human cancer treatment, might not negatively impact on organismal metabolism.

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.

Class III phosphoinositide 3-kinase/VPS34 and dynamin are critical for apical endocytic recycling.

Recycling is a limiting step for receptor‐mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan‐class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP‐2, causing surface down‐regulation. GFP‐(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002‐treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin‐GFP and dependence of dynamin‐GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K‐III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.

Environmental Stress Affects the Activity of Metabolic and Growth Factor Signaling Networks and Induces Autophagy Markers in MCF7 Breast Cancer Cells

Phosphoproteomic techniques are contributing to our understanding of how signaling pathways interact and regulate biological processes. This technology is also being used to characterize how signaling networks are remodeled during disease progression and to identify biomarkers of signaling pathway activity and of responses to cancer therapy. A potential caveat in these studies is that phosphorylation is a very dynamic modification that can substantially change during the course of an experiment or the retrieval and processing of cellular samples. Here, we investigated how exposure of cells to ambient conditions modulates phosphorylation and signaling pathway activity in the MCF7 breast cancer cell line. About 1.5% of 3,500 sites measured showed a significant change in phosphorylation extent upon exposure of cells to ambient conditions for 15 min. The effects of this perturbation in modifying phosphorylation patterns did not involve random changes due to stochastic activation of kinases and phosphatases. Instead, exposure of cells to ambient conditions elicited an environmental stress reaction that involved a coordinated response to a metabolic stress situation, which included: (1) the activation of AMPK; (2) the inhibition of PI3K, AKT, and ERK; (3) an increase in markers of protein synthesis inhibition at the level of translation elongation; and (4) an increase in autophagy markers. We also observed that maintaining cells in ice modified but did not completely abolish this metabolic stress response. In summary, exposure of cells to ambient conditions affects the activity of signaling networks previously implicated in metabolic and growth factor signaling. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000472.

A new tool to dissect the function of p70 S6 kinase.

Developing small-molecule inhibitors that are highly selective for specific protein kinases has been and remains a serious challenge. This especially applies to members of families of related kinases with overlapping substrate specificities, such as the serine/threonine kinases of the AGC family. In this issue of the Biochemical Journal, Dario Alessis group, in a collaboration with Pfizer, report on PF-4708671, a potent and highly selective inhibitor of S6K1 (p70 S6 kinase 1) in vitro and in cells. S6K1 is an AGC family member and a crucial effector of the mTORC1 (mammalian target of rapamycin complex 1) kinase. This is the first reported inhibitor that is highly selective for S6K1. This compound will help to understand the signalling and physiological roles of S6K1, and to dissect signalling downstream of mTORC1. S6K1 inhibitors may ultimately be useful in the treatment of diseases such as cancer where S6K1 is overexpressed, but most importantly in metabolic disease such as insulin resistance and obesity.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Cinderella finds her shoe: the first Vps34 inhibitor uncovers a new PI3K-AGC protein kinase connection.

Class II/III PI3Ks (phosphoinositide 3-kinases) produce the PtdIns(3)P lipid that is involved in intracellular vesicular trafficking. In contrast with class I PI3Ks, the potential signalling roles of class II/III PI3Ks are poorly understood. In a recent article in the Biochemical Journal, Bago and co-workers report that Vps34 (vacuolar protein sorting 34), the only class III PI3K, controls the activity of SGK3 (serum- and glucocorticoid-regulated protein kinase 3). Like other AGC kinases, the SGKs (SGK1, SGK2 and SGK3) are activated by dual phosphorylation. Unlike its cousins SGK1 and SGK2, SGK3 contains a PtdIns(3)P-binding domain, providing an additional element of regulation. The study by Bago et al. characterizes and makes extensive use of a Novartis Vps34 inhibitor (VPS34-IN1) that inhibits this PI3K isoform with nanomolar potency, without affecting other lipid kinases or more than 300 protein kinases. The authors show that this compound very rapidly reduced PtdIns(3)P levels at the endosome with concomitant loss of SGK3 phosphorylation. Co-inhibition of class I PI3Ks led to a further reduction in SGK3 activity, indicating that class I PI3Ks may also regulate SGK3 activity through an additional, currently unknown, mechanism. It remains to be assessed whether the novel PI3K-protein kinase connection established by this study is subject to acute cellular stimulation or is part of a constitutive housekeeping function. VPS34-IN1 will provide a useful tool to decipher the kinase-dependent functions of Vps34, with acute changes in SGK3 phosphorylation and subcellular localization being new biomarkers of Vps34 activity.

Inactivation of class II PI3K-C2α induces leptin resistance, age-dependent insulin resistance and obesity in male mice

Aims/hypothesisWhile the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice.MethodsWe have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a).ResultsWhile homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age.Conclusions/interpretationOur data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis.Access to research materialsAll reagents are available upon request.