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

Peter Storz, Heike Döppler, Judith Horn-Müller, Bernd Groner, Klaus Pfizenmaier, Gertraud Müller

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

A highly sensitive method for determination of insulin receptor (IR) kinase activity in whole cells, which is based on a STAT5 (signal transducer and activator of transcription 5)-dependent reporter gene assay, has been developed. We show in Rat1 fibroblasts stably overexpressing the human IR (Rat1-HIR-c15) an insulin-dependent direct association and phosphorylation of STAT5b by IR kinase. Rat1-HIR cells transfected with a luciferase gene reporter construct under control of a STAT5-inducible promoter showed insulin-mediated induction of STAT5-dependent luciferase activity, with peak activities around 8 h of insulin treatment over a wide dose range. Transient STAT5b but not STAT5a cotransfection significantly enhanced reporter gene activity, yielding up to a fivefold induction. Addition of the IR kinase inhibitor tyrphostin AG1024 down-regulated luciferase induction in a dose- dependent manner. 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 this cellular response, sensitivity range, and signal amplitude make it well suited for automation and offer the potential for establishing high-throughput screening systems for both insulin mimetic substances and IR kinase antagonists in a simple nonradioactive assay.

Original languageEnglish (US)
Pages (from-to)97-104
Number of pages8
JournalAnalytical Biochemistry
Volume276
Issue number1
DOIs
StatePublished - Dec 1 1999

Keywords

  • Insulin receptor kinase
  • STAT5 reporter gene assay

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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