The arginine methyltransferase PRMT5 and PRMT1 distinctly regulate the degradation of anti-apoptotic protein CFLAR_L in human lung cancer cells

Background: CFLAR_L, also known as c-FLIP_L, is a critical anti-apoptotic protein that inhibits activation of caspase 8 in mammalian cells. Previous studies have shown that arginine 122 of CFLAR_L can be mono-methylated. However, the precise role of arginine methyltransferase of CFLAR_L remains unknown. PRMT5 and PRMT1, which are important members of the PRMT family, catalyze the transfer of methyl groups to the arginine of substrate proteins. PRMT5 can monomethylate or symmetrically dimethylate arginine residues, while PRMT1 can monomethylate or asymmetrically dimethylate arginine residues.

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. CFLAR_L 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 CFLAR_L by decreasing the ubiquitination and increasing its protein level. Additionally, PRMT1 down-regulated the protein level of CFLAR_L by increasing the ubiquitination and degradation. The overexpression of PRMT5 can inhibit the interaction between CFLAR_L and ITCH, which has been identified as an E3 ubiquitin ligase of CFLAR_L, while overexpressed PRMT1 enhances the interaction between CFLAR_L and ITCH. Furthermore, we verified that dead mutations of PRMT5 or PRMT1 have the same effects on CFLAR_L as the wild-type ones have, suggesting it is the physical interaction between CFLAR and PRMT1/5 that regulates CFLAR_L 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 CFLAR_L in NSCLC cells.

Conclusions: PRMT5 and PRMT1 mediate the distinct effects on CFLAR_L 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 CFLAR_L degradation in human NSCLC cells.