Spontaneous autophosphorylation of Lyn tyrosine kinase at both its activation segment and C-terminal tail confers altered substrate specificity.

Two tyrosyl residues have been reported to play a crucial role in the regulation of protein tyrosine kinases of the Src family: autophosphorylation of Tyr416 (c-Src numbering) located in the catalytic domain correlates with enzyme activation, while Csk-mediated phosphorylation of the C-terminal tyrosine Tyr527 (c-Src numbering) gives rise to inactive forms of Src kinases. Here we show that the Src-related Lyn kinase undergoes spontaneous and stoichiometric autophosphorylation at both Tyr396 (homologous to c-Src Tyr416) and Tyr507 (homologous to c-Src Tyr527). Such a doubly autophosphorylated form of Lyn is hyperactive toward peptide substrates and insensitive to Csk-induced downregulation. In contrast, doubly autophosphorylated Lyn exhibits reduced activity toward protein substrates such as phospho-p50/HS1 (hematopoietic-lineage cell-specific protein) and p57/PDI (protein disulfide isomerase related protein), whose multiple sequential/processive phosphorylation relies on the accessibility of the SH2 domain of the kinase. These data disclose a novel conformation of Lyn that is catalytically active despite the presence of an intramolecular interaction between the phosphorylated tail and the SH2 domain. This enzyme conformation is expected to display a reduced oncogenic potential resulting from its defective recognition of a subset of protein substrates whose targeting is mediated by the Lyn SH2 domain.