title: Genetically encoded Förster resonance energy transfer sensors for the conformation of the Src family kinase Lck.
abstract: The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr(505) upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr(505) to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr(394) to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr(505) and Tyr(394) phosphorylation and conformational alterations.
authors.affiliation: Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Paster
authors.forename: Christian
authors.initials: C
authors.lastname: Paar
authors.forename: Paul
authors.initials: P
authors.lastname: Eckerstorfer
authors.forename: Andrea
authors.initials: A
authors.lastname: Jakober
authors.forename: Karel
authors.initials: K
authors.lastname: Drbal
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.forename: Hannes
authors.initials: H
authors.lastname: Stockinger
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D020836:Protein Structure, Quaternary / Q000502:physiology; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000737:chemistry / Q000201:enzymology*
chemical_list: D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)