title: De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling.
abstract: The enzymatic activity of the Src family tyrosine kinase p56Lck(Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394and Tyr505Phosphorylation of Tyr394and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr394to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr394plays a role in Lck function that goes beyond inducing an open conformation of the kinase.
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Philipsen
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Amarendra V
authors.initials: AV
authors.lastname: Reddycherla
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Hartig
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Gumz
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kästle
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kritikos
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany.
authors.forename: Mateusz P
authors.initials: MP
authors.lastname: Poltorak
authors.affiliation: Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany.
authors.forename: Yury
authors.initials: Y
authors.lastname: Prokazov
authors.affiliation: Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany.
authors.forename: Evgeny
authors.initials: E
authors.lastname: Turbin
authors.affiliation: Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Weber
authors.affiliation: Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Zuschratter
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. andreas.mueller@med.ovgu.de luca.simeoni@med.ovgu.de burkhart.schraven@med.ovgu.de.|Department of Immune Control, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Burkhart
authors.initials: B
authors.lastname: Schraven
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. andreas.mueller@med.ovgu.de luca.simeoni@med.ovgu.de burkhart.schraven@med.ovgu.de.
authors.forename: Luca
authors.initials: L
authors.lastname: Simeoni
authors.affiliation: Institute of Molecular and Clinical Immunology, Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany. andreas.mueller@med.ovgu.de luca.simeoni@med.ovgu.de burkhart.schraven@med.ovgu.de.|Intravital Microscopy in Infection and Immunity, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Müller
mesh_terms: D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)