A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal
| Autor: | Gertrude Bunt, Gisela Link, Klaus Pfizenmaier, Angelika Hausser, Oliver Schlicker, Stephan A. Eisler, Yannick F. Fuchs |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Recombinant Fusion Proteins
Molecular Sequence Data Mutant Golgi Apparatus Biology Biochemistry Cell Line Green fluorescent protein 03 medical and health sciences symbols.namesake chemistry.chemical_compound 0302 clinical medicine Genes Reporter Structural Biology Organelle Genetics Animals Humans Amino Acid Sequence RNA Small Interfering Molecular Biology Protein Kinase C 030304 developmental biology 0303 health sciences Gene knockdown Kinase Nocodazole Cell Biology Golgi apparatus musculoskeletal system Molecular biology Tubulin Modulators Cell biology Enzyme Activation Isoenzymes chemistry cardiovascular system symbols Phosphorylation 030217 neurology & neurosurgery |
| Zdroj: | Traffic. 10:858-867 |
| ISSN: | 1398-9219 |
| DOI: | 10.1111/j.1600-0854.2009.00918.x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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