Assaying AKT/Protein Kinase B Activity

Abstract

Akt/protein kinase B (PKB) is a serine/threonine kinase that mediates many of the anabolic actions of insulin (1), as well as the growth-promoting and/or antiapoptotic effects of other growth factors, cytokines, or transforming oncogenes (2). These agonists all stimulate Akt/PKB by promoting its phosphorylation on two regulatory residues (e.g., S473 and T308 for the Akt1 isoform), an event dependent on the prior activation of a signaling pathway initiated by the lipid kinase phosphatidylinositol 3-kinase. Once Akt/PKB is activated it phosphorylates numerous different substrates, including transcription factors (e.g., FKHR1, others), anti-apoptotic enzymes (e.g., Bad caspase-9), and metabolic enzymes (e.g., glycogen synthase kinase 3β), to regulate this diverse array of biological processes. Herein we describe a method for measuring the catalytic activity of Akt/PKB isolated from cell or tissue extracts by quantifying its ability to catalyze phosphate incorporation into an exogenous substrate (outlined in Fig. 1). This chapter illustrates techniques for immunoprecipitating Akt/PKB, choosing an appropriate substrate for the reaction, and optimizing the assay conditions.