REGULATION OF P53 STABILITY BY SER315 PHOSPHORYLATION

SER315 磷酸化对 P53 稳定性的调节

• 批准号: 6377345
• 负责人: MATS LJUNGMAN
• 金额: $ 20.87万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377345
• 关键词: CD14 molecule; DNA directed RNA polymerase; MCF7 cell; cell cycle proteins; cell free system; cyclin dependent kinase; enzyme activity; enzyme inhibitors; molecular site; monoclonal antibody; mutant; p53 gene /protein; phosphorylation; protein degradation; protein kinase C; protein purification; protein structure function; protein tyrosine phosphatase; tetracyclines; transfection /expression vector; ultraviolet radiation; western blottings

We have recently shown that blockage of RNA polymerase II is sufficient to cause the induction of p53 in mammalian cells and may be a common mechanism by which p53 is stabilized following various cellular stresses. The cellular proteins MDM2 and JNK are known to bind p53 and stimulate p53 degradation. Recent studies show that both the and C-terminal domains of p53 are required for efficient degradation of p53. We have obtained preliminary results suggesting that ser315 of p53 is important in the regulation of p53 stability. Firstly, phosphorylation of ser315 enhanced the degradation of p53 in vitro. Secondly, inhibition of the ser315 kinases Cdc2 and Cdk2 via inhibition of the CDK-activating kinase (CAK), caused a stabilization of p53 in cells. Thirdly, we have found a phosphatase, hCDC14, which binds p53 and specifically dephosphorylates the ser315 site of p53 in vitro. In this proposal, we will further explore the potential role of ser315 phosphorylation in the regulation of p53 degradation and how blockage of RNA polymerase II may attenuate ser315 phosphorylation. In specific aim number 1, we will utilize a cell-free extract system established in the lab to study the degradation of p53 in vitro. Recombinant wild-type p53 will be phosphorylated specifically on ser315 and its stability in cell extracts will be assessed. Preliminary results suggest that phosphorylation of ser315 enhances degradation in vitro. We will also assess the stability of recombinant p53 mutated at the 315 site, or containing deletions of the C- or N-terminal domains. In specific aim number 2, we will investigate the role of the Cdk- activating kinase (CAK), Cdk2 and Cdc2 in the regulation of p53 stability. CAK is a component of the RNA polymerase II holoenzyme, and as such, it may be a sensor for RNA polymerase II elongation. We hypothesize that under normal conditions CAK will activate Cdc2 and Cdk2 which in turn will phosphorylate p53 on ser315. Following inhibition of RNA polymerase II elongation, the activity of CAK is attenuated leading to increased stability of p53 by abrogation of ser315 phosphorylation of p53. We will use dominant negative CAK, Cdc2 and Cdk2 to specifically inhibit these enzymes in cells and study whether inhibition of these kinases causes an increased stability of p53. Finally, in specific aim number 3, we will explore the role of the novel human p53 phosphatase, hCDC14, in the regulation of p53 stability. Preliminary studies have shown that hCDC14 binds p53 and specifically dephosphorylates ser315 of p53. We will express wild-type or mutant CDC14 in MCF7 cells and study what effect it will have on basal and induced levels of p53.

我们最近的研究表明，RNA聚合酶II的阻断足以导致哺乳动物细胞中p53的诱导，并且可能是p53在各种细胞应激下稳定的共同机制。已知细胞蛋白MDM2和JNK与p53结合并刺激p53降解。最近的研究表明，p53的c端结构域和c端结构域都是有效降解p53所必需的。我们已经获得了初步结果，提示p53的ser315在p53的稳定性调控中起重要作用。首先，ser315的磷酸化增强了p53在体外的降解。其次，通过抑制cdk活化激酶（CAK）来抑制ser315激酶Cdc2和Cdk2，导致细胞中p53的稳定。第三，我们发现了一种磷酸酶hCDC14，它可以结合p53，并在体外特异性地使p53的ser315位点去磷酸化。在本提案中，我们将进一步探索ser315磷酸化在p53降解调控中的潜在作用，以及RNA聚合酶II阻断如何减弱ser315磷酸化。在具体目标1中，我们将利用实验室建立的无细胞提取系统在体外研究p53的降解。重组野生型p53将在ser315上特异性磷酸化，并评估其在细胞提取物中的稳定性。初步结果表明，ser315的磷酸化增强了体外降解。我们还将评估315位点突变或含有C端或n端结构域缺失的重组p53的稳定性。在具体目标2中，我们将研究Cdk活化激酶（CAK）、Cdk2和Cdc2在p53稳定性调控中的作用。CAK是RNA聚合酶II全酶的组成部分，因此，它可能是RNA聚合酶II延伸的传感器。我们假设在正常情况下，CAK会激活Cdc2和Cdk2，进而使p53的ser315磷酸化。在抑制RNA聚合酶II延伸后，CAK的活性减弱，通过取消p53的ser315磷酸化，从而增加了p53的稳定性。我们将使用显性阴性CAK， Cdc2和Cdk2特异性抑制细胞中的这些酶，并研究这些激酶的抑制是否会导致p53的稳定性增加。最后，在具体目标3中，我们将探讨新型人类p53磷酸酶hCDC14在p53稳定性调控中的作用。初步研究表明，hCDC14结合p53并特异性地使p53的ser315去磷酸化。我们将在MCF7细胞中表达野生型或突变型CDC14，并研究它对p53基础和诱导水平的影响。