LiPing Wang and Scott A Summers Introduction

Numerous growth factors, hormones, and transforming oncogenes activate phosphatidylinositol 3-kinase (PI3K), an enzyme that phosphorylates the 3'-hydroxyl position of phosphoinositides to generate the intracellular messengers phosphatidylinositol-3-phosphate [PI(3)P], phosphatidylinositol-3,4-bisphos-phate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] (reviewed in ref. 1). PI3K commonly participates in signaling pathways leading to increased cell survival, proliferation, and growth, but in some cells and tissues it regulates more specialized biological processes. Notably, PI3K is an obligate intermediate in the signaling pathway linking insulin's arrival at the cell surface to the regulation of glucose uptake and anabolic metabolism (reviewed in ref. 2). This chapter describes a method for quantifying the activation of the class 1A type PI3Ks by insulin.

Insulin stimulates all of its pleiotropic intracellular effects by activating the intrinsic tyrosine kinase activity of its receptor. The receptor phosphorylates several insulin receptor substrate proteins (IRS proteins) to produce phospho-tyrosine binding sites for numerous proteins containing src homology 2 (SH2) domains, including the regulatory subunit of PI3K (3). By interacting with these phosphotyrosine moieties, this regulatory PI3K subunit, which is either 55 or 85 kDa in size, induces a conformational shift in the PI3K catalytic subunit (110 kDa), leading to its activation. Assays designed to assess PI3K activity involve the isolation by immunoprecipitation of this multiprotein complex that contains IRS proteins, the regulatory and catalytic domains of PI3K, and

Fig. 1. Schematic diagram depicting the tyrosine phosphorylated IRS proteins recruiting and activating downstream effector molecules such as the adaptor protein Grb2, the tyrosine phosphatase SHPTP2, and PI3K.

others (see Fig. 1). Antibodies used for immunoprecipitation of this complex often include anti-phosphotyrosine or anti-IRS antibodies, which do not extract total cellular PI3K, but rather the amount of PI3K associated with newly tyro-sine-phosphorylated proteins. Alternatively, one can also use antibodies against the p85 subunit of PI3K, which will also precipitate PI3K not bound to IRS proteins, to obtain a more accurate reflection of the degree of cellular PI3K activity. The catalytic activity of PI3K is then determined by measuring the incorporation of 32P from [y-32P]ATP into an exogenous phosphoinositide substrate. Phosphorylated lipids are extracted in an acidified chloroform/methanol solution, resolved by thin-layer chromatography (TLC), and detected by autoradiography. The method described in this chapter is optimized to measure the IRS-bound PI3K activity isolated from 3T3-L1 adipocytes, a model tissue culture system for evaluating insulin action.

Supplements For Diabetics

Supplements For Diabetics

All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.

Get My Free Ebook

Post a comment