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

Wortmannin是一种有效的磷酸肌苷3-激酶抑制剂，利用其衍生物研究了该酶的功能。利用放射性标记的wortmannin衍生物分离了一个新的磷酸苷3-激酶催化亚基。该亚基的初级结构与磷酸肌肽3-激酶的β亚型非常相似。新亚型和β亚型被发现由酪氨酸磷酸化肽和gypp结合蛋白的β / γ亚基协同激活。α和γ亚型没有表现出类似的性质。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发挥作用。协同作用还观察到蛋白激酶B的细胞活性，蛋白激酶B是磷酸肌苷3激酶的下游靶点。因此，脂质激酶亚型之间的功能差异被新提出。Wortmannin被发现能抑制高转移肝癌细胞的体外粘附和运动。通过向细胞中引入磷酸肌苷3-激酶的显性阴性突变体，可以再现这种效果。因此，该酶可能调节肝癌细胞的转移活性。发现了缺乏与磷酸肌肽3-激酶共价结合能力的worwormannin衍生物。这些化合物有望用作亲和层析的配体。

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.