Sasaki Mark, Nie Linghu, Maki Carl G
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637, USA.
J Biol Chem. 2007 May 11;282(19):14626-34. doi: 10.1074/jbc.M610514200. Epub 2007 Mar 15.
p53 protein conformation is an important determinant of its localization and activity. Changes in p53 conformation can be monitored by reactivity with wild-type conformation-specific (pAb-1620) or mutant conformation-specific (pAb-240) p53 antibodies. Wild-type p53 accumulated in a mutant (pAb-240 reactive) form when its proteasome-dependent degradation was blocked during recovery from stress treatment and in cells co-expressing p53 and MDM2. This suggests that conformational change precedes wild-type p53 degradation by the proteasome. MDM2 binding to the p53 N terminus could induce a conformational change in wild-type p53. Interestingly, this conformational change was opposed by heat-shock protein 90 and did not require the MDM2 RING-finger domain and p53 ubiquitination. Finally, ubiquitinated p53 accumulated in a pAb-240 reactive form when p53 degradation was blocked by proteasome inhibition, and a p53-ubiquitin fusion protein displayed a mutant-only conformation in MDM2-null cells. These results support a model in which MDM2 binding induces a conformational change that is opposed by heat-shock protein 90 and precedes p53 ubiquitination. The covalent attachment of ubiquitin may "lock" p53 in a mutant conformation in the absence of MDM2-binding and prior to its degradation by the proteasome.
p53蛋白构象是其定位和活性的重要决定因素。p53构象的变化可通过与野生型构象特异性（pAb - 1620）或突变型构象特异性（pAb - 240）p53抗体的反应性来监测。当野生型p53在从应激处理恢复过程中其蛋白酶体依赖性降解被阻断时，以及在共表达p53和MDM2的细胞中，野生型p53以突变型（pAb - 240反应性）形式积累。这表明构象变化先于蛋白酶体对野生型p53的降解。MDM2与p53 N末端的结合可诱导野生型p53的构象变化。有趣的是，这种构象变化受到热休克蛋白90的抑制，并且不需要MDM2的环指结构域和p53的泛素化。最后，当蛋白酶体抑制阻断p53降解时，泛素化的p53以pAb - 240反应性形式积累，并且p53 - 泛素融合蛋白在缺失MDM2的细胞中呈现仅突变型的构象。这些结果支持了一个模型，即MDM2结合诱导一种构象变化，这种变化受到热休克蛋白90的抑制并且先于p53的泛素化。在没有MDM2结合且在其被蛋白酶体降解之前，泛素的共价连接可能将p53“锁定”在突变构象中。
