Browsing by Author "Kienzle, Christine"

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    Protein Kinase D controls mitotic Golgi complex fragmentation through a RAF-MEK1 pathway
    (2012) Kienzle, Christine; Pfizenmaier, Klaus (Prof. Dr. )
    The process of Golgi inheritance in mammalian cells involves multiple signaling pathways and proteins to ensure correct partitioning of Golgi membranes among dividing cells. As the Golgi apparatus is a single-copy organelle, the mechanism of separation is rather complex. First Golgi stacks become separated by the action of several key proteins such as BARS, MEK1 or GRASP-65. During cell cycle progression, Golgi stacks further break down into small vesicles that become dispersed throughout the cytoplasm. This fragmentation process is a prerequisite to allow equal distribution of Golgi membranes between daughter cells. Essential for mitotic entry is the cleavage of Golgi inter stack connections. Since the blocking of this particular event causes an arrest of cells in G2, it defines the Golgi mitotic checkpoint. In this work PKD was identified as a novel regulator in Golgi mitotic checkpoint control. The PKD family of protein kinases has a well characterized role at the trans-Golgi network regulating fission of cargo-containing vesicles en route to the plasma membrane. However, by now only few publications proposed a role for PKD in cell division. In this study we provide evidence that siRNA-mediated depletion of PKD 1 and 2 delays the passage of synchronized HeLa cells into M phase. Furthermore, a semi-intact assay approach identified PKD as a regulator of Golgi fragmentation, since PKD inhibition abolished dispersion of Golgi stacks. Detailed microscopic analyses such as mitotic index determinations and Golgi integrity measurements, respectively, demonstrate that PKD acts on the level of Golgi ribbon cleavage during G2. Finally, evidence is provided that PKD acts through a Raf-1-MEK pathway to exert its function during mitosis; however, Raf-1 appeared to be not a direct PKD substrate. Taken together, this study demonstrates a novel role of PKD in Golgi mitotic checkpoint control by acting upstream of Raf-1/MEK1. The data further emphasize the importance of PKD in the maintenance of the structural integrity of the Golgi complex.