A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C.
Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of approximately 10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
Violin
Jonathan D
JD
Department of Pharmacology, University of California, San Diego, La Jolla 92093, USA.
Zhang
Jin
J
Tsien
Roger Y
RY
Newton
Alexandra C
AC
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001683:Biological Clocks / Q000187:drug effects / Q000235:genetics*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D017930:Genes, Reporter / Q000235:genetics*; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D008954:Models, Biological; D010716:Phosphatidylinositols / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D015290:Second Messenger Systems / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D010738:Type C Phospholipases / Q000378:metabolism
D004075:Diglycerides; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D010716:Phosphatidylinositols; C118301:phosphoinositide-3,4-bisphosphate; D006632:Histamine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D010738:Type C Phospholipases