Tyrosine phosphorylation of Downstream of kinase 3 (Dok-3) plays crucial role in Leishmania donovani infection by regulating inhibitory proteins, SH-PTP-1 and SHIP-1 in macrophages

Downstream of kinase-3 (Dok-3) is a hematopoietic adaptor protein whose function depends on phosphorylation of conserved C-terminal tyrosine residues. Here, we examined the significance of Dok-3 tyrosine phosphorylation using RAW264.7 macrophages stably expressing wild-type Dok-3 (Dok-3_WT) or a phosphorylation-deficient mutant (Dok-3_4F), in which four conserved tyrosine residues (Y325, Y343, Y378, and Y399) were substituted with phenylalanine.

Biochemical analyses demonstrated that Dok-3 tyrosine phosphorylation promotes stable association with SHIP-1 and, for the first time, identifies SHPTP-1 as a Dok-3-interacting partner in macrophages. AlphaFold3-based structural modelling further predicted that phosphorylation markedly strengthens the Dok-3–SHIP-1 interaction by increasing interface size, hydrogen-bond formation, and binding stability, while exerting a comparatively modest effect on SHPTP-1 binding.