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Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases
J C Scimeca1, R Ballotti, C Filloux
1INSERM U 145, Faculté de Médecine, Nice, France.
Abstract:
Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.
Insights
Insulin and orthovanadate activate specific serine kinase activities in mouse cells. These enzymes, identified by their tyrosine phosphorylation, are found in the cytosol and are sensitive to insulin receptor signaling.
Area of Science:
- Molecular Biology
- Cell Signaling
- Enzymology
Background:
- Insulin signaling is crucial for cellular metabolism and growth.
- Serine kinases play key roles in signal transduction pathways.
- Understanding insulin-mediated kinase activation is vital for metabolic research.
Purpose of the Study:
- To investigate insulin-sensitive serine kinase activity in mouse fibroblasts.
- To characterize the properties and molecular weight of these enzymes.
- To explore the role of tyrosine phosphorylation in enzyme activation.
Main Methods:
- Utilized synthetic peptide substrate Kemptide and cytosolic extracts.
- Employed insulin and orthovanadate for enzyme stimulation.
- Used antiphosphotyrosine antibodies for immunoprecipitation and gel filtration chromatography.
Main Results:
- Identified an insulin- and orthovanadate-sensitive serine kinase activity.
- Enzyme activity was preserved by para-nitrophenylphosphate and phosphotyrosine.
- Multiple tyrosine-phosphorylated serine kinase activities (≤30 kDa) were stimulated by insulin and orthovanadate.
Conclusions:
- Insulin and orthovanadate enhance cytosolic serine kinase activities.
- These activities are characterized by tyrosine phosphorylation.
- Suggests a link between insulin receptor signaling and cytosolic serine kinases.