Use of Wortmannin and its Derivatives for the Study on Phosphoinositide 3-Kinase

Wortmannin是一种有效的磷酸肌醇3-激酶抑制剂，其衍生物被用于研究该酶的功能.利用放射性标记的渥曼青霉素衍生物分离了磷酸肌醇3-激酶的一个新的催化亚基。该亚基的一级结构与磷酸肌醇3-激酶的β亚型非常相似。新的亚型和β-亚型被发现是协同激活的酪氨酸磷酸化肽和β/γ亚基的GYP结合蛋白。α和γ亚型没有表现出相似的性质.上述体外协同作用在包括大鼠脂肪细胞在内的完整细胞系统中起作用。还观察到协同作用作为磷酸肌醇3-激酶的下游靶蛋白激酶B的细胞活性。因此，脂质激酶的亚型之间的功能差异是新的. Wortmannin被发现在体外抑制高转移性肝癌细胞的粘附和运动。通过将磷酸肌醇3-激酶的显性负突变体引入细胞来再现该效果。提示该酶可能参与调节肝癌细胞的转移活性.渥曼青霉素衍生物不能与磷脂酰肌醇3-激酶共价结合，有望作为亲和层析的配体。

Wortmannin, a potent inhibitor of phosphoinositide 3-kinase, and its derivatives were utilized to investigate the functions of the enzyme.1. A novel catalytic subunit of phosphoinoside 3-kinase was isolated by use of a radiolabeled derivative of wortmannin. This subunit had a primary structure very similar to the beta-subtype of phosphoinositide 3-kinase. The novel subtype and the beta-subtype were found to be synergistically activated by a tyrosine-phosphorlated peptide and beta/gamma subunits of GYP-binding proteins. The alpha and gamma subtypes did not show the similar property.2. The above synergism in vitro was operating in intact cell systems including rat adipocytes. The synergism was also observed as the cellular activity of protein kinase B, a downstream target of phosphoinositide 3-kinase. Thus a functional difference between the subtypes of the lipid kinase was newly suggested.3. Wortmannin was found to inhibit the in vitro adhesion and motility of highly metastatic hepatoma cells. The effect was reproduced by introducing a dominant-negative mutant of phosphoinositide 3-kinase to the cells. Thus the enzyme was suggested to regulate the metastatic activity of hepatoma cells.4. Derivatives of wortmannin lacking an ability to bind covalently to phosphoinositide 3-kinase were found. The compounds are expected to be utilized as ligands for affinity chromatography.