Abstract
Optogenetic approaches enable the control of biological processes in a time- and space-resolved manner. These light-based methods are noninvasive and by using light as sole activator minimize side effects in contrast to chemical inducers. Here, we provide a protocol for the targeted control of the activity of protein kinases in mammalian cells based on the photoreceptor cryptochrome 2 (CRY2) of Arabidopsis thaliana and its interaction partner CIB1. Blue light (450 nm)-induced binding of CRY2 to CIB1 allows the recruitment of a chimeric cytosolic protein kinase AKT1 to the plasma membrane accompanied with stimulation of its kinase activity. This protocol comprises the transient and stable implementation of the light-regulated system into mammalian cells and its stimulation by blue light-emitting diodes (450 nm) irradiation as well as analysis of the light-activated AKT1.
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Acknowledgments
This work was supported by the Excellence Initiative of the German Federal and State Governments (EXC-294, BIOSS – Centre for Biological Signaling). We thank J. Schmidt, D. Schächtele, and J. Meßmer (University of Freiburg) for designing and constructing the illumination boxes.
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Mühlhäuser, W.W.D., Hörner, M., Weber, W., Radziwill, G. (2017). Light-Regulated Protein Kinases Based on the CRY2-CIB1 System. In: Stein, V. (eds) Synthetic Protein Switches. Methods in Molecular Biology, vol 1596. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6940-1_16
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DOI: https://doi.org/10.1007/978-1-4939-6940-1_16
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