Rai S S, Wolff J
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0830, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4253-7. doi: 10.1073/pnas.95.8.4253.
Oligoanions such as sodium triphosphate or GTP prevent and/or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastine-induced tubulin polymerization and abolishes the anion effect. Only the beta-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the alpha-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 microM block cleavage of the alpha-tubulin C terminus in the polymer, whereas cleavage of the beta-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pH-dependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastine-induced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the beta-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.
诸如三磷酸钠或鸟苷三磷酸（GTP）之类的寡聚阴离子可阻止和/或逆转长春花碱诱导的微管蛋白聚合。我们现在表明，微管蛋白富含谷氨酸的极端C末端阴离子同样参与长春花碱作用的调节。用枯草杆菌蛋白酶进行有限的蛋白水解切割C末端可增强长春花碱诱导的微管蛋白聚合，并消除阴离子效应。只需去除β-微管蛋白的C末端即可实现这些变化，而随后切割α-微管蛋白的C末端几乎没有额外效果。实际上，长春花碱浓度>20 microM可阻止聚合物中α-微管蛋白C末端的切割，而在所用时间内β-微管蛋白C末端的切割不受阻碍地进行。长春花碱对微管蛋白聚合的影响在pH 6.5至7.5之间也高度依赖于pH；枯草杆菌蛋白酶处理后这种依赖性不太明显，但并非不存在。我们提出了一个工作模型，其中极端C末端附近的阴离子结构域必须与阳离子结构域相互作用，以使长春花碱促进聚合。外源性阴离子和极端C末端阴离子都与近端阴离子结构域竞争阳离子结构域，以防止长春花碱诱导的聚合。我们得出结论，长春花碱诱导的微管蛋白聚合的静电调节主要存在于β-微管蛋白的C末端，但微管蛋白分子中近端的额外调节也起作用。
