Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56 Lck regulates T-cell activation independently
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RESEARCH
Tyrosine 192 within the SH2 domain of the Src‑protein tyrosine kinase p56Lck regulates T‑cell activation independently of Lck/ CD45 interactions Matthias Kästle1,2, Camilla Merten1,2, Roland Hartig1,2, Thilo Kaehne3, Ardiyanto Liaunardy‑Jopeace4, Nadine M. Woessner5,6,7, Wolfgang W. Schamel6,7, John James4,8, Susana Minguet6,7, Luca Simeoni1,2*† and Burkhart Schraven1,2*†
Abstract Background: Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phos‑ phorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods: Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemi‑ cal characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results: Co-immunoprecipitation studies and biochemical analyses using T cells from L ckY192E knock-in mice revealed a diminished binding of L ckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborat‑ ing previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that L ckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an L ckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when L ckY192E was expressed in C D45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, L ckY19E was recruited less to CD3 after TCR stimulation. Conclusions: Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by prevent‑ ing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.
*Correspondence: [email protected]; Burkhart.schraven@med. ovgu.de † Luca Simeoni and Burkhart Schraven have contributed equally to this work 1 Institute of Molecular and Clinical Immunology, Medical Faculty, Ottovon-Guericke University, Leipziger Str.44, Building 26, 39120 Magdeburg, Germany Full list of author information is available at the end of the article © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licen
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