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* Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria;
Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Linz, Austria;
Competence Center for Biomolecular Therapeutics Research, Vienna, Austria; and
Biophysics Institute, Johannes Kepler University, Linz, Austria
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 Tyr505 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 Tyr505 to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr394 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 Tyr505 and Tyr394 phosphorylation and conformational alterations.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by the GEN-AU Program of the Austrian Federal Ministry of Science and Research, the Austrian Science Fund, and the PhD program CCHD.
2 W.P. and C.P. contributed equally to this work.
3 Present address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, U.K.
4 Address correspondence and reprint requests to Dr. Hannes Stockinger, Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Lazarettgasse 19, 1090 Vienna, Austria. E-mail address: hannes.stockinger{at}meduniwien.ac.at or Dr. Alois Sonnleitner, Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstrasse 6-8, 4020 Linz, Austria. E-mail address: alois.sonnleitner{at}uar.at
5 Abbreviations used in this paper: SFK, Src family kinase; Eapp, apparent FRET efficiency; CLckY, construct ECFP-Lck-EYFP; CLck-2, donor-only tagged Lck; LckY, acceptor-only tagged Lck; ECYFP, fused ECFP and EYFP; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, Förster resonance energy transfer; pTyr, phosphotyrosine; SH, Src homology (domain); TIRF, total internal reflection fluorescence.
6 The online version of this article contains supplemental material.
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