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 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-cl5) 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.
Storz
P
P
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, D-70569, Germany.
Döppler
H
H
Horn-Müller
J
J
Groner
B
B
Pfizenmaier
K
K
Müller
G
G
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000235:genetics*; D008894:Milk Proteins; D051381:Rats; D011972:Receptor, Insulin / Q000032:analysis / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins; D001616:beta-Galactosidase / Q000235:genetics
D004268:DNA-Binding Proteins; D007328:Insulin; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; C494094:Stat5a protein, rat; C494097:Stat5b protein, rat; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D008156:Luciferases; D011972:Receptor, Insulin; D001616:beta-Galactosidase