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

本构分泌是细胞在正常状态和死亡状态（如癌症）下维持组织稳态和细胞间通讯的重要机制。跨高尔基网络（TGN）的货物运输是通过囊泡出芽和裂变的渐进循环完成的。adp -核糖基化因子（ARF） gtpase和蛋白激酶D （PKD）家族的激酶是囊泡分裂的重要调节因子。我们现在的初步数据表明PKD2可能参与了TGN上肌动蛋白聚合的协调控制，这被认为是驱动成熟囊泡的最终分裂。我们发现PKD2在S298位点磷酸化皮质蛋白以破坏肌动蛋白聚合。Cdc42反过来使TGN上的PKD2失活，并驱动协同的contacttin - n- wasp介导的肌动蛋白聚合，以帮助在囊泡颈部的Dynamin2寡聚，从而挤压和切断囊泡。有趣的是，Dynamin2也是一种新的PKD2底物。因此，我们的提议旨在研究在囊泡裂变的最后步骤中PKD2对肌动蛋白聚合和囊泡挤压的协调调节。目的1：囊泡裂变过程中PKD2活性的上游调控。我们将评估CDC42-GTPase激活蛋白ARHGAP21在囊泡断裂过程中PKD2活性调节中的作用，以及这些过程与ARF1活性的联系。我们还将研究ARF1与rho家族gtpase的串扰如何在分子水平上介导。目的2:PKD2、皮质蛋白和动力蛋白在TGN肌动蛋白聚合和囊泡裂变中的作用。我们将研究由N-WASP、Arp2/3复合物、cortac和大GTPase Dynamin2介导的控制肌动蛋白驱动囊泡分离的复合物的分子组成。具体而言，我们将测试PKD2磷酸化的皮质蛋白是否会调节TGN上的定向肌动蛋白聚合，从而在囊泡分离的最后阶段对囊泡施加力。我们进一步建议研究新的PKD2底物Dynamin2在肌动蛋白动力学和囊泡挤压协调中的作用。目的3：体外重建pkd2依赖的裂变步骤。我们将建立体外系统，利用基于脂质体和巨型单层囊泡（GUVs）生成的纯化蛋白质来研究囊泡裂变的定义方面。通过这些实验，我们将研究PKD2、cortacn或Dynamin2如何与Bar结构域蛋白Amphiphysin2一起影响巨脂质体的微囊泡形成，该结构域蛋白在囊泡裂变过程中与Dynamin2相互作用。我们建议进一步研究在这些过程中膜曲率和GTPase激活的相关性。通过完成这些目标，我们希望阐明PKD2在囊泡裂变的不同阶段的不同作用，并为PKD2依赖于TGN组成分泌的调节提供新的见解。

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.