Regulation of G2M transition in budding yeast

在真核生物中，进入有丝分裂是由复杂的激酶和磷酸酶网络调节的，它们协调地带来各种亚细胞结构的重组。该事件的一个关键调控成分是保守的细胞周期蛋白B结合的CDC2。在裂解酵母和高等真核生物中，Cdc2在Tyr15处被磷酸化，并受到Wee1的负调控，这一事件被CDc25C磷酸酶的活性逆转。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。对发芽酵母等可遗传生物的G2/M调控的研究为真核生物如何在有丝分裂进入之前及时激活细胞周期蛋白B-CDc2活性提供了有价值的见解。在萌芽酵母中，Swe1(Wee1同源基因)通过磷酸化等效的Tyr19残基来负调控有丝分裂Clb(统称为B型周期蛋白-Clb1、Clb2、Clb3和Clb4)相关的CDc28(CDc2同源基因)，这一修饰被Mih1(CDC25同源基因)逆转。我们以前证明了Cla4(PAK同系物)和Cdc5(Polo同系物)以一种循序渐进的方式磷酸化Swe1。我们最近的研究发现，Hsl1(Nim1邻位同源基因)和Hsl7是Swe1定位到芽颈的关键，也是CDc5定位到芽颈和依赖于CDc5的Swe1磷酸化所必需的。有丝分裂Clb结合的CDc28，而不是G1或S细胞周期蛋白结合的CDc28，直接磷酸化Swe1，这一磷酸化步骤似乎对启动Swe1随后的CDC5依赖的Swe1磷酸化是重要的。我们想进一步研究Hsl1和Hsl7如何与多个激酶(Cla4、CDC28和CDC5)协同作用，促进Swe1过度磷酸化和随后在有丝分裂进入之前的降解。我们目前的模型是Swe1作为一个节点来整合多个依赖于激酶的信号，这些信号允许进入有丝分裂。在另一项单独的研究中，我们一直有兴趣了解一种萌芽酵母Nim1相关激酶Gin4的调节。Gin4在母芽颈隔素环的正确组织中起着重要作用，这种丝状结构对包括有丝分裂进入和胞质分裂在内的各种细胞过程的调节至关重要。在这里，我们展示了颈部相关的丝氨酸/苏氨酸激酶Elm1，它对Septin组装是重要的，对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，我们证明了Elm1直接磷酸化并激活Gin4，Gin4进而磷酸化Shs1。缺乏Elm1依赖的磷酸化位点的Gin4突变体似乎在定位上受损，并伴有降低的激酶活性。与这些观察结果一致的是，该突变体表现出轻微的生长缺陷，芽形态经常拉长。因此，我们认为Elm1通过直接调节依赖于Gin4的Shs1途径来参与正确的Septin组织。Elm1-Gin4-Shs1途径如何与其他途径协同调控Septins，最终实现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.