Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH2 or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca2+ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca2+, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.
Gallegos
Lisa L
LL
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.
Kunkel
Maya T
MT
Newton
Alexandra C
AC
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004075:Diglycerides / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D008954:Models, Biological; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D004075:Diglycerides; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D002118:Calcium