Chen C H, Wang H C, Chuang N N
Division of Biochemistry and Molecular Sciences, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan.
J Exp Zool. 2000 Nov 1;287(6):432-9. doi: 10.1002/1097-010x(20001101)287:6<432::aid-jez4>3.0.co;2-q.
BALB/3T3 cells were transformed by transfection with DNA encoding the mutated ras(Q(61)K) from shrimp Penaeus japonicus (Huang and Chuang. 1999. J Exp Zool 283:510-521). The caveolin-1 in the membrane fraction extractable with 2% octyl glucoside was significant reduced, compared to untransformed cells. To understand this in more detail, the interaction of S-Ras with caveolin was investigated using caveolin-1 purified from rat lungs. The purified caveolin-1 binds c-Src, suppressing its autophosphorylation. It also binds to phosphatidylserine-cholesterol liposomes. These reconstituted caveolin-phosphatidylserine-cholesterol vesicles, which act as a model of caveolae, recruit both bacterially expressed S-Ras and rat K(B)-Ras proteins, as demonstrated on western blots with antibodies against caveolin-1 and Ras. Caveolin-1 suppressed the intrinsic GTPase activity of S-Ras, sustaining it in the active GTP bound form. By contrast, caveolin-1 enhanced the intrinsic GTPase activity of K(B)-Ras, to convert it into the inactive GDP-bound form. These events suggest that caveolin may act as a docking site for Ras proteins and may be able to either maintain or alter their activity state. These events may be associated with the ability of S-ras(Q(61)K) to successfully transform cells.
用编码日本对虾突变型ras（Q(61)K）的DNA转染BALB/3T3细胞（Huang和Chuang，1999年，《实验动物学杂志》283:510 - 521）。与未转化细胞相比，用2%辛基葡糖苷可提取的膜组分中的小窝蛋白-1显著减少。为了更详细地了解这一点，使用从大鼠肺中纯化的小窝蛋白-1研究了S-Ras与小窝蛋白的相互作用。纯化的小窝蛋白-1结合c-Src，抑制其自身磷酸化。它还与磷脂酰丝氨酸-胆固醇脂质体结合。这些重构的小窝蛋白-磷脂酰丝氨酸-胆固醇囊泡作为小窝的模型，如用抗小窝蛋白-1和Ras抗体进行的蛋白质印迹所示，募集细菌表达的S-Ras和大鼠K(B)-Ras蛋白。小窝蛋白-1抑制S-Ras的内在GTP酶活性，使其维持在活性GTP结合形式。相比之下，小窝蛋白-1增强K(B)-Ras的内在GTP酶活性，将其转化为无活性的GDP结合形式。这些事件表明，小窝蛋白可能作为Ras蛋白的停靠位点，并且可能能够维持或改变它们的活性状态。这些事件可能与S-ras(Q(61)K)成功转化细胞的能力有关。
