Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.
Subscribe

Logo for

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.

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling.
B. H. Falkenburger, E. J. Dickson, and B. Hille (2013)
J. Gen. Physiol. 141, 537-555
   Abstract »    Full Text »    PDF »
P2Y1 receptor-dependent diacylglycerol signaling microdomains in {beta} cells promote insulin secretion.
A. Wuttke, O. Idevall-Hagren, and A. Tengholm (2013)
FASEB J 27, 1610-1620
   Abstract »    Full Text »    PDF »
Simultaneous assembly of two target proteins using split inteins for live cell imaging.
S. Wong, E. Mills, and K. Truong (2013)
Protein Eng. Des. Sel. 26, 207-213
   Abstract »    Full Text »    PDF »
Isozyme-specific Interaction of Protein Kinase C{delta} with Mitochondria Dissected Using Live Cell Fluorescence Imaging.
A. X. Wu-Zhang, A. N. Murphy, M. Bachman, and A. C. Newton (2012)
J. Biol. Chem. 287, 37891-37906
   Abstract »    Full Text »    PDF »
TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation.
M. Tauseef, N. Knezevic, K. R. Chava, M. Smith, S. Sukriti, N. Gianaris, A. G. Obukhov, S. M. Vogel, D. E. Schraufnagel, A. Dietrich, et al. (2012)
J. Exp. Med. 209, 1953-1968
   Abstract »    Full Text »    PDF »
Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing.
D. Liu, O. Davydenko, and M. A. Lampson (2012)
J. Cell Biol. 198, 491-499
   Abstract »    Full Text »    PDF »
Spontaneous transmitter release recruits postsynaptic mechanisms of long-term and intermediate-term facilitation in Aplysia.
I. Jin, H. Udo, J. B. Rayman, S. Puthanveettil, E. R. Kandel, and R. D. Hawkins (2012)
PNAS 109, 9137-9142
   Abstract »    Full Text »    PDF »
Cellular Pharmacology of Protein Kinase M{zeta} (PKM{zeta}) Contrasts with Its in Vitro Profile: IMPLICATIONS FOR PKM{zeta} AS A MEDIATOR OF MEMORY.
A. X. Wu-Zhang, C. L. Schramm, S. Nabavi, R. Malinow, and A. C. Newton (2012)
J. Biol. Chem. 287, 12879-12885
   Abstract »    Full Text »    PDF »
Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling.
M. J. Lohse, S. Nuber, and C. Hoffmann (2012)
Pharmacol. Rev. 64, 299-336
   Abstract »    Full Text »    PDF »
Neurexin-1{alpha} Contributes to Insulin-containing Secretory Granule Docking.
M. Mosedale, S. Egodage, R. C. Calma, N.-W. Chi, and S. D. Chessler (2012)
J. Biol. Chem. 287, 6350-6361
   Abstract »    Full Text »    PDF »
Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels.
H. Hallaq, D. W. Wang, J. D. Kunic, A. L. George Jr, K. S. Wells, and K. T. Murray (2012)
Am J Physiol Heart Circ Physiol 302, H782-H789
   Abstract »    Full Text »    PDF »
Systematic control of protein interaction using a modular ER/K {alpha}-helix linker.
S. Sivaramakrishnan and J. A. Spudich (2011)
PNAS 108, 20467-20472
   Abstract »    Full Text »    PDF »
Development of an optimized backbone of FRET biosensors for kinases and GTPases.
N. Komatsu, K. Aoki, M. Yamada, H. Yukinaga, Y. Fujita, Y. Kamioka, and M. Matsuda (2011)
Mol. Biol. Cell 22, 4647-4656
   Abstract »    Full Text »    PDF »
Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient.
E. Wang, E. R. Ballister, and M. A. Lampson (2011)
J. Cell Biol. 194, 539-549
   Abstract »    Full Text »    PDF »
Aurora B kinase activation requires survivin priming phosphorylation by PLK1.
Y. Chu, P. Y. Yao, W. Wang, D. Wang, Z. Wang, L. Zhang, Y. Huang, Y. Ke, X. Ding, and X. Yao (2011)
J Mol Cell Biol 3, 260-267
   Abstract »    Full Text »    PDF »
Protein Kinase C: The "Masters" of Calcium and Lipid.
P. Lipp and G. Reither (2011)
Cold Spring Harb Perspect Biol 3, a004556
   Abstract »    Full Text »    PDF »
Targeted in Vivo O-GlcNAc Sensors Reveal Discrete Compartment-specific Dynamics during Signal Transduction.
L. D. Carrillo, J. A. Froemming, and L. K. Mahal (2011)
J. Biol. Chem. 286, 6650-6658
   Abstract »    Full Text »    PDF »
Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells.
E. J. Weinman, D. Steplock, S. Shenolikar, and T. A. Blanpied (2011)
Am J Physiol Renal Physiol 300, F231-F235
   Abstract »    Full Text »    PDF »
Protein Kinase C {delta}-specific Activity Reporter Reveals Agonist-evoked Nuclear Activity Controlled by Src Family of Kinases.
T. Kajimoto, S. Sawamura, Y. Tohyama, Y. Mori, and A. C. Newton (2010)
J. Biol. Chem. 285, 41896-41910
   Abstract »    Full Text »    PDF »
GABAA Receptor Trafficking Is Regulated by Protein Kinase C{varepsilon} and the N-Ethylmaleimide-Sensitive Factor.
W.-H. Chou, D. Wang, T. McMahon, Z.-H. Qi, M. Song, C. Zhang, K. M. Shokat, and R. O. Messing (2010)
J. Neurosci. 30, 13955-13965
   Abstract »    Full Text »    PDF »
Calcium Transduces Plasma Membrane Receptor Signals to Produce Diacylglycerol at Golgi Membranes.
M. T. Kunkel and A. C. Newton (2010)
J. Biol. Chem. 285, 22748-22752
   Abstract »    Full Text »    PDF »
Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase C{alpha}-dependent NADPH Oxidase Activation.
M. Herrera, G. B. Silva, and J. L. Garvin (2010)
J. Biol. Chem. 285, 21323-21328
   Abstract »    Full Text »    PDF »
Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues.
D. M. Chudakov, M. V. Matz, S. Lukyanov, and K. A. Lukyanov (2010)
Physiol Rev 90, 1103-1163
   Abstract »    Full Text »    PDF »
Signaling Responses to Pulsatile Gonadotropin-releasing Hormone in L{beta}T2 Gonadotrope Cells.
R. Tsutsumi, D. Mistry, and N. J. G. Webster (2010)
J. Biol. Chem. 285, 20262-20272
   Abstract »    Full Text »    PDF »
{beta}-Arrestin-Biased Agonism of the Angiotensin Receptor Induced by Mechanical Stress.
K. Rakesh, B. Yoo, I.-M. Kim, N. Salazar, K.-S. Kim, and H. A. Rockman (2010)
Science Signaling 3, ra46
   Abstract »    Full Text »    PDF »
PKC-{alpha} mediates flow-stimulated superoxide production in thick ascending limbs.
N. J. Hong, G. B. Silva, and J. L. Garvin (2010)
Am J Physiol Renal Physiol 298, F885-F891
   Abstract »    Full Text »    PDF »
Ca2+ influx and protein scaffolding via TRPC3 sustain PKC{beta} and ERK activation in B cells.
T. Numaga, M. Nishida, S. Kiyonaka, K. Kato, M. Katano, E. Mori, T. Kurosaki, R. Inoue, M. Hikida, J. W. Putney Jr, et al. (2010)
J. Cell Sci. 123, 927-938
   Abstract »    Full Text »    PDF »
Protein kinase C: poised to signal.
A. C. Newton (2010)
Am J Physiol Endocrinol Metab 298, E395-E402
   Abstract »    Full Text »    PDF »
Stimulus-Specific Distinctions in Spatial and Temporal Dynamics of Stress-Activated Protein Kinase Kinase Kinases Revealed by a Fluorescence Resonance Energy Transfer Biosensor.
T. Tomida, M. Takekawa, P. O'Grady, and H. Saito (2009)
Mol. Cell. Biol. 29, 6117-6127
   Abstract »    Full Text »    PDF »
The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity.
M. T. Kunkel, E. L. Garcia, T. Kajimoto, R. A. Hall, and A. C. Newton (2009)
J. Biol. Chem. 284, 24653-24661
   Abstract »    Full Text »    PDF »
Visualization of Dopamine Transporter Trafficking in Live Neurons by Use of Fluorescent Cocaine Analogs.
J. Eriksen, S. G. F. Rasmussen, T. N. Rasmussen, C. B. Vaegter, J. H. Cha, M.-F. Zou, A. H. Newman, and U. Gether (2009)
J. Neurosci. 29, 6794-6808
   Abstract »    Full Text »    PDF »
Lipid activation of protein kinases.
A. C. Newton (2009)
J. Lipid Res. 50, S266-S271
   Abstract »    Full Text »    PDF »
Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes.
J. Goedhart and T. W.J Gadella Jr (2009)
J R Soc Interface 6, S27-S34
   Abstract »    Full Text »    PDF »
A genetically encoded fluorescent sensor of ERK activity.
C. D. Harvey, A. G. Ehrhardt, C. Cellurale, H. Zhong, R. Yasuda, R. J. Davis, and K. Svoboda (2008)
PNAS 105, 19264-19269
   Abstract »    Full Text »    PDF »
Regulation of PLC{beta}1a membrane anchoring by its substrate phosphatidylinositol (4,5)-bisphosphate.
M. J. W. Adjobo-Hermans, J. Goedhart, and T. W. J. Gadella Jr (2008)
J. Cell Sci. 121, 3770-3777
   Abstract »    Full Text »    PDF »
Dynamic Effect of Bortezomib on Nuclear Factor-{kappa}B Activity and Gene Expression in Tumor Cells.
M.-H. Sung, L. Bagain, Z. Chen, T. Karpova, X. Yang, C. Silvin, T. C. Voss, J. G. McNally, C. Van Waes, and G. L. Hager (2008)
Mol. Pharmacol. 74, 1215-1222
   Abstract »    Full Text »    PDF »
Structural Basis of Protein Kinase C Isoform Function.
S. F. Steinberg (2008)
Physiol Rev 88, 1341-1378
   Abstract »    Full Text »    PDF »
Counter Regulatory Effects of PKC{beta}II and PKC{delta} on Coronary Endothelial Permeability.
N. Gaudreault, R. M. Perrin, M. Guo, C. P. Clanton, M. H. Wu, and S. Y. Yuan (2008)
Arterioscler Thromb Vasc Biol 28, 1527-1533
   Abstract »    Full Text »    PDF »
PKC{gamma} mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling.
D. S. Verbeek, J. Goedhart, L. Bruinsma, R. J. Sinke, and E. A. Reits (2008)
J. Cell Sci. 121, 2339-2349
   Abstract »    Full Text »    PDF »
Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Forster resonance energy transfer.
K. Aoki, E. Kiyokawa, T. Nakamura, and M. Matsuda (2008)
Phil Trans R Soc B 363, 2143-2151
   Abstract »    Full Text »    PDF »
Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells.
M. Zhong, D. A. Murtazina, J. Phillips, C.-Y. Ku, and B. M. Sanborn (2008)
Biol Reprod 78, 1007-1017
   Abstract »    Full Text »    PDF »
Kinetic Analysis of the Interaction of the C1 Domain of Protein Kinase C with Lipid Membranes by Stopped-flow Spectroscopy.
D. R. Dries and A. C. Newton (2008)
J. Biol. Chem. 283, 7885-7893
   Abstract »    Full Text »    PDF »
Sarcoplasmic reticulum Ca2+ leak in heart failure: mere observation or functional relevance?.
C. H. George (2008)
Cardiovasc Res 77, 302-314
   Abstract »    Full Text »    PDF »
Phospholipase C{varepsilon} is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation.
S. Citro, S. Malik, E. A. Oestreich, J. Radeff-Huang, G. G. Kelley, A. V. Smrcka, and J. H. Brown (2007)
PNAS 104, 15543-15548
   Abstract »    Full Text »    PDF »
Calcium-dependent Regulation of Protein Kinase D Revealed by a Genetically Encoded Kinase Activity Reporter.
M. T. Kunkel, A. Toker, R. Y. Tsien, and A. C. Newton (2007)
J. Biol. Chem. 282, 6733-6742
   Abstract »    Full Text »    PDF »
A Single Residue in the C1 Domain Sensitizes Novel Protein Kinase C Isoforms to Cellular Diacylglycerol Production.
D. R. Dries, L. L. Gallegos, and A. C. Newton (2007)
J. Biol. Chem. 282, 826-830
   Abstract »    Full Text »    PDF »
G Protein-coupled Receptor Kinase and beta-Arrestin-mediated Desensitization of the Angiotensin II Type 1A Receptor Elucidated by Diacylglycerol Dynamics.
J. D. Violin, S. M. DeWire, W. G. Barnes, and R. J. Lefkowitz (2006)
J. Biol. Chem. 281, 36411-36419
   Abstract »    Full Text »    PDF »
beta-Arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes.
K. Rajagopal, E. J. Whalen, J. D. Violin, J. A. Stiber, P. B. Rosenberg, R. T. Premont, T. M. Coffman, H. A. Rockman, and R. J. Lefkowitz (2006)
PNAS 103, 16284-16289
   Abstract »    Full Text »    PDF »
Targeting Protein Kinase C Activity Reporter to Discrete Intracellular Regions Reveals Spatiotemporal Differences in Agonist-dependent Signaling.
L. L. Gallegos, M. T. Kunkel, and A. C. Newton (2006)
J. Biol. Chem. 281, 30947-30956
   Abstract »    Full Text »    PDF »
Protein Kinase C as a Therapeutic Target.
B. A. Teicher (2006)
Clin. Cancer Res. 12, 5336-5345
   Full Text »    PDF »
G-protein-coupled Receptor Kinase Specificity for beta-Arrestin Recruitment to the beta2-Adrenergic Receptor Revealed by Fluorescence Resonance Energy Transfer.
J. D. Violin, X.-R. Ren, and R. J. Lefkowitz (2006)
J. Biol. Chem. 281, 20577-20588
   Abstract »    Full Text »    PDF »
Annexin A4 Self-Association Modulates General Membrane Protein Mobility in Living Cells.
A. Piljic and C. Schultz (2006)
Mol. Biol. Cell 17, 3318-3328
   Abstract »    Full Text »    PDF »
Second Messenger Pas de Deux: The Coordinated Dance Between Calcium and cAMP.
L. N. Borodinsky and N. C. Spitzer (2006)
Sci. STKE 2006, pe22
   Abstract »    Full Text »    PDF »
A Spatiotemporally Coordinated Cascade of Protein Kinase C Activation Controls Isoform-Selective Translocation.
A. Collazos, B. Diouf, N. C. Guerineau, C. Quittau-Prevostel, M. Peter, F. Coudane, F. Hollande, and D. Joubert (2006)
Mol. Cell. Biol. 26, 2247-2261
   Abstract »    Full Text »    PDF »
TAB-1 Modulates Intracellular Localization of p38 MAP Kinase and Downstream Signaling.
G. Lu, Y. J. Kang, J. Han, H. R. Herschman, E. Stefani, and Y. Wang (2006)
J. Biol. Chem. 281, 6087-6095
   Abstract »    Full Text »    PDF »
Increased Membrane Affinity of the C1 Domain of Protein Kinase C{delta} Compensates for the Lack of Involvement of Its C2 Domain in Membrane Recruitment.
J. R. Giorgione, J.-H. Lin, J. A. McCammon, and A. C. Newton (2006)
J. Biol. Chem. 281, 1660-1669
   Abstract »    Full Text »    PDF »
Endothelial focal adhesions and barrier function.
M. H. Wu (2005)
J. Physiol. 569, 359-366
   Abstract »    Full Text »    PDF »
Rational Design of p53, an Intrinsically Unstructured Protein, for the Fabrication of Novel Molecular Sensors.
M. L. Geddie, T. L. O'Loughlin, K. K. Woods, and I. Matsumura (2005)
J. Biol. Chem. 280, 35641-35646
   Abstract »    Full Text »    PDF »
Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity.
B. Ananthanarayanan, Q. Ni, and J. Zhang (2005)
PNAS 102, 15081-15086
   Abstract »    Full Text »    PDF »
CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+.
Q. Liu, S. A. Walker, D. Gao, J. A. Taylor, Y.-F. Dai, R. S. Arkell, M. D. Bootman, H. L. Roderick, P. J. Cullen, and P. J. Lockyer (2005)
J. Cell Biol. 170, 183-190
   Abstract »    Full Text »    PDF »
Single Cell Analysis and Temporal Profiling of Agonist-mediated Inositol 1,4,5-Trisphosphate, Ca2+, Diacylglycerol, and Protein Kinase C Signaling using Fluorescent Biosensors.
P. J. Bartlett, K. W. Young, S. R. Nahorski, and R. A. J. Challiss (2005)
J. Biol. Chem. 280, 21837-21846
   Abstract »    Full Text »    PDF »
The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade.
S. Kupzig, S. A. Walker, and P. J. Cullen (2005)
PNAS 102, 7577-7582
   Abstract »    Full Text »    PDF »
Analysis by Fluorescence Resonance Energy Transfer of the Interaction between Ligands and Protein Kinase C{delta} in the Intact Cell.
D. C. Braun, S. H. Garfield, and P. M. Blumberg (2005)
J. Biol. Chem. 280, 8164-8171
   Abstract »    Full Text »    PDF »
Spatio-temporal Dynamics of Protein Kinase B/Akt Signaling Revealed by a Genetically Encoded Fluorescent Reporter.
M. T. Kunkel, Q. Ni, R. Y. Tsien, J. Zhang, and A. C. Newton (2005)
J. Biol. Chem. 280, 5581-5587
   Abstract »    Full Text »    PDF »
Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments.
L. M. DiPilato, X. Cheng, and J. Zhang (2004)
PNAS 101, 16513-16518
   Abstract »    Full Text »    PDF »
PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo.
J. J Eckert, A. McCallum, A. Mears, M. G Rumsby, I. T Cameron, and T. P Fleming (2004)
Reproduction 127, 653-667
   Abstract »    Full Text »    PDF »
Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses.
M. Zaccolo (2004)
Circ. Res. 94, 866-873
   Abstract »    Full Text »    PDF »
Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs.
G. Halet, R. Tunwell, S. J. Parkinson, and J. Carroll (2004)
J. Cell Biol. 164, 1033-1044
   Abstract »    Full Text »    PDF »
Modulation of Histamine-induced Ca2+ Release by Protein Kinase C: EFFECTS ON CYTOSOLIC AND MITOCHONDRIAL [Ca2+] PEAKS.
M. Montero, C. D. Lobaton, S. Gutierrez-Fernandez, A. Moreno, and J. Alvarez (2003)
J. Biol. Chem. 278, 49972-49979
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882