Bech-Otschir D, Kraft R, Huang X, Henklein P, Kapelari B, Pollmann C, Dubiel W
Division of Molecular Biology, Department of Surgery and Institute of Biochemistry, Medical Faculty Charité, Humboldt University, Monbijoustrasse 2, 10117 Berlin, Germany.
EMBO J. 2001 Apr 2;20(7):1630-9. doi: 10.1093/emboj/20.7.1630.
In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.
在高等真核细胞中，p53蛋白通过由Mdm2或人乳头瘤病毒E6蛋白介导的泛素-26S蛋白酶体系统降解。在此我们表明，COP9信号体（CSN）特异性磷酸化将人p53靶向泛素-26S蛋白酶体依赖性降解。通过电子显微镜观察，p53与天然CSN复合物高亲和力结合。如远缘印迹和下拉分析所示，p53通过其N末端与CSN亚基5/Jab1相互作用。CSN特异性磷酸化位点定位于p53的核心结构域，包括Thr155。磷酸化肽Deltap53(145-164)特异性抑制CSN介导的磷酸化和p53降解。姜黄素，一种CSN激酶抑制剂，可阻断网织红细胞裂解物中E6依赖性p53降解。将Thr155突变为缬氨酸足以稳定p53以抵抗网织红细胞裂解物中E6依赖性降解，并减少与Mdm2的结合。p53T155V突变体在HeLa和HL 60细胞中均积累，并呈现突变体（PAb 240+）构象。它诱导细胞周期蛋白依赖性抑制剂p21。在HeLa和MCF-7细胞中，姜黄素或Deltap53(145-164)对CSN激酶的抑制导致内源性p53的积累。
