Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases

✍ Scribed by Jean-Claude Scimeca; Robert Ballotti; Chantal Filloux; Emmanuel Obberghen

• Book ID: 104673761
• Publisher: Springer
• Year: 1992
• Tongue: English
• Weight: 792 KB
• Volume: 109
• Category: Article
• ISSN: 0300-8177
• DOI: 10.1007/bf00229768

✦ Synopsis

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.