Involvement of phosphatidylinositol 3-kinase in cell growth

从牛胸腺细胞质中分别纯化了两种类型的磷脂酰肌醇（PI） 3-激酶（PI3K），分别为6250倍（PI3KI）和1250倍（PI3KII）。经凝胶过滤纯化后的PI3KI和PI3KII的表观分子量分别为110 KDa和190 KDa。另一方面，在SDS凝胶电泳上，PI3KI的分子量也估计为110 KDa，但PI3KII有两条分子量为110 KDa和85 KDa的条带，表明PI3KII为异源二聚体形式。肽图谱分析还表明，PI3KII中含有的110 KDa蛋白与PI3KI相同。PI3KI的比活性为250 nmol/min/mg蛋白，PI3KII的比活性为25 nmol/min/mg蛋白，表明该单体活性高于异源二聚体。即使在PI3K未被磷酸化的情况下，PI3KII也与pp60^<v-src>和中间T/pp60^<c-src>共同免疫沉淀，这表明非磷酸化的PI3K识别pp60^<v-src>， PI3KII被pp60^<v-src>磷酸化并与pp60^<v-src>结合。在src转化细胞中沉淀PI3KII的抗p85 （PI3KII的85 Kda亚基）抗体共同免疫沉淀pp60^<v-src>，提示PI3KII结合pp60^<v-src>。这些数据表明这些pi3k是独立调控的。

Two types of phosphatidylinositol(PI) 3-kinase (PI3K) have been purified 6250-fold (PI3KI) and 1250-fold (PI3KII) from the cytosol fraction of bovine thymus, respectively. Purified PI3KI and PI3KII were found to be apparent molecular weight of 110 KDa and 190 KDa respectively by a gel filtration. On the other hand, on SDS gel electrophoresis molecular weight of PI3KI was also estimated as 110 KDa but PI3KII showed two bands of which molecular weight are 110 KDa and 85 KDa, suggesting a heterodimer form. Peptide mapping analysis also demonstrated that a 110 KDa protein contained in PI3KII is the same protein as PI3KI. Specific activity of PI3KI was calculated as 250 nmol/min/mg protein, while that of PI3KII was as 25 nmol/min/mg protein, indicating that monomer has higher activity than heterodimer. PI3KII but not PI3KI was co-immunoprecipitated with pp60^<v-src> and middle T/pp60^<c-src> even in the conditions where PI3Ks were not phosphorylated suggesting that non phosphorylated PI3K recognized pp60^<v-src> PI3KII was phosphorylated by pp60^<v-src> and bound to pp60^<v-src>. Anti-p85 (85 Kda subunit of PI3KII) antibody which precipitated PI3KII co-immunoprecipitated pp60^<v-src> in src-transformed cells suggesting that PI3KII bound to pp60^<v-src>. All these data shows that these PI3Ks are regulated independently.