Scimeca J C, Ballotti R, Filloux C, Van Obberghen E
INSERM U 145, Faculté de Médecine, Nice, France.
Mol Cell Biochem. 1992 Feb 12;109(2):139-47. doi: 10.1007/BF00229768.
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.
利用合成肽底物Kemptide以及用人类胰岛素受体cDNA构建体转染的小鼠成纤维细胞的胞质提取物，我们研究了一种胰岛素敏感的丝氨酸激酶活性。这种活性可被胰岛素迅速刺激（5分钟内达到最大值），也可被原钒酸盐刺激。在细胞提取物制备过程中，对硝基苯磷酸酯和磷酸酪氨酸能够保留酶活性，而磷酸苏氨酸和磷酸丝氨酸则不能。使用抗磷酸酪氨酸抗体，对这种对胰岛素和原钒酸盐敏感的丝氨酸激酶进行了特异性免疫沉淀。然后，我们通过凝胶过滤色谱法分析了从未受刺激、经胰岛素和钒酸盐处理的细胞中获得的含有酪氨酸磷酸化蛋白的洗脱液。我们发现，分子量估计为30 kDa及更小的几种活性受到胰岛素和原钒酸盐两者的刺激。总体而言，我们的数据表明，胰岛素和原钒酸盐至少增强了2或3种含磷酸酪氨酸的丝氨酸激酶活性的胞质含量。
