A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal.
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.
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Yannick F
YF
Fuchs
Stephan A
SA
Eisler
Gisela
G
Link
Oliver
O
Schlicker
Gertrude
G
Bunt
Klaus
K
Pfizenmaier
Angelika
A
Hausser
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
D007527:Isoenzymes; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D050257:Tubulin Modulators; C089212:protein kinase D; D011493:Protein Kinase C; D015739:Nocodazole