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

结构性分泌是细胞在正常条件下和癌症等死亡状态下维持组织内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萌发和分裂的渐进循环完成的。ADP-核糖化因子(ARF)、GTP酶和蛋白激酶D(PKD)家族是囊泡分裂的重要调节因子。我们的初步数据现在表明，PKD2可能参与了TGN的肌动蛋白聚合的协调控制，这被认为是驱动成熟小泡最终断裂的原因。我们发现，PKD2在S298处使Cortactin磷酸化，从而削弱肌动蛋白聚合。Cdc42反过来使TGN的PKD2失活，并驱动Cortactin-N-WASP介导的协同肌动蛋白聚合，以帮助囊泡颈部寡聚的Dynamin2夹住和切断小泡。有趣的是，Dynamin2也是一种新的PKD2底物。因此，我们的建议旨在研究在囊泡分裂的最后步骤中，PKD2对肌动蛋白聚合和囊泡收缩的协调调节。目的：小泡分裂过程中PKD2活性的上游调控。我们将评估CDC42-GTPase激活蛋白ARHGAP21在囊泡断裂过程中调节PKD2活性的可能作用，以及这些过程与ARF1活性的关系。我们还将研究ARF1对Rho家族GTP酶的串扰是如何在分子水平上调节的。AIM2：PKD2、Cortactin和Dynamin在TGN肌动蛋白聚合和囊泡分裂中的作用。我们将研究控制N-WASP、Arp2/3复合体、Cortactin和大GTP酶Dynamin2介导的肌动蛋白驱动的囊泡分离的复合体的分子组成。特别是，我们将测试Cortactin及其被PKD2磷酸化是否会调节TGN的直接肌动蛋白聚合，以便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新的PKD2底物Dynamin2在协调肌动蛋白动力学和囊泡收缩中的作用。目的：体外重建依赖于PKD2的分裂步骤。我们将建立体外系统，利用基于脂质体和巨大单层囊泡(GUV)生成的纯化蛋白来研究囊泡分裂的特定方面。通过这些分析，我们将研究PKD2、Cortactin或Dynamin2如何影响巨型脂质体的微囊泡形成，以及被证明在囊泡分裂过程中与Dynamin2相互作用的Bar结构域蛋白AmphiPhyin2。通过这些研究，我们希望阐明PKD2在囊泡分裂的不同阶段中的不同作用，并为PKD2依赖于TGN结构性分泌的调控提供新的见解。

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.