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.
%0 Journal Article
%1 Fuchs2009
%A Fuchs, Yannick F.
%A Eisler, Stephan A.
%A Link, Gisela
%A Schlicker, Oliver
%A Bunt, Gertrude
%A Pfizenmaier, Klaus
%A Hausser, Angelika
%D 2009
%J Traffic
%K 2009 hausser izi pfizenmaier
%N 7
%P 858--867
%R 10.1111/j.1600-0854.2009.00918.x
%T A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal
%U https://www.ncbi.nlm.nih.gov/pubmed/19416469
%V 10
%X 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.
%Z Fuchs, Yannick FEisler, Stephan ALink, GiselaSchlicker, OliverBunt, GertrudePfizenmaier, KlausHausser, AngelikaengResearch Support, Non-U.S. Gov'tEnglandTraffic. 2009 Jul;10(7):858-67. doi: 10.1111/j.1600-0854.2009.00918.x. Epub 2009 Apr 25.
%7 2009/05/07
%@ 1600-0854 (Electronic)$\backslash$r1398-9219 (Linking)
@article{Fuchs2009,
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.},
added-at = {2023-06-29T13:07:55.000+0200},
annote = {Fuchs, Yannick FEisler, Stephan ALink, GiselaSchlicker, OliverBunt, GertrudePfizenmaier, KlausHausser, AngelikaengResearch Support, Non-U.S. Gov'tEnglandTraffic. 2009 Jul;10(7):858-67. doi: 10.1111/j.1600-0854.2009.00918.x. Epub 2009 Apr 25.},
author = {Fuchs, Yannick F. and Eisler, Stephan A. and Link, Gisela and Schlicker, Oliver and Bunt, Gertrude and Pfizenmaier, Klaus and Hausser, Angelika},
biburl = {https://puma.ub.uni-stuttgart.de/bibtex/2fb8ce96199c629bca215d6f0d71aa3b9/fabian},
doi = {10.1111/j.1600-0854.2009.00918.x},
edition = {2009/05/07},
interhash = {b77ee765db2cc3305eac6b30cadd460b},
intrahash = {fb8ce96199c629bca215d6f0d71aa3b9},
isbn = {1600-0854 (Electronic)$\backslash$r1398-9219 (Linking)},
issn = {13989219},
journal = {Traffic},
keywords = {2009 hausser izi pfizenmaier},
number = 7,
pages = {858--867},
pmid = {19416469},
timestamp = {2023-06-29T13:07:55.000+0200},
title = {{A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal}},
url = {https://www.ncbi.nlm.nih.gov/pubmed/19416469},
volume = 10,
year = 2009
}