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

项目总结 CDC34是细胞周期G1期至S期检查点1-3的关键调控因子。它起到了E2泛素的作用 结合酶，首先由E1酶Uba1与泛素充电，然后与SCF一起起作用 E3酶家族，用泛素修饰其他细胞周期蛋白，通过 蛋白水解酶4，5。自发现以来的几年中，CDC34失调与几种类型的 癌症患者6-8岁。Cdc34的许多下游靶点是细胞周期蛋白依赖的激酶抑制物，如p27和p40 对癌症的进展至关重要。总之，这些发现导致了对cdc34的调查。 作为癌症治疗的靶点，但更有针对性，例如专门针对CDC34 需要与其酶伙伴相互作用。然而，对分子的详细结构理解 它的E1配对、Uba1和E3连接酶配对不能识别CDC34。此外，监管 对CDC34的了解还不完全。先前的研究表明，CDC34通过 磷酸化；然而，我们有令人兴奋的初步数据表明残基丝氨酸的磷酸化。 10作为cdc34活性的负调节因子，这在文献中得到了磷酸蛋白质组质量的支持。 鉴定vivo15-18中该残基的光谱分析。此外，根据我们认为的结构性预测， 与E1和E3酶相互作用的CDC34表面的Ser10残基。因此，我们的目标是确定 CDC34的酶对分子识别中的关键残基和假设 Ser10的磷酸化将通过空间碰撞和 静电斥力。 我们的假设将通过以下具体目标得到解决。目标1将确定关键残留物 CDC34与E1和E3酶对相互作用。我们将使用x射线结晶学与 生物化学和基因技术来实现这一目标。目标2将阐明cdc34的调节作用。 Ser10残基的磷酸化。我们将在体内确认Ser10的磷酸化并检测其影响 S10D拟磷突变对其酶对CDC34分子识别的影响 生化分析和活体酵母菌遗传学。

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.