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Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLC\u03b31 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70C564R, is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70C564A displayed the hyperphosphorylation of TCR-\u03b6 and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.<\/jats:p>","DOI":"10.3390\/cells11172723","type":"journal-article","created":{"date-parts":[[2022,9,1]],"date-time":"2022-09-01T21:27:17Z","timestamp":1662067637000},"page":"2723","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["A Cysteine Residue within the Kinase Domain of Zap70 Regulates Lck Activity and Proximal TCR Signaling"],"prefix":"10.3390","volume":"11","author":[{"given":"Annika","family":"Schultz","sequence":"first","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marvin","family":"Schnurra","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ali","family":"El-Bizri","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0650-8406","authenticated-orcid":false,"given":"Nadine M.","family":"Woessner","sequence":"additional","affiliation":[{"name":"Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79085 Freiburg, Germany"},{"name":"Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 79085 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sara","family":"Hartmann","sequence":"additional","affiliation":[{"name":"Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79085 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3706-7458","authenticated-orcid":false,"given":"Roland","family":"Hartig","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8211-5538","authenticated-orcid":false,"given":"Susana","family":"Minguet","sequence":"additional","affiliation":[{"name":"Faculty of Biology, Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79085 Freiburg, Germany"},{"name":"Center of Chronic Immunodeficiency CCI, University Clinics and Medical Faculty, 79106 Freiburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4321-7405","authenticated-orcid":false,"given":"Burkhart","family":"Schraven","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"},{"name":"Health Campus Immunology, Infectiology and Inflammation (GC-I3), Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"},{"name":"Center for Health and Medical Prevention (CHaMP), Otto-von-Guericke-University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luca","family":"Simeoni","sequence":"additional","affiliation":[{"name":"Institute of Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"},{"name":"Health Campus Immunology, Infectiology and Inflammation (GC-I3), Medical Faculty, Otto-von-Guericke University, 39120 Magdeburg, Germany"},{"name":"Center for Health and Medical Prevention (CHaMP), Otto-von-Guericke-University, 39120 Magdeburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.tibs.2017.11.008","article-title":"TCR Signaling: Mechanisms of Initiation and Propagation","volume":"43","author":"Courtney","year":"2018","journal-title":"Trends Biochem. 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