Cuesta-Hernández, Hipólito NicolásContreras, JuliaSoriano Maldonado, PabloSánchez Wandelmer, JanaYeung, WaylandMartín-Hurtado, AnaMuñoz, Inés G.Kannan, NatarajanLlimargas, MartaMuñoz, JavierPlaza-Menacho, Iván2023-12-152023-12-1520232041-1723https://hdl.handle.net/10641/3593Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phosphomotif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants.Weshow that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrateand enzyme-acting kinases during autophosphorylation.engAtribución-NoComercial-SinDerivadas 3.0 Españahttp://creativecommons.org/licenses/by-nc-nd/3.0/es/journal articleopen access10.1038/s41467-023-41890-7