Fig. 1

CARM1 methylates and stabilizes PI3KC2α at R175 residue. A and B The interaction between PI3KC2α and CARM1 as well as the methylation of PI3KC2α by CARM1 were confirmed by performing immunoprecipitation (IP) of anti-CARM1 antibody from CARM1 wild-type (WT), knockout (KO), or CARM1 R169A knock-in (KI) MEFs (A). CARM1-PI3KC2α interaction was detected by immunoblotting after immunoprecipitation of PI3KC2α in CARM1-KD cells, and the methylation level of PI3KC2α was measured using an anti-ADMA⁵⁸²⁵ antibody (B). C to F CARM1 methylates the R175 site of PI3KC2α. An in vitro methylation assay was performed using GST-fusion peptides from PI3KC2α as CARM1 substrates (C and D). GST-PI3KC2α (101–220) was methylated in vitro by CARM1 but not by PRMT1, PRMT5, or PRMT6 (E). GST-PI3KC2α RK mutants (residues 101–220; R134K, R139K, R175K, and R216K represent the only arginine residues present between 101–220) were methylated in vitro by CARM1, but the methylation signal was reduced only in R175K (F). G MS/MS spectra of R175-containing peptides. Compared with unmodified peptides, monomethylated and dimethylated peptides had masses of 14 and 28 Da, respectively. The mass difference of b3 ions, which are indicative of the third amino acid residue, R175, also showed the same mass difference as the parent mass. H After myc-PI3KC2α WT or R175K mutant construct was transfected into MCF7 cells, myc-PI3KC2α was immunoprecipitated. I CARM1 stabilizes the PI3KC2α protein by methylating the R175 residue. Cycloheximide (CHX, 50 μg/ml) was added to MCF7 cells transfected with control or CARM1 siRNA for 72 h. Error bar represents the standard deviation of three independent experiments