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A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal
Authors:Yannick F Fuchs  Stephan A Eisler  Gisela Link  Oliver Schlicker  Gertrude Bunt  Klaus Pfizenmaier  Angelika Hausser
Institution:Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany; Laboratory for Molecular Cellular Systems, Department of Neuro- and Sensory Physiology, University Medicine Göttingen, Humboldtallee 23, 37073 Göttingen, Germany; DFG Research Center for Molecular Physiology of the Brain (CMPB), Göttingen, Germany
Abstract:The protein kinase D (PKD) family comprises multifunctional serine/threonine-specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral-targeted transport carrier fission from the trans -Golgi network (TGN). To visualize site-specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi-specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD-specific pharmacological inhibitors and siRNA-mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole-induced Golgi complex break-up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant-negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.
Keywords:PKD                        Golgi complex                        reporter                        nocodazole                        fragmentation
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