Lavan B E, Lienhard G E
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844.
J Biol Chem. 1993 Mar 15;268(8):5921-8.
Insulin stimulates the tyrosine phosphorylation of a 60-kDa protein (pp60) in rat adipocytes. After insulin treatment of these cells, pp60, as well as the 160-kDa insulin receptor substrate-1 (IRS-1), were found to be associated with the enzyme phosphatidylinositol 3-kinase (PtdIns-3-kinase) in separate complexes. By contrast, pp60 was not detected in insulin-treated mouse 3T3-L1 adipocytes, which contain abundant IRS-1. PtdIns-3-kinase complex. The pp60.PtdIns 3-kinase complex was located in both the soluble and membrane fractions of the rat adipocytes. Fusion proteins containing the isolated src homology 2 domains from the 85-kDa subunit of PtdIns-3-kinase bound to pp60 in lysates of insulin-treated rat adipocytes. This finding indicates that the most likely mode of association of pp60 with PtdIns-3-kinase is through binding of phosphotyrosine residues in pp60 to these domains. By immunoaffinity chromatography on a monoclonal antibody against phosphotyrosine, pp60 was purified in high percentage yield from insulin-stimulated rat adipocytes, but the low amount of the protein obtained (about 3 ng from the adipocytes of one rat) precluded sequence analysis.
胰岛素可刺激大鼠脂肪细胞中一种60 kDa蛋白（pp60）的酪氨酸磷酸化。在用胰岛素处理这些细胞后，发现pp60以及160 kDa的胰岛素受体底物-1（IRS-1）与磷脂酰肌醇3激酶（PtdIns-3激酶）以不同的复合物形式存在。相比之下，在含有丰富IRS-1的胰岛素处理的小鼠3T3-L1脂肪细胞中未检测到pp60。PtdIns-3激酶复合物。pp60·PtdIns 3激酶复合物存在于大鼠脂肪细胞的可溶性和膜部分。含有从PtdIns-3激酶85 kDa亚基分离的src同源2结构域的融合蛋白与胰岛素处理的大鼠脂肪细胞裂解物中的pp60结合。这一发现表明，pp60与PtdIns-3激酶最可能的结合方式是通过pp60中的磷酸酪氨酸残基与这些结构域结合。通过用抗磷酸酪氨酸单克隆抗体进行免疫亲和层析，从胰岛素刺激的大鼠脂肪细胞中以高百分比产率纯化了pp60，但获得的蛋白量很少（一只大鼠的脂肪细胞中约为3 ng），无法进行序列分析。
