The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi-network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(-induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G-PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi-specific PKD activity in living cells we now refined G-PKDrep to generate G-PKDrep-live. Specifically, phosphorylation of G-PKDrep-live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof-of-principle experiment using phorbolester treatment, we demonstrate the reporter's capability to track rapid activation of PKD at the TGN. Furthermore, activation-induced FRET changes are reversed by treatment with PKD-specific pharmacological inhibitors. Thus, the newly developed reporter G-PKDrep-live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.
%0 Journal Article
%1 Eisler2012
%A Eisler, Stephan A.
%A Fuchs, Yannick F.
%A Pfizenmaier, Klaus
%A Hausser, Angelika
%D 2012
%J Biotechnology Journal
%K 2012 hausser izi pfizenmaier
%N 1
%P 148--154
%R 10.1002/biot.201100273
%T G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi-network
%U http://www.ncbi.nlm.nih.gov/pubmed/21898831
%V 7
%X The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi-network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(-induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G-PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi-specific PKD activity in living cells we now refined G-PKDrep to generate G-PKDrep-live. Specifically, phosphorylation of G-PKDrep-live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof-of-principle experiment using phorbolester treatment, we demonstrate the reporter's capability to track rapid activation of PKD at the TGN. Furthermore, activation-induced FRET changes are reversed by treatment with PKD-specific pharmacological inhibitors. Thus, the newly developed reporter G-PKDrep-live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.
@article{Eisler2012,
abstract = {The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi-network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(-induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G-PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi-specific PKD activity in living cells we now refined G-PKDrep to generate G-PKDrep-live. Specifically, phosphorylation of G-PKDrep-live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof-of-principle experiment using phorbolester treatment, we demonstrate the reporter's capability to track rapid activation of PKD at the TGN. Furthermore, activation-induced FRET changes are reversed by treatment with PKD-specific pharmacological inhibitors. Thus, the newly developed reporter G-PKDrep-live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.},
added-at = {2018-02-01T16:21:30.000+0100},
author = {Eisler, Stephan A. and Fuchs, Yannick F. and Pfizenmaier, Klaus and Hausser, Angelika},
biburl = {https://puma.ub.uni-stuttgart.de/bibtex/2bfa6002b317c3aa9b040559903f0d488/cristiano},
doi = {10.1002/biot.201100273},
interhash = {74475a00f79590f4090534ef5e1a1ce0},
intrahash = {bfa6002b317c3aa9b040559903f0d488},
issn = {18606768},
journal = {Biotechnology Journal},
keywords = {2012 hausser izi pfizenmaier},
month = jan,
number = 1,
pages = {148--154},
pmid = {21898831},
timestamp = {2019-01-17T13:18:28.000+0100},
title = {{G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi-network}},
url = {http://www.ncbi.nlm.nih.gov/pubmed/21898831},
volume = 7,
year = 2012
}