Andresson T, Sparkowski J, Goldstein D J, Schlegel R
Department of Pathology, Georgetown University Medical School, Washington, D.C. 20007.
J Biol Chem. 1995 Mar 24;270(12):6830-7. doi: 10.1074/jbc.270.12.6830.
The 16K subunit of the vacuolar H(+)-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R. (1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.
液泡H(+)-ATP酶的16K亚基特异性结合牛乳头瘤病毒（BPV）和人乳头瘤病毒（HPV）的E5癌蛋白，有人认为这种相互作用可能与细胞转化有关（戈尔茨坦，D.J.，和施莱格尔，R.（1990）《欧洲分子生物学组织杂志》9，137 - 146；戈尔茨坦，D.J.，芬博，M.E.，安德松，T.，麦克林，P.，史密斯，K.，巴布，V.J.，和施莱格尔，R.（1991）《自然》352，347 - 349；康拉德，M.，巴布，V.J.，和施莱格尔，R.（1993）《病毒学杂志》67，6170 - 6178；戈尔茨坦，D.J.，远山，R.，施莱格尔，R.，和达尔，R.（1992）《病毒学》190，889 - 893）。我们在16K蛋白内产生突变，以确定BPV - 1 E5的结合结构域，并表征16K在细胞转化中的作用。16K主要由4个跨膜（TM）结构域组成。我们发现TM4结构域内的突变严重抑制E5结合。更具体地说，TM4内的谷氨酸143转换为精氨酸会严重降低16K/E5结合，这表明带电相互作用促进了有效结合。通过证明E5中的互补电荷突变可以恢复与有缺陷的16K精氨酸突变体的结合，证实了这一假设；E5谷氨酰胺17转换为谷氨酸或天冬氨酸增强了与16K精氨酸突变体的相互作用。令人惊讶的是，TM4中的突变体不仅与野生型E5结合不佳，而且被转化为癌蛋白并诱导NIH 3T3细胞的锚定非依赖性生长。这些数据确定了16K TM4结构域中的谷氨酸143和E5中的谷氨酰胺17是E5/16K结合的重要贡献者，并表明16K蛋白在细胞增殖调节中起作用。
