REGULATION OF P53 STABILITY BY SER315 PHOSPHORYLATION

我们最近已经表明，RNA聚合酶II的阻断足以引起哺乳动物细胞中p53的诱导，并且可能是在各种细胞应激后稳定p53的常见机制。 已知细胞蛋白MDM 2和JNK结合p53并刺激p53降解。 最近的研究表明， 并且p53的C-末端结构域是有效降解p53所必需的。 我们已经获得的初步结果表明，p53的ser 315是重要的调节p53的稳定性。 首先，ser 315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ser 315激酶Cdc 2和Cdk 2，引起细胞中p53的稳定。 第三，我们已经发现了一种磷酸酶，hCDC 14，它结合p53，并在体外特异性地去磷酸化p53的ser 315位点。 在这个建议中，我们将进一步探讨ser 315磷酸化在调节p53降解中的潜在作用，以及RNA聚合酶II的阻断如何减弱ser 315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来研究p53的体外降解。 重组野生型p53将在ser 315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ser 315的磷酸化增强了体外降解。 我们还将评估在315位点突变或含有C-或N-末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激活激酶（CAK）、Cdk 2和Cdc 2在调节p53稳定性中的作用。CAK是RNA聚合酶II全酶的组分，因此，它可以是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Cdc 2和Cdk 2，这反过来又会磷酸化p53的ser 315。在RNA聚合酶II延长的抑制之后，CAK的活性减弱，导致通过消除p53的ser 315磷酸化而增加的p53的稳定性。 我们将使用显性负性CAK，Cdc 2和Cdk 2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，我们将探索新的人p53磷酸酶hCDC 14在调节p53稳定性中的作用。 初步研究表明，hCDC 14结合p53并特异性地使p53的ser 315去磷酸化。 我们将在MCF 7细胞中表达野生型或突变型CDC 14，并研究它对p53的基础和诱导水平的影响。

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.