Abstract
The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.