Elucidating the role of TRAIL-R2 tyrosine phosphorylation in mediating trail resistance in the K562 cell line

Objective: Death receptors are the key mediators used by immune system to eliminate malignant cells. TRAIL, effectively induces apoptosis in various human cancer cells through its receptors. However, TRAIL-resistance in some tumor cells, or development of resistance following prolonged exposure, allow cells to evade apoptosis. K562 cells are normally TRAIL-resistant, but can acquire TRAIL-sensitivity following prolonged treatment with imatinib. Therefore, tyrosine phosphorylation of TRAIL-R2, potentially mediated by an as-yet unidentified kinase, may contribute to the activation of proliferative pathways and TRAIL-resistance. In this study, we aimed to investigate the tyrosine phosphorylation of TRAIL-R2 in K562 cells, to identify the tyrosine kinase responsible, and to evaluate the impact of this phosphorylation on TRAIL sensitivity. Method: TRAIL-R2 tyrosine phosphorylation in K562 and HeLa, and potential associated kinases were assessed through immunoprecipitation using TRAIL-R2 and PY20 antibodies, followed by western blotting. The effects of sodium orthovanadate, inhibitors and TRAIL-R2 mutants mimicking or blocking tyrosine phosphorylation, on TRAIL sensitivity were evaluated via Resazurin assay. Results: TRAIL-R2 tyrosine phosphorylation in K562 cells remained constant upon TRAIL treatment. TRAIL-R2 is found to be part of complexes with c-Src, JAK2, and Abl. However, tyrosine phosphorylation appears to be largely regulated by JAK2 and Src family kinases, rather than directly by Abl. Treatment with imatinib, dasatinib, JAK inhibitors, and PP1 resulted in significant decreased phosphorylation. In contrast, Abl activity was found to be low in imatinib-resistant cells, yet tyrosine phosphorylation of TRAIL-R2 was preserved. Moreover, co-treatment with Na₃VO₄ and TRAIL or imatinib significantly enhanced cell death in wild-type cells, while no such effect was observed in TRAIL-R2 knockout cells. Conclusion: In K562 cells, TRAIL-R2 tyrosine phosphorylation is constitutive, predominantly regulated by Src and JAK2 kinases rather than Abl. The cytotoxic effect of Na₃VO₄ in K562 cells appears to involve mechanism dependent on TRAIL-R2 and Abl tyrosine kinase activity. Key words: TRAIL-R2, tyrosine phosphorylation, TRAIL resistance in K562 cells.