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Roles of Protein Kinase D2 during actin-driven vesicle scission at the Trans-Golgi-network (TGN).

蛋白激酶 D2 在肌动蛋白驱动的跨高尔基体网络 (TGN) 囊泡分裂过程中的作用。

基本信息

批准号：
380319649
负责人：
Dr. Tim Eiseler, since 1/2019
金额：
--
依托单位：
Department of Cell Biology
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2021-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/380319649?language=en
关键词：
Roles Protein Kinase D2 during

项目摘要

Constitutive secretion is an essential mechanism for cells to maintain tissue homeostasis and cell-cell communication under normal conditions and in decease states, such as cancer. The trafficking of cargo from the Trans-Golgi-network (TGN) is accomplished by progressive cycles of vesicle budding and fission. ADP-ribosylation factor (ARF) GTPases and kinases of the Protein Kinase D (PKD) family are important regulators of vesicle fission. Our preliminary data now indicate that PKD2 could be involved in the coordinated control of actin polymerization at the TGN that is suggested to drive the final scission of mature vesicles. We show that PKD2 phosphorylates Cortactin at S298 to impair actin polymerization. Cdc42 in-turn inactivates PKD2 at the TGN and drives synergistic Cortactin-N-WASP-mediated actin polymerization to aid oligomerized Dynamin2 at the vesicle neck in pinching and severing vesicles. Interestingly, Dynamin2 is also a novel PKD2 substrate. Therefore, our proposal aims to investigate the coordinated regulation of actin polymerization and vesicle pinching by PKD2 during the final steps of vesicle fission. Aim1: Upstream regulation of PKD2 activity during vesicle fission steps. We will evaluate a putative role of the CDC42-GTPase activating protein ARHGAP21 in the modulation of PKD2 activity during vesicle scission and the connection of these processes to ARF1 activity. We will also investigate how crosstalk of ARF1 to Rho-familiy GTPases could be mediated on a molecular level. Aim2: Role of PKD2, Cortactin and Dynamin during actin polymerization and vesicle fission at the TGN. We will investigate the molecular composition of complexes that control actin-driven vesicle separation mediated by N-WASP, the Arp2/3 complex, Cortactin and the large GTPase Dynamin2. In particular, we will test whether Cortactin and its phosphorylation by PKD2 will modulate directed actin polymerization at the TGN to apply force at vesicles during final stages of vesicle separation. We further propose to study the role of the novel PKD2 substrate Dynamin2 in the coordination of actin dynamics and vesicle pinching. Aim3: In vitro reconstitution of PKD2-dependent fission steps. We will establish in vitro systems to investigate defined aspects of vesicle fission utilizing purified proteins that are based on the generation of liposomes and Giant Unilamellar Vesicles (GUVs). With these assays we will investigate how PKD2, Cortactin or Dynamin2 will affect micro-vesiculation of giant liposomes together with the Bar domain protein Amphiphysin2, that was shown to interact with Dynamin2 during vesicle fission. We further propose to study the relevance of membrane curvature and GTPase activation during these processes.By completing these aims we hope to elucidate the different roles of PKD2 during different stages of vesicle fission and provide novel insights in a PKD2-dependent regulation of constitutive secretion from the TGN.

组成性分泌是细胞在正常情况下和癌症等疾病状态下维持组织稳态和细胞间通讯的重要机制。跨高尔基体网络（TGN）的货物运输是通过囊泡出芽和分裂的渐进循环完成的。ADP-核糖基化因子（ARF）GTP酶和蛋白激酶D（PKD）家族的激酶是囊泡分裂的重要调节剂。我们的初步数据表明，PKD 2可能参与协调控制肌动蛋白聚合在TGN，建议驱动成熟囊泡的最终分裂。我们发现PKD 2在S298处磷酸化Corpine以损害肌动蛋白聚合。Cdc 42反过来使TGN处的PKD 2失活，并驱动协同Cortactin-N-WASP介导的肌动蛋白聚合，以帮助囊泡颈部的寡聚化动力蛋白2夹紧和切断囊泡。有趣的是，Dynamin 2也是一种新型PKD 2底物。因此，我们的建议的目的是调查协调调节肌动蛋白聚合和囊泡捏PKD 2在囊泡分裂的最后步骤。目的1：囊泡分裂步骤中PKD 2活性的上游调控。我们将评估CDC 42-GTP酶激活蛋白ARHGAP 21在囊泡断裂过程中PKD 2活性调节中的假定作用，以及这些过程与ARF 1活性的联系。我们还将研究如何在分子水平上介导ARF 1与Rho家族GTP酶的串扰。目的2：PKD 2，Cordyn和Dynamin在TGN的肌动蛋白聚合和囊泡分裂过程中的作用。我们将研究复合物的分子组成，控制肌动蛋白驱动的囊泡分离介导的N-WASP，Arp 2/3复合物，Corpyn和大的GT3-Dynamin 2。特别是，我们将测试Cordyn和PKD 2磷酸化是否会调节TGN的定向肌动蛋白聚合，以在囊泡分离的最后阶段向囊泡施加力。我们进一步建议研究的作用，新的PKD 2底物Dynamin 2的协调肌动蛋白动力学和囊泡捏。目的3：PKD 2依赖性分裂步骤的体外重建。我们将建立体外系统，利用基于脂质体和巨单层囊泡（GUV）生成的纯化蛋白质来研究囊泡分裂的定义方面。通过这些试验，我们将研究PKD 2，Corpedin或Dynamin 2如何与Bar结构域蛋白Amphiphysin 2一起影响巨脂质体的微囊泡形成，该蛋白在囊泡分裂期间与Dynamin 2相互作用。我们进一步提出研究在这些过程中膜曲率和GT3激活的相关性，通过完成这些目标，我们希望阐明PKD 2在囊泡分裂的不同阶段中的不同作用，并提供新的见解，在PKD 2依赖的组成性分泌从TGN的调节。