Regulation of G2M transition in budding yeast

芽殖酵母 G2M 转变的调控

• 批准号: 7291870
• 负责人: Kyung Lee
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7291870
• 关键词: Regulation; G2M; transition; budding; yeast

Entry into mitosis in eukaryotic organisms is regulated by an intricate network of kinases and phosphatases that coordinately bring about reorganization of various subcellular structures. A key regulatory component for this event is the conserved cyclin B-bound Cdc2. In fission yeast and higher eukaryotes, Cdc2 is phosphorylated at Tyr15 and negatively regulated by Wee1, an event that is reversed by the activity of Cdc25C phosphatase. These critical steps at mitotic entry appear to be largely conserved throughout evolution. Studies on the G2/M regulation in genetically-amenable organisms such as budding yeast have provided valuable insights into how eukaryotic organisms bring about timely activation of cyclin B-Cdc2 activity prior to mitotic entry. In budding yeast, Swe1 (Wee1 ortholog) negatively regulates mitotic Clb (collectively for the B-type cyclins - Clb1, Clb2, Clb3, and Clb4) associated-Cdc28 (Cdc2 homolog) by phosphorylating the equivalent Tyr19 residue, a modification that is reversed by Mih1 (Cdc25 ortholog). We previously showed that Cla4 (PAK homolog) and Cdc5 (Polo homolog) phosphorylate Swe1 in a step-wise manner. Our recent study found that Hsl1 (Nim1 ortholog) and Hsl7, which are critical for Swe1 localization to the bud-neck, are also required for proper localization of Cdc5 to the bud-neck and the Cdc5-dependent Swe1 phosphorylation. Mitotic Clb-bound Cdc28, but not G1 or S cyclin-bound Cdc28, directly phosphorylated Swe1 and this phosphorylation step appears to be important to prime Swe1 for the subsequent Cdc5-dependent Swe1 phosphorylation. We would like to further investigate the mechanism of how Hsl1 and Hsl7 cooperate with multi-kinases (Cla4, Cdc28, and Cdc5) to facilitate Swe1 hyperphosphorylation and subsequent degradation prior to mitotic entry. Our current model is that Swe1 functions as a nodal point to integrate multi-kinase-dependent signals that license passage into mitosis.In a separate study, we have been interested in understanding the regulation of one of the budding yeast Nim1-related kinases Gin4. Gin4 plays an important role in proper organization of septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including the regulation of mitotic entry and cytokinesis. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. Using purified recombinant proteins, we demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. A Gin4 mutant lacking the Elm1-dependent phosphorylation sites appeared to be impaired in localization with a diminished kinase activity. Consistent with these observations, this mutant exhibited mild growth defect with frequently elongated bud morphology. Thus, we propose that Elm1 contributes to proper septin organization by directly regulating the Gin4-dependent Shs1 pathway. How the Elm1-Gin4-Shs1 pathway cooperates with other pathways leading to the regulation of septins and ultimately G2/M transition will be an intriguing question that requires further investigation.

在真核生物中，进入有丝分裂受激酶和磷酸酶的复杂网络调节，这些网络协调地引起各种亚细胞结构的重组。此事件的一个关键调控成分是保守的细胞周期蛋白B结合Cdc 2。在裂殖酵母和高等真核生物中，Cdc 2在Tyr 15处被磷酸化，并由Wee 1负调控，这一事件由Cdc 25 C磷酸酶的活性逆转。有丝分裂进入的这些关键步骤似乎在整个进化过程中基本上是保守的。研究G2/M期调控在遗传上适合的生物体，如芽殖酵母提供了宝贵的见解，真核生物如何带来及时激活细胞周期蛋白B-Cdc 2活性有丝分裂进入之前。在芽殖酵母中，Sve 1（Wee 1直系同源物）通过磷酸化等效Tyr 19残基（这种修饰被Mih 1（Cdc 25直系同源物）逆转）来负调节与Cdc 28（Cdc 2同源物）相关的有丝分裂Clb（B型细胞周期蛋白-Clb 1、Clb 2、Clb 3和Clb 4的统称）。我们以前表明，Cla 4（PAK同源物）和Cdc 5（波罗同源物）磷酸化Swe 1在一个逐步的方式。我们最近的研究发现，Hsl 1（Nim 1的直系同源物）和Hsl 7，这是关键的Swe 1定位到芽颈，也需要适当的定位Cdc 5的芽颈和Cdc 5依赖的Swe 1磷酸化。有丝分裂Clb结合的Cdc 28，而不是G1或S细胞周期蛋白结合的Cdc 28，直接磷酸化的Swe 1和这个磷酸化步骤似乎是重要的启动Swe 1随后Cdc 5依赖的Swe 1磷酸化。我们希望进一步研究Hsl 1和Hsl 7如何与多激酶（Cla 4、Cdc 28和Cdc 5）合作以促进Swe 1过度磷酸化和随后的有丝分裂进入前的降解的机制。我们目前的模型是，Swe 1作为一个节点的功能，以整合多激酶依赖的信号，许可证通过到有丝分裂。在一个单独的研究中，我们一直有兴趣了解芽殖酵母Nim 1相关激酶Gin 4的调节。Gin 4在母芽颈处的隔蛋白环的适当组织中起重要作用，所述母芽颈是对包括有丝分裂进入和胞质分裂的调节在内的多种细胞过程至关重要的丝状结构。在这里，我们表明，颈部相关的丝氨酸/苏氨酸激酶Elm 1，这是很重要的隔蛋白组装，是至关重要的适当修改的Gin 4和其生理底物Shs 1。使用纯化的重组蛋白，我们证明了Elm 1直接磷酸化和激活Gin 4，这反过来磷酸化Shs 1。缺乏Elm 1依赖性磷酸化位点的Gin 4突变体似乎在激酶活性降低的情况下在定位中受损。与这些观察结果一致，该突变体表现出轻微的生长缺陷，经常延长芽形态。因此，我们建议Elm 1有助于通过直接调节Gin 4依赖性Shs 1通路来适当地组织septin。Elm 1-Gin 4-Shs 1通路如何与其他通路合作，导致septins的调节，并最终G2/M转换将是一个有趣的问题，需要进一步研究。