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
Description
Leishmania donovani subverts macrophage signaling to establish intracellular infection; however, the contribution of adaptor proteins regulating phosphotyrosine-dependent signaling remains poorly understood. 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. Functionally, macrophages expressing Dok-3_4F harboured significantly higher intracellular parasite burdens than Dok-3_WT cells. Loss of Dok-3 phosphorylation was accompanied by impaired activation of the p38 MAPK–STAT1 pathway, enhanced ERK1/2 signaling, and altered endosomal trafficking characterized by reduced association of parasites with EEA1-positive compartments and increased localization within Rab7-positive compartments. Collectively, these findings identify Dok-3 tyrosine phosphorylation as a previously unrecognized regulator of macrophage defence against L. donovani. By coordinating phosphatase recruitment, host signaling, and intracellular trafficking, phosphorylated Dok-3 functions as a critical molecular checkpoint restricting parasite survival and represents a potential target for host-directed therapeutic strategies against visceral leishmaniasis.
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Steps to reproduce
The tertiary structure of mouse Dok3 protein was generated using the AlphaFold Server (https://alphafoldserver.com), powered by AlphaFold3, with the FASTA sequence obtained from the UniProt database (ID: Q9QZK7). The phosphorylated Dok3 was generated using the ‘Post-Translational Modifications’ module in AlphaFold3 by introducing ‘O-Phospho-L-tyrosine’ modifications at residues Y325, Y343, Y378, and Y399. A phosphorylation deficient mutant (Dok3_4F) was generated by substituting these four tyrosine (Y) residues with phenylalanine residues (F) in protein sequence. To evaluate the protein-protein interactions, separate complexes of wild-type, phosphorylated and mutant Dok3 proteins were generated with SHIP1 (Uniprot ID: Q9ES52) and SHPTP (Uniprot ID: P29351) using AlphaFold 3 with their respective FASTA sequences. Multiple random seeds were run for each structure complex using the default parameters to improve the overall model quality. For each protein complex, five models were generated for each complex, and the top-ranked model was selected for further analyses based on the highest prediction confidence.
Institutions
- University of CalcuttaWest Bengal, Kolkata
- St. Xavier's College (Autonomous), KolkataWest Bengal, Kolkata