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

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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.