A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

doi:10.1083/jcb.200302125

Published online 2 June 2003. doi:10.1083/jcb.200302125

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© The Rockefeller University Press, 0021-9525/2003/6/899 $5.00
The Journal of Cell Biology, Volume 161, Number 5, 899-909

Article

A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C

Jonathan D. Violin¹^,2, Jin Zhang¹, Roger Y. Tsien¹^,3 and Alexandra C. Newton¹

¹ Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093
² Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA 92093
³ Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093

Address correspondence to Alexandra C. Newton, 9500 Gilman Dr., La Jolla, CA 92093-0640. Tel.: (858) 534-4527. Fax: (858) 534-6020. E-mail: anewton{at}ucsd.edu

Signals transduced by kinases depend on the extent and durationof substrate phosphorylation. We generated genetically encodedfluorescent reporters for PKC activity that reversibly respondto stimuli activating PKC. Specifically, phosphorylation ofthe reporter expressed in mammalian cells causes changes influorescence resonance energy transfer (FRET), allowing realtime imaging of phosphorylation resulting from PKC activation.Targeting of the reporter to the plasma membrane, where PKCis activated, reveals oscillatory phosphorylation in HeLa cellsin response to histamine. Each oscillation in substrate phosphorylationfollows a calcium oscillation with a lag of $~$ 10 s. Novel FRET-basedreporters for PKC translocation, phosphoinositide bisphosphateconversion to IP₃, and diacylglycerol show that in HeLa cellsthe oscillatory phosphorylations correlate with Ca²⁺-controlledtranslocation of conventional PKC to the membrane without oscillationsof PLC activity or diacylglycerol. However, in MDCK cells stimulatedwith ATP, PLC and diacylglycerol fluctuate together with Ca²⁺and phosphorylation. Thus, specificity of PKC signaling dependson the local second messenger-controlled equilibrium betweenkinase and phosphatase activities to result in strict calcium-controlledtemporal regulation of substrate phosphorylation.

Key Words: calcium; fluorescence resonance energy transfer; oscillation; phosphatase; PKC

^* Abbreviations used in this paper: AKAR, A kinase activity reporter;CICR, calcium-induced calcium release; CKAR, C kinase activityreporter; CYPHR, cyan/yellow PH domain reporter; DAG, diacylglycerol;DAGR, DAG receptor; FRET, fluorescence resonance energy transfer;IP₃, inositol 1,4,5-trisphosphate; mCFP, monomeric CFP; mYFP,monomeric YFP; MyrPalm, myristoylated and palmitoylated; PDBu,phorbol dibutyrate;PIP₂, phosphoinositide bisphosphate.

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