Lymphocyte-specific tyrosine-protein kinase Lck dimers boost T-cell antigen receptor signaling by trans-activatory auto-phosphorylation
Philipp Schatzlmaier1, Florian Baumgart2, Paul Eckerstorfer1, Sophie Kraupp1, Gerhard Schütz2, Hannes Stockinger1
1Institut für Hygiene und Angewandte Immunologie, Medizinische Universität Wien, Wien, Österreich
2Institut für Angewandte Physik, Technische Universität Wien, Wien, Österreich
Engagement of the T-cell antigen receptor (TCR) initiates a signaling cascade resulting in T-cell activation and differentiation. Intracellular lymphocyte-specific kinase (Lck) plays a pivotal role in this process, transducing TCR/CD3 stimulation into tyrosine phosphorylation, calcium fluxing, synapse formation, and altered gene expression. Lck activity is regulated on multiple intercalated levels, including its subcellular localization, lipid anchorage, 2-D micro-domain distribution within the plasma membrane, and its phosphorylation status that is directly linked to its enzymatic activity. Another potential mechanism of Lck regulation conceptualized in reviews but not fully investigated is its homo-dimerization leading to trans-activatory auto-phosphorylation.
Employing Blue Native gel electrophoresis, we found a fraction of endogenous Lck signals at dimeric and higher-order complex size. Co-immunoprecipitation experiments with differently tagged Lck proteins confirmed self-association of membrane-anchored Lck molecules. Via TOCCSL, a super-resolution imaging technique, we depicted a significant amount of Lck-mEGFP dimers in living T-cells. To further investigate the role of Lck homo-dimers during T-cell signaling, we established an inducible Lck-dimerization system in human Jurkat T-cells after CRISPR/Cas9 knock-out of endogenous Lck. Controlled and specific dimerization of Lck by a membrane-permeable X-linking agent significantly altered its phospho-status and enzymatic activity in a titratable fashion, modulating early TCR signaling events like calcium fluxing as well as late-stage CD69 surface expression. In conclusion, homo-dimerization of Lck represents a novel regulatory mechanism for controlling Lck kinase activity and thus thresholds for T-cell signaling.
Keywords: Adaptive immunity, Cell signalling, Molecular immunologyback to: Institute for Hygiene and Applied Immunology