Buschmann T, Minamoto T, Wagle N, Fuchs S Y, Adler V, Mai M, Ronai Z
Ruttenberg Cancer Center, New York, NY, 10029, USA.
J Mol Biol. 2000 Jan 28;295(4):1009-21. doi: 10.1006/jmbi.1999.3387.
Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.
Mdm2和JNK作为靶向野生型p53降解的蛋白质被鉴定出来，这促使我们研究它们对突变型p53的影响，突变型p53具有延长的半衰期。在研究的五种与靶向分子相关的突变型p53形式中，有两种不再与Mdm2和JNK结合。三种表现出高表达水平的突变形式与Mdm2和JNK结合的亲和力较低，这与它们对p14(ARF)的亲和力更高相一致，p14(ARF)是稳定p53的分子之一。体外监测突变型p53的稳定性证实，虽然某些形式的突变型p53不再受JNK或Mdm2的影响，但其他形式的突变型p53会被JNK/Mdm2靶向降解，尽管与野生型p53相比效率较低。野生型p53在肿瘤细胞中的表达显示出较短的半衰期，这表明靶向分子具有功能。在p53缺失的细胞中强制表达突变型p53证实了与JNK/Mdm2的结合模式和延长的半衰期，这与在肿瘤细胞中发现的情况一致。在肿瘤细胞（确实表达内源性功能性Mdm2）或p53缺失的细胞中过表达Mdm2会降低突变型p53的稳定性，这表明尽管Mdm2/JNK有表达，但它们（数量/亲和力）不足以靶向突变型p53降解。基于体外和体内分析，我们得出结论，突变型p53延长的半衰期取决于突变的性质，突变要么改变了与靶向分子的结合、p53与靶向/稳定分子之间的比例，要么改变了靶向效率。
