Phospotyrosine proteomics reveals novel Zap70 and Itk pathway targets downstream of TCR and CAR in Jurkat T cells

{zeta}-associated protein of 70 kDa (Zap70) and interleukin-2-inducible T cell kinase (Itk) propagate the primary and CD28-integrated phosphotyrosine (pY) signalling, respectively, to achieve full T cell activation. Despite their canonical roles in T cell activation, our understanding of how each kinase controls canonical and noncanonical pY signalling is incomplete. Here, using three T cell activation methods (soluble antibodies, APC- pMHC/TCR, and CD19-CAR/Raji), we evaluated the effects of two novel inhibitors, RDN2150 (RDN, Zap70) and Soquelitinib (Soq, Itk), on T cell activation. We validated the published working concentrations of RDN and Soq on phosphorylation of key T cell signalling proteins and on T cell activation markers, finding that RDN provides more complete inhibition of T cell signalling and activation. We used LC-MS/MS to evaluate how RDN and Soq treatment affected the phosphotyrosine (pY) signalling and proteome of T cells, finding that RDN, as opposed to Soq, completely downregulated the TCR signalling pathway. Finally, we identified new, noncanonical pY sites responsive to RDN and Soq, providing new insights into the pathways regulated by Zap70 and Itk. Together, our work provides a basis for further study on RDN and Soq, as well as a molecular roadmap for the effects of these inhibitors.