An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

• Published in: Nature communications Vol. 14; no. 1; p. 6548
• Main Authors: Cuesta-Hernández, Hipólito Nicolás, Contreras, Julia, Soriano-Maldonado, Pablo, Sánchez-Wandelmer, Jana, Yeung, Wayland, Martín-Hurtado, Ana, Muñoz, Inés G, Kannan, Natarajan, Llimargas, Marta, Muñoz, Javier, Plaza-Menacho, Iván
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 17.10.2023; Nature Publishing Group UK; Nature Portfolio
• Subjects: Allosteric properties; Colorectal carcinoma; Crystallography; Enzymes; Kinases; Kinetics; Perturbation; Phosphorylation; Protein kinase; Proteins; Src protein; Substrate inhibition; Substrates; X-ray crystallography
• DOI: 10.1038/s41467-023-41890-7
• ISSN: 2041-1723

Abstract Autophosphorylation 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 phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show 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 substrate- and enzyme-acting kinases during autophosphorylation.