Korgaonkar Chandrashekhar, Zhao Lili, Modestou Modestos, Quelle Dawn E
Department of Pharmacology. Molecular Biology Graduate Program, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Mol Cell Biol. 2002 Jan;22(1):196-206. doi: 10.1128/MCB.22.1.196-206.2002.
It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal (31)RRPR(34) did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.
一般认为，ARF肿瘤抑制因子通过将p53拮抗剂Mdm2隔离在核仁中来诱导p53依赖的生长停滞。先前对小鼠ARF的诱变研究表明，第1至14位和第26至37位残基对Mdm2结合至关重要，而后一个结构域也决定了ARF的核仁定位。我们发现，小鼠ARF的第6至10位和第21至25位残基是ARF诱导生长停滞所必需的，而第1至5位和第29至34位残基则是可有可无的。删除假定的核仁定位信号（31）RRPR（34）并不妨碍核仁定位。令人惊讶的是，与野生型ARF不同，生长抑制突变体D1-5和D29-34未能稳定p53，但在报告基因检测中诱导了其转录激活。这表明p53稳定化对于ARF介导的p53激活并非必不可少。与野生型ARF一样，这两个突变体也表现出p53非依赖性功能，因为它们能够使p53/Mdm2缺失细胞停滞生长。值得注意的是，其他缺乏保守的第6至10位或第21至25位残基的突变体，尽管具有核仁定位和导入Mdm2的能力，但仍无法抑制p53阳性细胞的生长。这些观察结果与野生型ARF在某些细胞中阻止生长而不将内源性Mdm2重新定位到核仁的能力形成明显对比。总之，这些数据表明ARF活性与Mdm2重新定位之间缺乏相关性，这表明ARF功能需要除Mdm2导入之外的其他事件。
