The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P-3. PI(3,4,5)P-3 can be dephosphorylated by 3-or 5-phosphatases, the latter producing PI(3,4)P-2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P-3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4) P-2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4) P-2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P-2 4-phosphatase, leads to synergistic accumulation of PI(3,4) P-2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P-2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P-2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4) P-2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

Cell migration is controlled by PI3Ks, which generate lipid messengers phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and consequently recruit pleckstrin homology (PH) domain–containing signaling proteins. PI3K inhibition impairs migration of normal and transformed B cells, an effect thought to partly underlie the therapeutic efficacy of PI3K inhibitors in treatment of B cell malignancies such as chronic lymphocytic leukemia. Although a number of studies have implicated phosphatidylinositol-3,4,5-trisphosphate in cell migration, it remains unknown whether PI(3,4)P2 plays a distinct role. Using the PI(3,4)P2-specific phosphatase inositol polyphosphate 4-phosphatase, we investigate the impact of depleting PI(3,4)P2 on migration behavior of malignant B cells. We find that cells expressing wild-type, but not phosphatase dead, inositol polyphosphate 4-phosphatase show impaired SDF-induced PI(3,4)P2 responses and reduced migration in Transwell chamber assays. Moreover, PI(3,4)P2 depletion in primary chronic lymphocytic leukemia cells significantly impaired their migration capacity. PI(3,4)P2 depletion reduced both overall motility and migration directionality in the presence of a stable chemokine gradient. Within chemotaxing B cells, the PI(3,4)P2-binding cytoskeletal regulator lamellipodin (Lpd) was found to colocalize with PI(3,4)P2 on the plasma membrane via its PH domain. Overexpression and knockdown studies indicated that Lpd levels significantly impact migration capacity. Moreover, the ability of Lpd to promote directional migration of B cells in an SDF-1 gradient was dependent on its PI(3,4)P2-binding PH domain. These results demonstrate that PI(3,4)P2 plays a significant role in cell migration via binding to specific cytoskeletal regulators such as Lpd, and they suggest that impairment of PI(3,4)P2-dependent processes may contribute to the therapeutic efficacy of PI3K inhibitors in B cell malignancies.

Introduction: GSK2269557 was investigated as a dry powder formulation in a double blind, placebo controlled study in healthy cigarette smokers. Safety, tolerability, PK and PD data were collected following SD (100, 500, 2000 and 3000 µg) and RD (2000 µg for 14 days). Plasma and lung PK (from bronchoalveolar lavage) and PD (PIP3 proportion [using PIP2] from induced sputum) were measured. Lung PK was the drug concentration in the cellular fraction (CF) and epithelial lining fluid (ELF). PD was measured to demonstrate target engagement. Results: Adverse Events (AE's) were few, of mild to moderate intensity and not different from placebo. SD plasma Cmax (Tmax approx 5 min) dropped rapidly (< 6 hours) followed by a slower elimination (T1/2 was approx 20h). Cmax and AUC(0-t) were 0.04 to 2.6 ng/mL and 0.14 to 33 ng.h/mL for 100 to 3000 µg respectively. RD showed accumulation in plasma with increased peak (3-fold) and trough (4.5-fold) levels. Lung ELF and CF concentrations at 24h post day 14 dose were 55 ng/mL (ELF:plasma ratio 32:1) and 366 ng/mL (CF:plasma ratio 214:1) respectively. PIP3 proportion levels in sputum were reduced by 6.7%, 8.4% and 18.7% at 3hrs post dose, day 1 for 100, 500 and 2000µg respectively, and by 35.7% at 3 hours post dose, day 12 for 2000µg. Conclusions: GSK2269557 was well tolerated with a low incidence of AE's which was not different from placebo. Plasma PK was well defined with lung data confirming higher levels of drug compared to plasma. Target engagement was confirmed by sputum PIP3 data.

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.

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.

, 863 (2013); 342 Science et al. Vilasack Thammavongsa Promote Immune Cell Death Degrades Neutrophil Extracellular Traps to Staphylococcus aureus This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): November 23, 2013 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/342/6160/863.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2013/11/13/342.6160.863.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/342/6160/863.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/342/6160/863.full.html#ref-list-1 , 10 of which can be accessed free: cites 23 articles This article

We disrupted the gene encoding lysophosphatidylinositol-acyltransferase-1 (LPIAT1) in the mouse with the aim of understanding its role in determining cellular phosphoinositide content. LPIAT1(-/-) mice were born at lower than Mendelian ratios and exhibited a severe developmental brain defect. We compared the phospholipid content of livers and brains from LPIAT1(-/-) and LPIAT1(+/+) littermates by LC-ESI/MS. In accord with previous studies, the most abundant molecular species of each phosphoinositide class (PtdIns, PtdInsP, PtdInsP2 and PtdInsP3) possessed a C38∶4 complement of fatty-acyl esters (C18∶0 and C20∶4 are usually assigned to the sn-1 and sn-2 positions, respectively). LPIAT1(-/-) liver and brain contained relatively less of the C38∶4 species of PtdIns, PtdInsP and PtdInsP2 (dropping from 95-97% to 75-85% of the total species measured for each lipid class) and relatively more of the less abundant species (PtdInsP3 less abundant species were below our quantification levels). The increases in the less abundant PtdIns and PtdInsP2 species did not compensate for the loss in C38∶4 species, resulting in a 26-44% reduction in total PtdIns and PtdInsP2 levels in both brain and liver. LPIAT1(-/-) brain and liver also contained increased levels of C18∶0 lyso-PtdIns (300% and 525% respectively) indicating a defect in the reacylation of this molecule. LPIAT1(-/-) brain additionally contained significantly reduced C38∶4 PC and PE levels (by 47% and 55% respectively), possibly contributing to the phenotype in this organ. The levels of all other molecular species of PC, PE, PS and PA measured in the brain and liver were very similar between LPIAT1(-/-) and LPIAT1(+/+) samples. These results suggest LPIAT1 activity plays a non-redundant role in maintaining physiological levels of PtdIns within an active deacylation/reacylation cycle in mouse tissues. They also suggest that this pathway must act in concert with other, as yet unidentified, mechanisms to achieve the enrichment observed in C38∶4 molecular species of phosphoinositides.

The phosphoinositide family of phospholipids, defined here as PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, play pivotal roles in organising the location and activity of many different proteins acting on biological membranes, including those involved in vesicle and protein trafficking through the endolysosomal system and receptor signal transduction at the plasma membrane. Accurate measurement of the cellular levels of these lipids, particularly the more highly phosphorylated species, is hampered by their high polarity and low cellular concentrations. Recently, much progress has been made in using mass spectrometry to measure many different lipid classes in parallel, an approach generally referred to as ‘lipidomics’. Unfortunately, the acidic nature of highly phosphorylated phosphoinositides makes them difficult to measure using these methods, because they yield low levels of useful ions; this is particularly the case with PtdIns(3,4,5)P3. We have solved some of these problems by methylating the phosphate groups of these lipids with TMS-diazomethane and describe a simple, integrated approach to measuring PtdIns, PtdInsP, PtdInsP2 and PtdInsP3 classes of lipids, in parallel with other phospholipid species, in cell and tissue extracts. This methodology is sensitive, accurate and robust, and also yields fatty-acyl compositions, suggesting it can be used to further our understanding of both the normal and pathophysiological roles of these important lipids.