SPOP磷酸化及蛋白相互作用在前列腺癌DNA损伤应答中的作用

Speckle-type POZ protein (SPOP) is an adaptor of the cullin 3 (CUL3)-based ubiquitin ligases responsible for the degradation of the oncoprotein SRC-3. Recent studies have found that SPOP is the most frequent mutated gene in prostate cancer with chromosomal aberrations, indicating a role of SPOP in maintaining genome stability. We recently reported that SPOP was a component of the DNA damage response (DDR), a major mechanism for the maintenance of genome stability in human cells. Our preliminary studies demonstrate a link between ATM and SPOP in prostate cancer cells and we have identified a list of SPOP interaction proteins in response to DNA damage. Based on these results, we aim to dissect SPOP signaling pathways in the DDR in prostate cancer cells and elucidate the functional significance of SPOP in the process. First we will investigate the functional significance of ATM-mediated SPOP phosphorylation. Our preliminary data demonstrate that ATM interacts with SPOP and phosphorylates it on Serine 119 after DNA damage. Since Serine 119 is a frequent mutated site in prostate cancer and that we have observed S199N mutation resulting in a suboptimal DDR, we hypothesize that ATM-mediated SPOP phosphorylation is critical for activation of the DDR. To test this, we will investigate the functional significance of ATM-mediated SPOP Serine 119 phosphorylation and identify downstream targets of SPOP phosphorylation. Second we will determine the functional role of altered SPOP interactions with proteins in the DDR. By mass spectrometry we identified a list of proteins that displayed alterations in association with SPOP in response to DNA damage. We will map SPOP interaction domains on SMC1, UBR5, MCM5 and XRCC6 and study functional significance of altered SPOP interaction with these proteins. Completion of these studies will reveal the molecular mechanism of SPOP in the DDR as well as insights into processes involved in prostate cancer initiation and progression.

SPOP是前列腺癌中最常见的体细胞突变基因，其突变导致基因组不稳定性。SPOP是CUL3泛素化酶适配器，参与癌蛋白SRC-3的降解。我们发现SPOP是DNA损伤应答（DDR）的关键蛋白，而DDR是防止基因组不稳定性的主要机制。前期研究表明在前列腺癌细胞中ATM蛋白激酶可磷酸化SPOP，同时我们通过质谱法鉴别出SPOP结合蛋白。基于这些结果，我们将深入研究SPOP通路在前列腺癌DDR中的功能。首先将研究ATM介导的SPOP丝氨酸119磷酸化的功能，并确定磷酸化后的下游蛋白。其次将研究SPOP结合蛋白在前列腺癌DDR的功能。DNA损伤后出现改变的SPOP结合蛋白包括SMC1、UBR5、MCM5和XRCC6。我们将找出这些蛋白与SPOP的结合域并研究其在S期检测点及非同源末端连接DNA修复的功能。完成这些研究将揭示SPOP在DDR的功能并深入了解前列腺癌的发生发展机制。

共济失调毛细血管扩张突变（ATM）激酶可以磷酸化多种靶点以促进DNA损伤反应。然而，很少发现这些磷酸化位点在癌症中发生突变。我们报告了前列腺癌相关的突变SPOP S119N（119位丝氨酸突变成天冬氨酸）导致延长DNA损伤修复时间并促进放射敏感性。我们证明了丝氨酸119位点是SPOP与ATM相互作用所必需的，并且证明ATM磷酸化SPOP的S119位点。我们发现ATM介导的SPOP磷酸化对放射敏感性、细胞周期检查点和DNA损伤修复有重要作用。此外，我们还鉴定了SPOP蛋白的相互作用蛋白复合物MCM5-MCM3，该复合物对DNA损伤应答有重要作用。其中，SPOP丝氨酸119磷酸化是SPOP-MCM3解离所必需的，并且SPOP磷酸化抑制MCM3泛素化降解。在我们研究中，我们发现了一个新的由ATM磷酸化SPOP介导的DNA损伤修复途径。此外，携带SPOP S119突变的前列腺癌患者可能对放射治疗更加敏感。这也是我们临床上发现的与ATM磷酸化相关的第一个病理生理性突变。