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rdf:type |
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lifeskim:mentions |
umls-concept:C0017262,
umls-concept:C0034818,
umls-concept:C0178539,
umls-concept:C0181904,
umls-concept:C0205548,
umls-concept:C0335038,
umls-concept:C0596972,
umls-concept:C0728873,
umls-concept:C0805732,
umls-concept:C1150423,
umls-concept:C1334435,
umls-concept:C1335960,
umls-concept:C1366753,
umls-concept:C1510438,
umls-concept:C1521743,
umls-concept:C1527178,
umls-concept:C1548425,
umls-concept:C1704646,
umls-concept:C1705938
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pubmed:issue |
1
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pubmed:dateCreated |
2000-2-14
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pubmed:abstractText |
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.
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Insulin,
http://linkedlifedata.com/resource/pubmed/chemical/Luciferases,
http://linkedlifedata.com/resource/pubmed/chemical/Milk Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Receptor, Insulin,
http://linkedlifedata.com/resource/pubmed/chemical/STAT5 Transcription Factor,
http://linkedlifedata.com/resource/pubmed/chemical/STAT5A protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/STAT5B protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Stat5a protein, rat,
http://linkedlifedata.com/resource/pubmed/chemical/Stat5b protein, rat,
http://linkedlifedata.com/resource/pubmed/chemical/Trans-Activators,
http://linkedlifedata.com/resource/pubmed/chemical/Tumor Suppressor Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/beta-Galactosidase
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pubmed:status |
MEDLINE
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pubmed:month |
Dec
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pubmed:issn |
0003-2697
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pubmed:author |
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pubmed:copyrightInfo |
Copyright 1999 Academic Press.
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pubmed:issnType |
Print
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pubmed:day |
1
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pubmed:volume |
276
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
97-104
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pubmed:dateRevised |
2011-11-17
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pubmed:meshHeading |
pubmed-meshheading:10585749-Animals,
pubmed-meshheading:10585749-Cell Line,
pubmed-meshheading:10585749-DNA-Binding Proteins,
pubmed-meshheading:10585749-Gene Expression,
pubmed-meshheading:10585749-Genes, Reporter,
pubmed-meshheading:10585749-Humans,
pubmed-meshheading:10585749-Insulin,
pubmed-meshheading:10585749-Kinetics,
pubmed-meshheading:10585749-Luciferases,
pubmed-meshheading:10585749-Milk Proteins,
pubmed-meshheading:10585749-Rats,
pubmed-meshheading:10585749-Receptor, Insulin,
pubmed-meshheading:10585749-STAT5 Transcription Factor,
pubmed-meshheading:10585749-Sensitivity and Specificity,
pubmed-meshheading:10585749-Signal Transduction,
pubmed-meshheading:10585749-Trans-Activators,
pubmed-meshheading:10585749-Transfection,
pubmed-meshheading:10585749-Tumor Suppressor Proteins,
pubmed-meshheading:10585749-beta-Galactosidase
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pubmed:year |
1999
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pubmed:articleTitle |
A cellular reporter assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression.
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pubmed:affiliation |
Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, D-70569, Germany.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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