Helt A M, Galloway D A
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
J Virol. 2001 Aug;75(15):6737-47. doi: 10.1128/JVI.75.15.6737-6747.2001.
The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of E7 has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor repressor (Rb) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for E7 to induce proliferation. We tested the hypothesis that binding to Rb is not sufficient because conserved region 1 and/or the C terminus are required for E7 to functionally inactivate Rb and thus induce proliferation. One mechanism proposed for how E7 inactivates Rb is by blocking Rb-E2F binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro. While all full-length E7 proteins with mutations outside of the LxCxE motif inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, demonstrating that blocking Rb-E2F binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserved region 1 or the LxCxE motif prevented E7 from reducing the half-life of Rb. Though no specific C-terminal residues of E7 were essential for destabilizing Rb, a novel class of mutations that uncouple the destabilization of Rb from the deregulation of keratinocyte proliferation was discovered. Destabilization of Rb correlated with the abrogation of Rb-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 required for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family members is not sufficient to deregulate keratinocyte cell cycle control.
人乳头瘤病毒16型的E7癌蛋白在存在抗增殖信号的情况下促进细胞增殖。E7的诱变表明，这种活性需要三个区域，即保守区域1和2以及一个C端锌指。通过保守区域2中的LxCxE基序与视网膜母细胞瘤肿瘤抑制因子（Rb）结合对于E7诱导增殖是必要的，但并不充分。我们检验了以下假设：与Rb结合不充分是因为保守区域1和/或C端对于E7在功能上使Rb失活从而诱导增殖是必需的。提出的E7使Rb失活的一种机制是通过阻断Rb-E2F结合。保守区域1或C端与LxCxE基序结合，对于E7在体外阻断Rb-E2F结合是必需的。虽然所有在LxCxE基序之外有突变的全长E7蛋白都抑制Rb-E2F结合，但有些未能消除细胞周期停滞，这表明阻断Rb-E2F结合不足以消除抗增殖信号。提出的E7使Rb失活的另一种机制是通过促进Rb蛋白的不稳定。保守区域1或LxCxE基序中的突变阻止了E7降低Rb的半衰期。虽然E7的任何特定C端残基对于使Rb不稳定都不是必需的，但发现了一类新的突变，这些突变使Rb的不稳定与角质形成细胞增殖的失调脱钩。Rb的不稳定与Rb诱导的静止状态的消除相关，但不足以克服DNA损伤诱导的细胞周期停滞或增加角质形成细胞寿命。最后，使Rb不稳定所需的E7相同区域对于降低p107和p130水平也是必需的。总之，这些结果表明，使所有三个Rb家族成员失活不足以解除对角质形成细胞周期的控制。
