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Src Regulates Tyr20 Phosphorylation of Transferrin Receptor-1 and Potentiates Breast Cancer Cell Survival*

Background: The role of transferrin receptor-1 (TfR1) in cell signaling remains unknown. Results: Using gambogic acid, we found that TfR1 is bound to Src and its phosphorylation at Tyr20 potentiates breast cancer cell survival. Conclusion: In addition to its iron uptake function, TfR1 is involved in cell survival. Significance: High expression of TfR1 in cancer cells presents a therapeutic target for gambogic acid and Src inhibitor. Transferrin receptor 1 (TfR1) is a ubiquitous type II membrane receptor with 61 amino acids in the N-terminal cytoplasmic region. TfR1 is highly expressed in cancer cells, particularly under iron deficient conditions. Overexpression of TfR1 is thought to meet the increased requirement of iron uptake necessary for cell growth. In the present study, we used transferrin (Tf), a known ligand of TfR1, and gambogic acid (GA), an apoptosis-inducing agent and newly identified TfR1 ligand to investigate the signaling role of TfR1 in breast cancer cells. We found that GA but not Tf induced apoptosis in a TfR1-dependent manner in breast cancer MDA-MB-231 cells. Estrogen receptor-positive MCF-7 cells lack caspase-3 and were not responsive to GA treatment. GA activated the three major signaling pathways of the MAPK family, as well as caspase-3, -8, and Poly(ADP-ribose)polymerase apoptotic pathway. Interestingly, only Src inhibitor PP2 greatly sensitized the cells to GA-mediated apoptosis. Further investigations by confocal fluorescence microscopy and immunoprecipitation revealed that Src and TfR1 are constitutively bound. Using TfR1-deficient CHO TRVB cells, point mutation studies showed that Tyr20 within the 20YTRF23 motif of the cytoplasmic region of TfR1 is the phosphorylation site by Src. TfR1 Tyr20 phosphomutants were more sensitive to GA-mediated apoptosis. Our results indicate that, albeit its iron uptake function, TfR1 is a signaling molecule and tyrosine phosphorylation at position 20 by Src enhances anti-apoptosis and potentiates breast cancer cell survival.