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J. Cell Biol. 161 (5): 899-909

Copyright © 2003 by the Rockefeller University Press.


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

Abstract: 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 ~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.

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 activity reporter; 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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