Molecular determinants of differential substrate selection between the Src family kinases Lck and Src.

Src family kinases (SFKs) share highly conserved catalytic domains yet display distinct biological functions, raising the question of how substrate specificity is achieved. Here, we investigate the basis of differential ITAM recognition by Lck and Src, using complementary cellular and computational approaches. In-cell assays demonstrated that, contrary to Lck, Src was completely incapable of phosphorylating the TCR ITAMs when ectopically expressed in a T cell environment. Domain-swapping experiments further revealed that substitution of the Src kinase domain with that of Lck was sufficient to confer ITAM phosphorylation and trigger downstream TCR signaling responses, whereas exchange of adaptor domains had minimal effect. To gain structural insight into this selectivity, molecular dynamics simulations followed by ensemble docking were performed using conformations sampled from simulations of the Lck and Src kinase domains together with an ITAM peptide. The analyses revealed that Lck consistently accommodates the ITAM peptide with higher shape complementarity than Src, supporting a more favorable structural environment for ITAM recognition. Our findings demonstrate that subtle differences within the kinase domain of closely related SFKs can profoundly influence substrate recognition, with significant consequences for signaling responses in living cells.