Suppression Of PRMT6-mediated Arginine Methylation Of P16Protein Potentiates Its Ability To Arrest A549Cell Proliferation

As a cyclin-dependent kinase inhibitor, the tumor suppressor p16INK4A(p16) forms acomplex with CDK4and CDK6to prevent them from interacting with cyclin D, thusinhibiting the phosphorylation of the retinoblastoma protein and blocking the cell cycleprogression. We describe here a novel aspect of the posttranslational control that has animportant functional consequence on p16protein. We first discovered that the p16protein wasmethylated in various cell lineages. We then determined that the arginine22,131and138ofp16were the methylation sites, because mutations of these arginine residues to lysine resultedin hypomethylation of p16. Western blotting and TUNEL analyses revealed that the p16protein bearing these point mutations (p16KKK) induced a higher apoptosis ratio thanwild-type p16in A549cells. Furthermore, co-immunoprecipitation assays suggested thatdecrease of the p16arginine methylation level promoted the association of p16with CDK4.Additionally, we determined that the protein arginine methyltransferase6(PRMT6) wasresponsible for the p16arginine methylation. Results from flow cytometric analysis (FACS)demonstrated that PRMT6overexpression counteracted the cell cycle arrest at G1phaseinduced by wild-type p16in A549cells. We also showed that PRMT6was able to interactwith p16, and that the intensity of p16-CDK4association was reduced upon PRMT6overexpression. Consequently, PRMT6inhibited cell apoptosis via increase of p16argininemethylation. Together, data presented in this report establish that methylation at specificarginine residues of p16protein by PRMT6may be critical for the activity of p16.