TY - JOUR
T1 - A cellular reporter assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression
AU - Storz, Peter
AU - Döppler, Heike
AU - Horn-Müller, Judith
AU - Groner, Bernd
AU - Pfizenmaier, Klaus
AU - Müller, Gertraud
N1 - Funding Information:
The authors thank Dr. Axel Ullrich, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany, for the Rat1-HIR-cl5 cells. This work was supported in part by Boehringer Ingelheim Pharma KG and Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Germany (FKZ 0316500C, B.3.10.E) as well as by the Deutsche Forschungsgemeinschaft, Grant Mu625/5. At the German Patentamt, Munich, we applied for a patent on this method (Registered No. 199 37 235.7).
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 1999/12/1
Y1 - 1999/12/1
N2 - 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.
AB - 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.
KW - Insulin receptor kinase
KW - STAT5 reporter gene assay
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U2 - 10.1006/abio.1999.4345
DO - 10.1006/abio.1999.4345
M3 - Article
C2 - 10585749
AN - SCOPUS:0345148324
SN - 0003-2697
VL - 276
SP - 97
EP - 104
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -