Abstract
Protein kinase C (PKC) signaling drives many important cellular processes and its dysregulation results in pathophysiologies such as cancer (Gokmen-Polar et al., Cancer Res 61:1375–1381, 2001). Because PKC is activated acutely and allosterically, it is difficult to monitor the cellular activity of endogenous PKC by conventional methodologies (Newton, Methods Enzymol 345:499–506, 2002). Rather, PKC signaling is best studied in situ using biosensors such as FRET-based reporters. We have generated several FRET-based reporters for studying PKC signaling in real time in live cells (Violin and Newton, IUBMB Life 55:653–660, 2003). Using these reporters, we have demonstrated phase-locked oscillations in Ca2+ release and membrane-localized endogenous PKC activity in response to histamine (Violin et al., J Cell Biol 161:899–909, 2003), as well as distinct signatures of endogenous PKC signaling at specific organelles in response to uridine-5′-triphosphate (UTP; Gallegos et al., J Biol Chem 281:30947–30956, 2006). Here we describe methods to image cells expressing the reporters and elaborate on data analyses, control experiments, and variations for imaging the activity of expressed PKC.
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References
Manning, B. D. (2009) Challenges and opportunities in defining the essential cancer kinome. Sci Signal 2, pe15.
Newton, A. C. (2003) Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J 370, 361–71.
Suzuki, A., and Ohno, S. (2006) The PAR-aPKC system: lessons in polarity. J Cell Sci 119, 979–87.
Gallegos, L. L., and Newton, A. C. (2008) Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm. IUBMB Life 60, 782–9.
Newton, A. C. (2008) Protein kinase C. IUBMB Life 60, 765–8.
Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 161, 899–909.
Gallegos, L. L., Kunkel, M. T., and Newton, A. C. (2006) Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling. J Biol Chem 281, 30947–56.
Mattingly, R. R. (2003) Mitogen-activated protein kinase signaling in drug-resistant neuroblastoma cells. Methods Mol Biol 218, 71–83.
Newton, A. C. (2002) Analyzing protein kinase C activation. Methods Enzymol 345, 499–506.
Pan, T. T., Neo, K. L., Hu, L. F., Yong, Q. C., and Bian, J. S. (2008) H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes. Am J Physiol Cell Physiol 294, C169-77.
Hosoda, K., Saito, N., Kose, A., Ito, A., Tsujino, T., Ogitat, K., Kikkawat, U., Onot, Y., Igarashif, K., Nishizukat, Y., and Tanaka, C. (1989) Immunocytochemical localization of the beta I subspecies of protein kinase C in rat brain. Proc Natl Acad Sci U S A 86, 1393–7.
Saito, N., Kose A, Ito A, Hosoda, K., Mori, M., Hirata, M., Ogitat, K., Kikkawat, U., Onot, Y., Igarashif, K., Nishizukat, Y., and Tanaka, C. (1989) Immunocytochemical localization of beta II subspecies of protein kinase C in rat brain. Proc Natl Acad Sci U S A 86, 3409–13.
Miyawaki, A. (2008) Green fluorescent protein glows gold. Cell 135, 987–90.
Sakai, N., Sasaki, K., Ikegaki, N., Shirai, Y., Ono, Y., and Saito, N. (1997) Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J Cell Biol 139, 1465–76.
Oancea, E., and Meyer, T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307–18.
Wang, Q. J., Bhattacharyya, D., Garfield, S., Nacro, K., Marquez, V. E., and Blumberg, P. M. (1999) Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein. J Biol Chem 274, 37233–9.
Zhang, J., Ma, Y., Taylor, S. S., and Tsien. R. Y. (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98, 14997–5002.
Wang, J., Gambhir, A., Hangyas-Mihalyne, G., Murray, D., Golebiewska, U., and McLaughlin, S. (2002) Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J Biol Chem 277, 34401–12.
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., and Tsien, R. Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–72.
Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J Biol Chem 282, 826–30.
Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis. Cancer Res 61, 1375–81.
Violin, J. D., and Newton, A. C. (2003) Pathway illuminated: visualizing protein kinase C signaling. IUBMB Life 55, 653–60.
Kajimoto, T., Sawamura, S., Tohyama, Y., Mori, U., and Newton AC. (2010) Protein kinase C δ-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases. J Biol Chem 285, 41896–910.
Acknowledgments
We thank Maya Kunkel for critical reading of this manuscript. This work was supported by National Institutes of Health grants GM43154 and PO1 DK54441.
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Gallegos, L.L., Newton, A.C. (2011). Genetically Encoded Fluorescent Reporters to Visualize Protein Kinase C Activation in Live Cells. In: Luttrell, L., Ferguson, S. (eds) Signal Transduction Protocols. Methods in Molecular Biology, vol 756. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-160-4_17
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DOI: https://doi.org/10.1007/978-1-61779-160-4_17
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