title: Mimicking transient activation of protein kinases in living cells.
abstract: Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two protein-engineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1- and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612.
authors.forename: Jennifer E
authors.identifier: 0000-0003-1781-648X
authors.initials: JE
authors.lastname: Klomp
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612.
authors.forename: Vincent
authors.initials: V
authors.lastname: Huyot
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Ray
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612.
authors.forename: Kerrie B
authors.initials: KB
authors.lastname: Collins
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612.
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.affiliation: Department of Pharmacology, University of Illinois, Chicago, IL 60612 karginov@uic.edu.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Karginov
mesh_terms: D000081247:CSK Tyrosine-Protein Kinase; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015202:Protein Engineering; D015398:Signal Transduction; D058615:Synthetic Biology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000081247:CSK Tyrosine-Protein Kinase; D019869:Phosphatidylinositol 3-Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases; C000631346:CSK protein, human
keywords: Src; kinase; phosphorylation; signaling; synthetic biology