FOXOs (class O Forkhead transcription factors) stimulate transcription of proapoptotic genes, cell cycle inhibitory genes, and genes encoding enzymes that detoxify reactive oxygen species. FOXO proteins are acetylated, as well as subject to phosphorylation by at least two kinases, MST1 and Akt (see Michalek and Rathmell). Phosphorylation by Akt inhibits FOXO activity by promoting its nuclear export and proteasome-mediated degradation. Yamagata et al. now show that FOXO1 is methylated at several arginine residues by protein methyltransferase1 (PRMT1) in vitro. Two of the primary methylation sites (R248 and R250, amino acid numbering from mouse) occur within the consensus Akt phosphorylation site. In vitro assays with FOXO1, FOXO3, FOXO4, or FOXO6 peptides suggested that arginine methylation prevented Akt phosphorylation. Silencing of PRMT1 increased the phosphorylation of FOXO1 at the Akt site, increased the cytoplasmic localization of FOXO1, and, in the presence of proteasome inhibitors, increased the abundance of ubiquitinated FOXO1. Using a methylation-specific antibody, methylated FOXO1 was only detected in the nucleus, and the abundance of methylated FOXO1 was decreased when PRMT1 was knocked down. Reporter gene assays suggested that arginine methylation of FOXO1 promoted its transcriptional activity. Knockdown of PRMT1 or FOXO1 increased cell survival in response to oxidative stress, presumably due to decreased stimulation of proapoptotic genes, a conclusion supported by the reduction in the mRNA and protein abundance of the proapoptotic protein BIM in the PRMT1 or FOXO1 knockdown cells. Methylation of FOXO1 was stimulated by hydrogen peroxide, while Akt-phosphorylated FOXO1 was decreased. These results suggest that protein methylation and protein phosphorylation are coordinately regulated. In the case of FOXO1, these two posttranslational modifications produce opposing results: Methylation promotes the apoptotic response, whereas Akt phosphorylation promotes a survival response.
K. Yamagata, H. Daitoku, Y. Takahashi, K. Namiki, K. Hisatake, K. Kako, H. Mukai, Y. Kasuya, A. Fukamizu, Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol. Cell 32, 221-231 (2008). [PubMed]
R. D. Michalek, J. C. Rathmell, Methed-up FOXOs can’t in-Akt-ivate. Mol. Cell 32, 160-162 (2008). [PubMed]