Measurement of Phosphoinositide 3-Kinase Activity

Abstract

The term “PI 3-kinase” is now applied to a growing family of proteins that are able to convert phosphatidylinositol (PtdIns), PtdIns(4)P and PtdIns(4,5)P₂ into PtdIns(3)P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ respectively, by phosphorylating the D-3 position of the inositol head groups of phosphoinositide lipids (collectively known as D-3 phosphoinositide lipids shown in Fig. 1) (1,2). PtdIns(3)P is constitutively present in eukaryotic cells and its levels are largely unaltered upon cellular stimulation. In contrast, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ are generally absent from resting cells, but their intracellular concentration rises markedly upon stimulation via a variety of receptors suggesting a likely function as a second messenger (1,2). PI 3-kinases can be divided into three main classes on the basis of their in vitro lipid substrate specificity, structure and likely mode of regulation. Class I PI 3-kinases phos-phorylate PtdIns, PtdIns(4)P and PtdIns(4,5)P₂, interact with Ras and form heterodimeric complexes with adaptor proteins that link them to different upstream signaling events (1). The prototypical class IA PI 3-kinase is a heterodimer consisting of the 85 kDa regulatory subunit (responsible for protein-protein interactions either via protein tyrosine phosphate-binding SH2 domains or SH3 domains and/or proline rich regions) and a catalytic 110 kDa subunit. The class IB PI 3-kinases are stimulated by G protein αγ subunits and do not interact with the SH2-containing adaptors that bind class IA PI 3-kinases. Instead, the first identified member of this family p1 10γ, associates with a unique p101 adaptor molecule (1). The class II PI 3-kinases are characterized by the presence of a C-2 domain at the carboxy terminus and utilize predominantly PtdIns and PtdIns(4)P as substrates (e.g., PI3K-C2α), whereas the class III PI 3-kinases utilize only PtdIns as a substrate (e.g., mammalian PtdIns 3-kinase and yeast Vps34p) (1). Generally, PI 3 kinases are now regarded as an important intracellular signal upstream of a variety of biochemical (e.g., activation of Akt/protein kinase B and/or p70 S6 kinase and inhibition of glycogen synthase kinase-3) and functional responses (membrane trafficking of proteins such as the glucose transporter GLUT4, postendocytic sorting of ligand-stimulated receptors such as the Platelet-derived growth factor (PDGF) receptor, membrane ruffling, superoxide production, and chemotaxis) (1,2).