Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity

Abstract

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. Mass spectrometry identifies novel autophosphorylation sites in seven Src family kinases. A phosphomimetic mutant of Fyn at Tyr440 lowers Fyn kinase activity. Fyn SH2 phosphomimetics increase the phosphorylation of cellular substrates. Fyn SH2 autophosphorylation reduces its phosphotyrosine-dependent interactions. Homologous SH2 tyrosines are commonly phosphorylated when in cis with kinase domains.