Cell Cycle G2/M Pathway Modulated by Viral Protein R

描述（由申请人提供）：细胞从G2期的进展 细胞周期到有丝分裂是一个严格调控的过程， Cdc2激酶，它决定了所有真核细胞有丝分裂的开始。 在裂殖酵母（裂殖酵母）中，Cdc2的活性受到调节， 通过Cdc2上酪氨酸15（Tyr 15）的磷酸化状态， 在G2晚期被Wee1和Mik1酪氨酸激酶磷酸化， 通过Cdc25磷酸酶去磷酸化以触发进入有丝分裂。cdc25， Wee1和Mik1是两种充分表征的调节途径的靶标， DNA损伤和DNA复制检查点，它们都引起细胞周期 主要通过抑制Cdc2上Tyr15的磷酸化来阻止 抑制Cdc25。HIV-1的病毒蛋白R（Vpr）诱导G2期阻滞， 来自远亲真核生物的细胞，包括裂殖酵母和人类。 类似于两个检查点途径，Vpr也诱导裂变中的G2停滞 酵母通过抑制Tyr15的磷酸化。但是，Vpr不使用 检查点路径中的任何早期或晚期步骤，相反， 主要通过激活Wee 1，蛋白磷酸酶2A（PP2A）和一个 Cdc25的抑制程度较小，表明Vpr诱导G2期阻滞 通过一种新的PP2A介导的调节途径。这项提案的目的是 使用裂变酵母作为模型系统来测试这一假设，并阐明 这条新的G2/M调节通路。三个具体目标是：1）确定 当Vpr诱导G2期阻滞时，PP2A对Wee 1和Cdc 25的调节作用; 2） 表征Vpr与PP2A和酪蛋白激酶II的潜在蛋白复合物 α（CKIIa），并确定该复合物在Vpr诱导的G2期阻滞中的作用，以及 3)表征四个多拷贝Vpr特异性抑制子（Wos 2和 Vsp24C8）/增强子（Rad25和Sum1）及其在Vprinduced G2阻滞中的作用。 这些拟议的研究将为这种新细胞提供一个全面的框架 循环控制途径，并提供深入了解这一新的 真核细胞的G2/M监视系统。

DESCRIPTION (provided by applicant): Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In fission yeast (Schizosaccharomyces pombe), the activity of Cdc2 is regulated by the phosphorylation status of tyrosine 15 (Tyr 15) on Cdc2, which is phosphorylated by Wee1 and Mik1 tyrosine kinases during late G2 and is rapidly dephosphorylated by the Cdc25 phosphatase to trigger entry into mitosis. Cdc25, Wee1 and Mik1 are the targets of two well-characterized regulatory pathways, the DNA damage and DNA replication checkpoints, which both cause cell cycle arrest by inhibitory phosphorylation of Tyr15 on Cdc2 primarily through inhibition of Cdc25.The viral protein R (Vpr) of HIV-1 induces G2 arrest in cells from distantly related eukaryotes, including fission yeast and human. Similar to the two checkpoint pathways, Vpr also induces G2 arrest in fission yeast through inhibitory phosphorylation of Tyr15. However, Vpr does not use any of the early or late steps in the checkpoint pathways, and instead it acts primarily through activation of Wee1, protein phosphatase 2A (PP2A) and to a lesser extent inhibition of Cdc25, suggesting that Vpr induces G2 arrest through a novel PP2A-mediated regulatory pathway. The goal of this proposal is to use fission yeast as a model system to test this hypothesis and to elucidate this new G2/M regulatory pathway. The three specific aims are to 1) define the regulatory effects of PP2A on Wee1 and Cdc25 when Vpr induces G2 arrest; 2) characterize a potential protein complex of Vpr with PP2A and casein kinase II alpha (CKIIa) and define the role of this complex in Vpr-induced G2 arrest, and 3) characterize four multicopy Vpr-specific suppressors (Wos2 and Vsp24C8)/enhancers (Rad25 and Sum1) and their role in Vprinduced G2 arrest. These proposed studies will provide a comprehensive framework for this new cell cycle control pathway and provide insights into fundamental aspects of this new G2/M surveillance system of eukaryotic cells.