title: G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi-network.
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.
authors: Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring, Stuttgart, Germany.
authors: Stephan A
authors: SA
authors: Eisler
authors: Yannick F
authors: YF
authors: Fuchs
authors: Klaus
authors: K
authors: Pfizenmaier
authors: Angelika
authors: A
authors: Hausser
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D021601:trans-Golgi Network / Q000201:enzymology / Q000378:metabolism*
chemical_list: C089212:protein kinase D; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C