Class IA phosphoinositide 3-kinases (PI3-kinase) generate the secondary messenger PI [3,4,5]P3, which plays an important role in many cellular responses. The accumulation of PI [3,4,5]P3 in cell membranes is routinely measured using GFP-labeled PH domains. However, the kinetics of membrane PI[3,4,5]P3 synthesis and turnover as detected by PH domains has not been validated using an independent method. In the present study, we measured EGF-stimulated membrane PI [3,4,5]

Phosphoinositides (PIPs) are an integral part of multiple cell signaling pathways and are metabolized by kinases, phosphatases, and phospholipases. PIPs produced by the activities of phosphoinositide 3-kinase (PI3K) serve as second messengers in cell survival, growth, motility, immune response, inflammation, and apoptosis. Conversely, abnormal production of these signaling lipids leads to proliferative, metabolic, and inflammatory disorders. It is critical to know the absolute and relative concentration of individual PIPs in cell samples, however this analysis is difficult due to their low abundance and the inherent physical properties of lipids. Experiments to measure PIPs have involved radiolabeling cells, extraction of radioactive products, and separation using thin-layer chromatography. The amount of PIPn extracted from cell lysates was determined by means of a standard competitive ELISA format, eliminating the need for radioactivity. Using specific antibodies and binding proteins we developed assays able to differentiate closely related PIPs. These nonradioactive assays employ synthetic di-C16 lipids as standards and are able to measure as little as 0.1 pmol of lipid. Cell-extracted PIPs were added as competitors in these quantitative ELISAs. We used the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient) as a model system to evaluate multiple activators of PI3K pathways by looking at levels of PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. Using growth factors, hyperosmolarity, and oxidative stress we found that different stimulants targeted different classes of PI3K. All three classes of PI3K were more active in PTEN deficient cell line (468 cells) than PTEN+/+ (231 cells). We found that all stimulants used increased PI3K products. Pre-incubation with wortmannin, a known PI3K inhibitor, decreased PIPn levels. We propose that hyperactivation of class I and class II PI3K in the absence of PTEN regulation (468 cells) leads to excessive 3’ phosphorylated phosphoinositide levels that are known to activate Akt, We expect the ability to quantify individual phosphoinositides from cells will help assign specific biological functions to these important lipid second messengers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2011-3835

No abstract

Phosphatidylinositol 4‐phosphate (PI4P) produced by Phosphatidylinositol 4‐Phosphate kinases is an abundant Golgi phosphoinositide functioning in lipid exchange and vesicle transport within mammalian cells. Researchers wanting to quantify PI4P use the Bligh and Dyer method for extracting and separating phospholipids in cells and biological tissues. This common lipid extraction procedure is known to have inherent variation. Using a human promyelocytic leukemia cell line and an ELISA that is sensitive and specific for PI4P, we examine sources of variation in bioassays including sample (HL‐60 cells), the extraction procedure, and lipid detection. We determined a PI4P Mass ELISA has low coefficient of variation (<10%) if the extracted sample volume is held constant and samples are prepped and lipids presented with standardized procedures. Thus, the organic solvent extraction process is the major source of variation in typical Bligh‐Dyer preparations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is a 3′ phosphoinositide phosphatase that converts PI(3,4,5)P3 to PI(4,5)P2 thus opposing PKB/Akt activation by PI 3-Kinase. PTEN is involved in neuronal stem cell proliferation and self-renewal, cardiac myocyte hypertrophy and contractility, and a wide range of developmental processes. However, PTEN is best known for its role as a tumor suppressor. Loss of PTEN activity results in accumulation of PI(3,4,5)P3, abnormal activation of PKB/Akt, unregulated cell growth, suppression of apoptosis, and increased tumorigenesis in a number of human tissues. It has also been proposed that PTEN is a candidate for targeted chemotherapy because certain anti-cancer agents preferentially destroy tumors with PTEN mutations. In addition to this direct role in cancer, PTEN has recently been shown to regulate cancer-associated pathways including VEGF-mediated angiogenesis and others. Consequently interest in PTEN as a cancer target is still high. Traditionally, quantification of PTEN phosphatase activity has required the use of radioactivity, organic solvents, and chromatography. In addition, detection of free phosphate released from the PTEN activity is commonly used for quantification. However, this latter method has limited sensitivity and dynamic range, and is susceptible to contamination from other sources of inorganic phosphate which can give incorrect results. To address the need for a better quantification method, we have developed a PTEN activity assay using a standard competitive ELISA format. Rather than detecting free phosphate, the assay detects the PTEN product, PI(4,5)P2, giving much more accurate quantification, improved sensitivity, and eliminates possible errors from phosphate contamination. We have shown that the assay can successfully quantify PTEN activity using both recombinant and immunoprecipitated enzyme. Several cell lines were tested for PTEN activity including the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 (PTEN deficient). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 231. doi:1538-7445.AM2012-231