A cellular reporter assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression

A highly sensitive method for determination of insulin receptor (IR) kinase activity in whole cells, which is based on a STATE (signal transducer and activator of transcription 5)-dependent reporter gene assay, has been devel oped, We show in Rat1 fibroblasts stably overexpressing the human IR (Rat1- HIR-cl5) an insulin-dependent direct association and phosphorylation of STA T5b by IR kinase. Rat1-HIR cells transfected with a luciferase gene reporte r construct under control of a STATE-inducible promoter showed insulin-medi ated induction of STATE-dependent luciferase activity, with peak activities around 8 h of insulin treatment over a wide dose range. Transient STAT5b b ut not STAT5a cotransfection significantly enhanced reporter gene activity, yielding up to a fivefold induction. Addition of the IR kinase inhibitor t yrphostin AG1024 down-regulated luciferase induction in a dose-dependent ma nner. This is the first assay allowing determination of IR kinase activity in intact cells in a 24-well culture and a microtiter format. Kinetics of t his cellular response, sensitivity range, and signal amplitude make it well suited for automation and offer the potential for establishing high-throug hput screening systems for both insulin mimetic substances and IR kinase an tagonists in a simple nonradioactive assay. (C) 1999 Academic Press.