257
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_16, © Springer Science+Business Media LLC 2017
Chapter 16
Light-Regulated Protein Kinases Based
on the CRY2-CIB1 System
Wignand W.D. Mühlhäuser, Maximilian Hörner, Wilfried Weber,
and Gerald Radziwill
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.
Key words Optogenetics, Signal transduction, Protein kinases, Membrane recruitment, AKT, CRY2
1 Introduction
Protein kinases play a key role in orchestrating the complex cellular
signal transduction machinery. By phosphorylation they can influence activity, localization, and/or the interactome of these signaling complexes. Thereby, protein kinases significantly influence cell
fate decisions (e.g., proliferation vs. differentiation; survival vs.
apoptosis). For this reason, it is not surprising that protein kinases
themselves underlie a tight regulation. Reoccurring mechanisms
comprise regulation by other kinases, by local recruitment to
specific subcellular compartments or clustering (e.g., dimerization) of kinases [1].
Conventional approaches, which try to mimic activation
mechanisms, often employ chemical inducers or mutants. Major
drawbacks of those approaches lie in their poor or altogether missing spatiotemporal resolution and, in certain cases, unwanted or
unknown side effects in the case of chemical inducers. Therefore,
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_16, © Springer Science+Business Media LLC 2017
Chapter 16
Light-Regulated Protein Kinases Based
on the CRY2-CIB1 System
Wignand W.D. Mühlhäuser, Maximilian Hörner, Wilfried Weber,
and Gerald Radziwill
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.
Key words Optogenetics, Signal transduction, Protein kinases, Membrane recruitment, AKT, CRY2
1 Introduction
Protein kinases play a key role in orchestrating the complex cellular
signal transduction machinery. By phosphorylation they can influence activity, localization, and/or the interactome of these signaling complexes. Thereby, protein kinases significantly influence cell
fate decisions (e.g., proliferation vs. differentiation; survival vs.
apoptosis). For this reason, it is not surprising that protein kinases
themselves underlie a tight regulation. Reoccurring mechanisms
comprise regulation by other kinases, by local recruitment to
specific subcellular compartments or clustering (e.g., dimerization) of kinases [1].
Conventional approaches, which try to mimic activation
mechanisms, often employ chemical inducers or mutants. Major
drawbacks of those approaches lie in their poor or altogether missing spatiotemporal resolution and, in certain cases, unwanted or
unknown side effects in the case of chemical inducers. Therefore,