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A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole‐Induced Golgi Dispersal
Author(s) -
Fuchs Yannick F.,
Eisler Stephan A.,
Link Gisela,
Schlicker Oliver,
Bunt Gertrude,
Pfizenmaier Klaus,
Hausser Angelika
Publication year - 2009
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2009.00918.x
Subject(s) - nocodazole , golgi apparatus , biology , microbiology and biotechnology , microtubule , endoplasmic reticulum , cell , genetics , cytoskeleton
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.