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Involvement of phosphatidylinositol 3-kinase in cell growth

磷脂酰肌醇 3-激酶参与细胞生长

基本信息

批准号：
03454163
负责人：
TAKENAWA Tadaomi
金额：
$ 4.54万
依托单位：
The University of Tokyo (1992)Tokyo Metropolitan Institute of Gerontology (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454163/
关键词：
PI3 Kinase Tyrosine kinase Purification SH_2 Tyrosine phosphorylation PI3キナ-ゼ SH_3 αーアクチニン細胞骨格系

项目摘要

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.

两种类型的磷脂酰肌醇 (PI) 3-激酶 (PI3K) 从牛胸腺的胞质部分中分别纯化了 6250 倍 (PI3KI) 和 1250 倍 (PI3KII)。通过凝胶过滤发现纯化的PI3KI和PI3KII的表观分子量分别为110KDa和190KDa。另一方面，在SDS凝胶电泳上，PI3KI的分子量也估计为110KDa，但PI3KII显示两条分子量为110KDa和85KDa的条带，表明其为异二聚体形式。肽图分析还表明，PI3KII 中包含的一个 110 KDa 的蛋白质与 PI3KI 是相同的蛋白质。 PI3KI的比活性计算为250 nmol/min/mg蛋白质，而PI3KII的比活性为25 nmol/min/mg蛋白质，表明单体比异二聚体具有更高的活性。 PI3KII 但不是 PI3KI 与 pp60^<v-src> 和中间 T/pp60^<c-src> 共免疫沉淀，即使在 PI3K 未磷酸化的条件下，这表明非磷酸化 PI3K 识别 pp60^<v-src> PI3KII 被 pp60^<v-src> 磷酸化，并且绑定到 pp60^<v-src>。沉淀 PI3KII 的抗 p85（PI3KII 的 85 Kda 亚基）抗体在 src 转化细胞中免疫共沉淀 pp60^<v-src>，表明 PI3KII 与 pp60^<v-src> 结合。所有这些数据表明这些 PI3K 是独立调节的。