title: Light-Regulated Protein Kinases Based on the CRY2-CIB1 System.
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.
authors.affiliation: Faculty of Biology and BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 18, 79104, Freiburg, Germany.
authors.forename: Wignand W D
authors.initials: WW
authors.lastname: Mühlhäuser
authors.affiliation: Faculty of Biology and BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 18, 79104, Freiburg, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Hörner
authors.affiliation: Faculty of Biology and BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 18, 79104, Freiburg, Germany.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weber
authors.affiliation: Faculty of Biology and BIOSS - Centre for Biological Signalling Studies, University of Freiburg, Schänzlestr. 18, 79104, Freiburg, Germany. gerald.radziwill@bioss.uni-freiburg.de.
authors.forename: Gerald
authors.initials: G
authors.lastname: Radziwill
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D056931:Cryptochromes / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008027:Light; D061986:MCF-7 Cells; D008322:Mammals / Q000378:metabolism; D062308:Optogenetics / Q000379:methods; D015221:Potassium Channels / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism*; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14 / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D051792:Basic Helix-Loop-Helix Proteins; D056931:Cryptochromes; D015221:Potassium Channels; D011494:Protein Kinases; D053326:Tumor Necrosis Factor Ligand Superfamily Member 14; C533832:CIB1 protein, Arabidopsis; C103618:CRY2 protein, Arabidopsis; C074500:AKT1 protein, Arabidopsis
keywords: AKT; CRY2; Membrane recruitment; Optogenetics; Protein kinases; Signal transduction