Wesierska-Gadek Józefa, Wojciechowski Jacek, Schmid Gerald
Cell Cycle Regulation Unit, Institute of Cancer Research, University of Vienna, Vienna, Austria.
J Cell Biochem. 2003 Aug 15;89(6):1260-84. doi: 10.1002/jcb.10569.
We recently characterized the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1, whereas the amino-terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP-1 and overexpression of PARP-1. Our results unequivocally show that the presence of PARP-1 regulates the basal expression of wt p53 in unstressed cells.
我们最近对聚（ADP - 核糖）聚合酶 -1（PARP -1）与肿瘤抑制基因p53的产物之间的相互作用进行了表征。我们研究了人PARP -1和人野生型（wt）p53的哪些结构域参与了这种蛋白质 - 蛋白质相互作用。我们构建了杆状病毒表达载体，编码这两种蛋白质的全长或不同的功能结构域。全长PARP -1与全长wt p53蛋白或其不同的截短片段在昆虫细胞中同时共表达，反之亦然。对Sf9细胞裂解物进行相互免疫沉淀显示，p53的中央和羧基末端片段足以与PARP -1结合，而含有反式激活功能结构域的氨基末端部分则是不必要的。另一方面，PARP -1的氨基末端和中央片段对于与p53蛋白形成复合物是必需的。由于p53蛋白的最重要特征受磷酸化调节，我们探讨了其磷酸化对于这两种蛋白质之间结合是否必不可少的问题。杆状病毒表达的wt p53进行了翻译后修饰。在对杆状病毒表达的wt p53蛋白进行二维分离后，通过免疫印迹解析出至少六种不同的p53异构体。使用特异性磷酸丝氨酸抗体，我们鉴定出杆状病毒表达的p53蛋白在五个不同位点的磷酸化。为了确定p53磷酸化的作用，使用未处理和去磷酸化的p53蛋白进行了下拉试验。去磷酸化的p53无法结合PARP -1，表明这两种蛋白质之间的复合物形成受p53磷酸化调节。在未受应激的细胞中观察到p53在Ser392处有明显的磷酸化，这表明p53磷酸化的羧基末端部分与PARP -1形成复合物，导致核输出信号（NES）被掩盖，从而阻止其输出。在两种不同条件下研究了这两种蛋白质之间相互作用的功能意义：PARP -1失活和PARP -1过表达。我们的结果明确表明，PARP -1的存在调节未受应激细胞中wt p53的基础表达。
