METHODS: Lung cancer cells were cultured following the standard protocol and the cell lysates were prepared to detect the given proteins by Western Blot analysis, and the protein interaction was assayed by co-immunoprecipitation (Co-IP) or GST pull-down assay. CFLARL ubiquitination level was evaluated by proteasomal inhibitor treatment combined with HA-Ub transfection and WB assay. PRMT1 and PRMT5 genes were knocked down by siRNA technique.
RESULTS: We show that PRMT5 up-regulated the protein levels of CFLARL by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLARL by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLARL and ITCH, which has been identified as an E3 ubiquitin ligase of CFLARL, while overexpressed PRMT1 enhances the interaction between CFLARL and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLARL as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLARL degradation other than its enzymatic activity. Finally, we showed that PRMT5 and PRMT1 could suppress or facilitate apoptosis induced by doxorubicin or pemetrexed by affecting CFLARL in NSCLC cells.
CONCLUSIONS: PRMT5 and PRMT1 mediate the distinct effects on CFLARL degradation by regulating the binding of E3 ligase ITCH in NSCLC cells. This study identifies a cell death mechanism that is fine-tuned by PRMT1/5 that modulate CFLARL degradation in human NSCLC cells.