Figure 4

Local arginine methylation of hnRNPK inhibits S379 phosphorylation and also regulates migration of MDA-MB-231 cells. (a) PRMT1-mediated arginine methylation of hnRNPK inhibits the same proteins S379 phosphorylation. The His-hnRNPKs were incubated with GST-PRMT1 in the absence or presence of SAM at 30 °C for 16 h and this was followed by isolation of the resulting hnRNPKs using NTA-beads. These re-purified proteins (unmethylated and methylated hnRNPKs, respectively) were further incubated with Aurora-A in the absence or presence of ATP for 16 h as part of the in vitro kinase assay. The phosphorylation levels of hnRNPK S379 were detected by the use of the specific antibodies. (b) Quantification of in vitro S379 phosphorylation on hnRNPKs without or with pre-methylation. Data is shown as mean ± SD, and * indicates significant difference compared to control (**p < 0.01, n = 6). (c) Establishment of MDA-MB-R296K/R299K cell line. MDA-MB-231 cells were first overexpressed with an Flag-R296K/R299K hnRNPK mutant and this was followed by the knockdown of endogenous hnRNPK using shRNA. The stable clone of MDA-MB-231 cells expressing the Flag-R296K/R299K hnRNPK mutant was obtained and this was named MDA-MB-R296K/R299K. (d) Loss of hnRNPK Arg296/299 methylation in MDA-MB-R296K/R299K cells results in higher levels of in vivo S379 phosphorylation than in the control cells. Flag-tagged hnRNPKs in each stable clone were immunoprecipitated by Flag antibody and then the levels of hnRNPK S379 phosphorylation were measured using the specific antibodies. (e) Loss of hnRNPK Arg296/299 methylation produces lower levels of migration than by the control cells. Comparison of cell migration ability between MDA-MB-WT and MDA-MB-R296K/R299K cells was performed. (f) Measurement of the migration abilities of MDA-MB-WT and MDA-MB-R296K/R299K cells. Data is shown as mean ± SD, and *indicates significant difference compared to wild type (*p < 0.05, n = 5). (g) Determination of the migration abilities of MDA-MB-R296K/R299K and MDA-MB-WT cells in the wound healing assay. Each stable clone was independently seeded into 6-well plate for 24 h of incubation and this was followed by scratching an area on the plate using a 10 μL tip. The migrated cells were photographed at 48 h after the scratch using a microscope. The full blot images are shown in Supplementary Fig. S7.