Fig. 7.

PRMT5 suppresses FOXO1 and CREB phosphorylation by PKA and CREB target genes. A: alignment of FOXO1 and CREB sequences showing conserved arginine residues around the target serine residues and the PKA consensus sequence. B: peptide sequences showing the arginines surrounding the serine phosphorylation sites S133 and S253 for unmodified peptides CREB and FOXO1, respectively. Unmodified peptides and methylated peptides, methyl-CREB (symmetrically dimethylated at R130 and R131), and methyl-FOXO1 (symmetrically dimethylated at R248 and R250) were used for in vitro kinase assay using recombinant PKACα. C: in vitro kinase assay using recombinant PKACα and [γ-³²P]adenosine 5′-triphosphate ([γ-³²P]ATP) shows PKA-mediated phosphate incorporation in kemptide (positive control) and unmodified CREB and FOXO1 peptides but not in methyl-CREB and methyl-FOXO1 peptides (results are expressed as means ± SE of two similar experiments analyzed using Student’s t-test; *P < 0.05 and **P < 0.01). Representative Western blots (D) and quantitation (E) showing PRMT5 knockdown in MEFs increased FOXO1 phosphorylation upon forskolin (10 μM) treatment (F) and increased CREB phosphorylation in MEFs (G) Values are expressed as means ± SE of two similar experiments. Data were analyzed using Student’s t-test, #P < 0.001 and §P < 0.05. H and I: PRMT5 knockdown in MEFs leads to increased CREB target gene, IRS2 and NR4A2 transcription (three similar experiments, §P < 0.05 and ¶P < 0.01).