Regulation of G2M transition in budding yeast

真核生物进入有丝分裂是由一个复杂的激酶和磷酸酶网络调节的，这些网络协调地带来各种亚细胞结构的重组。这一事件的关键调控成分是保守的细胞周期蛋白b结合的Cdc2。在裂变酵母和高级真核生物中，Cdc2在Tyr15位点被磷酸化，并受到Wee1的负调控，这一事件被Cdc25C磷酸酶的活性逆转。在整个进化过程中，有丝分裂进入的这些关键步骤似乎在很大程度上是保守的。对芽殖酵母等遗传易感生物中G2/M调控的研究，为真核生物如何在有丝分裂进入前及时激活细胞周期蛋白B-Cdc2活性提供了有价值的见解。在出芽酵母中，Swe1 （Wee1同源）通过磷酸化对应的Tyr19残基（Cdc25同源）负调控有丝分裂Clb （b型细胞周期蛋白Clb1、Clb2、Clb3和Clb4）相关的cdc28 （Cdc2同源），这一修饰被Mih1 （Cdc25同源）逆转。我们之前发现cl4 （PAK同源物）和Cdc5 （Polo同源物）可以逐步磷酸化Swe1。我们最近的研究发现，Hsl1 （Nim1同源物）和Hsl7对于Swe1定位到芽颈至关重要，也需要Cdc5正确定位到芽颈和Cdc5依赖的Swe1磷酸化。有丝分裂clb结合的Cdc28，而不是G1或S周期蛋白结合的Cdc28，直接磷酸化了Swe1，这一磷酸化步骤似乎对随后cdc5依赖性的Swe1磷酸化至关重要。我们希望进一步研究Hsl1和Hsl7如何与多激酶（cl4、Cdc28和Cdc5）合作，促进Swe1在有丝分裂进入前的过度磷酸化和随后的降解。我们目前的模型是，Swe1作为一个节点，整合多激酶依赖的信号，使其进入有丝分裂。在另一项研究中，我们对芽殖酵母nim1相关激酶Gin4的调控很感兴趣。Gin4在芽母颈septin环的正常组织中起着重要作用，芽母颈septin环是一种丝状结构，对包括有丝分裂进入和细胞质分裂的调节在内的多种细胞过程至关重要。在这里，我们发现颈相关的丝氨酸/苏氨酸激酶Elm1对septin组装很重要，对于Gin4及其生理底物Shs1的适当修饰至关重要。使用纯化的重组蛋白，我们证明Elm1直接磷酸化并激活Gin4，而Gin4又磷酸化Shs1。缺乏elm1依赖性磷酸化位点的Gin4突变体似乎在定位上受损，激酶活性降低。与这些观察结果一致，该突变体表现出轻微的生长缺陷，芽形态经常拉长。因此，我们提出Elm1通过直接调节依赖gin4的Shs1通路来促进正确的septin组织。Elm1-Gin4-Shs1通路如何与其他通路合作导致septin调控并最终向G2/M转变将是一个有趣的问题，需要进一步研究。

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.