Investigation of Cdc34 Molecular Recognition by its Enzyme Partners, Uba1 and SCF Ligases.

项目摘要 Cdc34是细胞周期进程中G1至S期检查点的关键调节因子1 - 3。它作为E2泛素 首先通过E1酶Uba1与泛素结合，然后与SCF一起发挥功能的结合酶 E3酶家族用泛素修饰其他细胞周期蛋白，以通过靶向降解 蛋白酶体4，5。自从发现Cdc34以来，Cdc34失调已经涉及几种类型的肿瘤。 癌症6 - 8 Cdc34的许多下游靶点是细胞周期蛋白依赖性激酶抑制剂，如p27和p40， 对癌症进展至关重要9 - 14。这些发现共同引发了对Cdc 34的调查 作为癌症治疗的靶点，但更直接的方法，如特异性靶向Cdc34 需要与其酶伙伴的相互作用。然而，对分子结构的详细了解 Cdc34通过其E1配偶体、Uba1和E3连接酶配偶体的识别是不可用的。此外，监管 cdc34还没有被完全理解。以前的研究表明，Cdc34的正调控， 然而，我们有令人兴奋的初步数据表明，丝氨酸残基的磷酸化 10作为Cdc34活性的负调节剂，这在文献中得到磷酸化蛋白质组质量的支持。 在体内鉴定该残留物的光谱分析筛选15 - 18。此外，根据结构预测，我们认为 Cdc34表面的Ser10残基与E1和E3酶相互作用。因此，我们的目标是确定 Cdc34通过其酶伴侣分子识别中的关键残基，并假设 Ser10的磷酸化将通过空间冲突负调节这种分子识别， 静电排斥 我们的假设将通过以下具体目标来解决。目标1将确定关键残留物， Cdc34与E1和E3酶伴侣的相互作用。我们将使用X射线晶体学， 生物化学和遗传技术来实现这一目标。目的2将阐明Cdc34的调节作用 丝氨酸10残基的磷酸化。我们将在体内确认Ser10的磷酸化，并检查其作用 S10D拟磷酸化突变对Cdc34分子识别的影响 生物化学测定和体内使用酵母遗传学。

PROJECT SUMMARY Cdc34 is a key regulator in cell cycle progression at the G1 to S phase checkpoint1-3. It acts as an E2 ubiquitin conjugating enzyme that is first charged with ubiquitin by E1 enzyme, Uba1, and then functions with the SCF family of E3 enzymes to modify other cell cycle proteins with ubiquitin for targeted degradation by the proteasome4,5. In years since its discovery, Cdc34 dysregulation has been implicated in several types of cancer6-8. Many downstream targets of Cdc34 are cyclin-dependent kinase inhibitors such as p27 and p40 that are critically important for cancer progression9-14. Together, these findings have led to investigations of Cdc34 as a target for cancer therapeutics but a more directed approach, such as specifically targeting Cdc34 interactions with its enzyme partners is needed. However, a detailed structural understanding for molecular recognition of Cdc34 by its E1 partner, Uba1, and E3 ligase partners is not available. Further, regulation of Cdc34 is not completely understood. Previous studies have shown positive regulation of Cdc34 by phosphorylation; however, we have exciting preliminary data that implicates phosphorylation of residue serine 10 as a negative regulator of Cdc34 activity and this is supported in the literature by a phosphoproteome mass spectrometry screen that identified this residue in vivo15-18. Further, based on structural predictions we believe Ser10 residues on the surface of Cdc34 that interacts with both E1 and E3 enzymes. Thus, we aim to identify key residues in the molecular recognition of Cdc34 by its enzyme partners and hypothesize that phosphorylation of Ser10 will negatively regulate this molecular recognition through steric clash and electrostatic repulsions. Our hypothesis will be addressed through the following Specific Aims. Aim 1 will identify key residues for Cdc34 interaction with E1 and E3 enzyme partners. We will use x-ray crystallography in concert with biochemical and genetic techniques to accomplish this aim. Aim 2 will elucidate the regulatory effects of Cdc34 phosphorylation at residue Ser10. We will confirm the phosphorylation of Ser10 in vivo and examine the effects of S10D phosphomimetic mutation on molecular recognition of Cdc34 by its enzyme partners in vitro with biochemical assays and in vivo using yeast genetics.