title: Genetically encoded fluorescent reporters to visualize protein kinase C activation in live cells.
abstract: Protein kinase C (PKC) signaling drives many important cellular processes and its dysregulation results in pathophysiologies such as cancer (Gokmen-Polar et al., Cancer Res 61:1375-1381, 2001). Because PKC is activated acutely and allosterically, it is difficult to monitor the cellular activity of endogenous PKC by conventional methodologies (Newton, Methods Enzymol 345:499-506, 2002). Rather, PKC signaling is best studied in situ using biosensors such as FRET-based reporters. We have generated several FRET-based reporters for studying PKC signaling in real time in live cells (Violin and Newton, IUBMB Life 55:653-660, 2003). Using these reporters, we have demonstrated phase-locked oscillations in Ca2+ release and membrane-localized endogenous PKC activity in response to histamine (Violin et al., J Cell Biol 161:899-909, 2003), as well as distinct signatures of endogenous PKC signaling at specific organelles in response to uridine-5'-triphosphate (UTP; Gallegos et al., J Biol Chem 281:30947-30956, 2006). Here we describe methods to image cells expressing the reporters and elaborate on data analyses, control experiments, and variations for imaging the activity of expressed PKC.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Gallegos
authors.forename: Alexandra C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D004075:Diglycerides; D008164:Luminescent Proteins; D011493:Protein Kinase C