Serunian L A, Haber M T, Fukui T, Kim J W, Rhee S G, Lowenstein J M, Cantley L C
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
J Biol Chem. 1989 Oct 25;264(30):17809-15.
The ability of three pure types of bovine brain phospholipase C (PLC) and one pure rat liver PLC to utilize as substrates the recently discovered phosphatidylinositol 3-phosphate (PI-3-P), a putative phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3) was investigated. PI-3-P, PI-3,4-P2, and PIP3 are the products of phosphorylation of PI, PI-4-P, and PI-4,5-P2, respectively, by phosphoinositide 3-kinase activities that are associated with certain protein-tyrosine kinases. Although these new phospholipids have been found in intact cells, PI-3,4-P2 and PIP3 appear only after stimulation of quiescent cells with growth factors such as platelet-derived growth factor (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175) and after transformation by certain oncoproteins (L. A. Serunian, K. R. Auger, T. M. Roberts, and L. C. Cantley, manuscript in preparation). Mixtures of [3H]PI-4-P plus [32P]PI-3-P or [3H]PI-4,5-P2 plus [32P]PI-3,4-P2 or PIP3 alone were used as substrates for PLCs in vitro. After incubation with enzyme followed by extraction with chloroform/methanol/HCl, the ratio of 3H/32P in the aqueous layer revealed the selective hydrolysis of PI-4-P and PI-4,5-P2 over PI-3-P and PI-3,4-P2. High performance liquid chromatography analysis of the aqueous layer containing reaction products confirmed that only PI-4-P and PI-4,5-P2, were hydrolyzed to inositol 1,4-P2 and inositol 1,4,5-P3, respectively. These findings suggest that the turnover of PI-3-P, PI-3,4-P2, and PIP3 occurs independently of the turnover of PI-4-P and PI-4,5-P2.
研究了三种纯型牛脑磷脂酶C（PLC）和一种纯大鼠肝PLC将最近发现的磷脂酰肌醇3-磷酸（PI-3-P）、一种假定的磷脂酰肌醇3,4-二磷酸（PI-3,4-P2）和磷脂酰肌醇三磷酸（PIP3）用作底物的能力。PI-3-P、PI-3,4-P2和PIP3分别是PI、PI-4-P和PI-4,5-P2通过与某些蛋白酪氨酸激酶相关的磷酸肌醇3-激酶活性磷酸化的产物。尽管这些新的磷脂已在完整细胞中被发现，但PI-3,4-P2和PIP3仅在静止细胞被生长因子如血小板衍生生长因子刺激后（奥格，K.R.，塞鲁尼亚，L.A.，索尔托夫，S.P.，利比，P.，和坎特利，L.C.（1989年）《细胞》57卷，167 - 175页）以及在被某些癌蛋白转化后（L.A.塞鲁尼亚，K.R.奥格，T.M.罗伯茨，和L.C.坎特利，正在准备的手稿）才出现。[3H]PI-4-P加[32P]PI-3-P或[3H]PI-4,5-P2加[32P]PI-3,4-P2或单独PIP3的混合物在体外用作PLC的底物。在用酶孵育后接着用氯仿/甲醇/盐酸萃取，水层中3H/32P的比值显示PI-4-P和PI-4,5-P2相对于PI-3-P和PI-3,4-P2被选择性水解。对含有反应产物的水层进行高效液相色谱分析证实，只有PI-4-P和PI-4,5-P2分别被水解为肌醇1,4-P2和肌醇1,4,5-P3。这些发现表明PI-3-P、PI-3,4-P2和PIP3的周转独立于PI-4-P和PI-4,5-P2的周转。
