Ser²¹⁷ phosphorylation does not affect CARM1 dimerization or coactivator association. A, CARM1 dimerization is not affected by Ser²¹⁷ phosphorylation. Myc-tagged wild-type CARM1 was coexpressed with HA-tagged wild-type (WT) CARM1 or the S217E, S217A, or S229E mutant in HEK293T cells. Following immunoprecipitation (IP) with antibody against the Myc tag, the precipitated proteins were separated on SDS-polyacrylamide gel and analyzed by Western blot analysis with antibody against the HA tag. B, CARM1 coactivator association is not affected by Ser²¹⁷ phosphorylation. HEK293T cells were transfected with the indicated plasmids. Following immunoprecipitation with anti-FLAG or anti-Myc antibodies, the precipitated proteins were separated on SDS-polyacrylamide gel, and associated coactivators were determined by Western blot analysis. vec, vector.

Phosphorylation of CARM1 at Ser²¹⁷ abolishes its AdoMet binding activity. A, the crystal structures of the CARM1 catalytic core in the apo (left panel) and holo (middle and right panels) states. The right panel shows a close look at the hydrogen bond between Ser²¹⁷ and Tyr¹⁵⁴. B, Ser²¹⁷ mutants lost AdoMet binding ability. CARM1 proteins were prepared as described for Fig. 2A. Their AdoMet binding activities were visualized by autoradiography. C, conservation of Tyr¹⁵⁴ shown by the primary sequence alignment of the type I PRMT proteins. D, Tyr¹⁵⁴ mutants lost methyltransferase activity and AdoMet binding activity. CARM1 proteins were prepared as described for Fig. 2A. Core histones were used as substrates for the methyltransferase assay. Purified CARM1 proteins were determined by Western blot analysis. WT, wild-type CARM1; h, human; m, mouse.

Human CARM1 is phosphorylated at Ser²¹⁷ in MCF-7 cells. A, purification of CARM1 protein from MCF-7 cells. Endogenous CARM1 protein was immunoprecipitated from MCF-7 whole cell lysate using anti-CARM1 antibody. Following SDS-PAGE, the gel was stained with GelCode blue stain reagent to illustrate the precipitated bands. B, schematic representation of mouse CARM1 functional domains conserved in the PRMT family of enzymes. C, primary sequence alignment of the PRMT family of proteins. The accession numbers for the PRMTs are as follows (where h is human and m is mouse): hPRMT1 (AAF62893), hPRMT2 (AAH00727), hPRMT3 (AAC39837), hCARM1 (NP_954592), hPRMT5 (AAF04502), hPRMT6 (Q96LA8), hPRMT7 (NP_061896), mPRMT8 (DAA01382), and hPRMT9 (AAH64403). D, primary sequence alignment of CARM1 proteins from different species. The accession numbers for CARM1 sequences are as follows: Homo sapiens CARM1 (NP_954592), Canis lupus CARM1 (XP_853774), Mus musculus CARM1 (NP_067506), Rattus norvegicus CARM1 (NP_001029258), Danio rerio CARM1 (NP_001003645), Drosophila melanogaster CARM1 (NP_649963), Anopheles gambiae CARM1 (XP_318375), Arabidopsis thaliana CARM1 (NP_974913), and Oryza sativa CARM1 (NP_001060600).

Phosphorylation of Ser²¹⁷ abrogates CARM1 methyltransferase activity. A, Ser²¹⁷ mutants of CARM1 lost methyltransferase activity. Wild-type (WT), S217E, and S217A CARM1 were exogenously expressed in 293T cells. CARM1 proteins were purified by immunoprecipitation followed by elution with HA peptide. Their methyltransferase activities were analyzed by in vitro methylation assay. Purified core histones, recombinant SRC-3, and p300 were used as substrates. Western blot analysis was used to determine the amount of CARM1 proteins used for the methylation assay. B, characterization of CARM1 Ser²¹⁷ phosphospecific antibody. Exogenously expressed wild-type and S217A mutant CARM1 were purified from HEK293T cells as described in A. The pSer²¹⁷ and CARM1 protein levels were determined by Western blot analysis. C, Ser²¹⁷ phosphorylation abrogates CARM1 methyltransferase activity. Total CARM1 protein or CARM1 specifically phosphorylated at Ser²¹⁷ was immunoprecipitated (IP) from HeLa cell lysate with the indicated antibodies (Ab). Their activities were analyzed by in vitro methylation assay using core histones as substrate. Western blot analysis showed the amount of CARM1 protein used in the assay. IB, immunoblot. D, Ser²¹⁷ phosphorylation inhibits CARM1 methyltransferase activity. Endogenous Ser²¹⁷-phosphorylated CARM1 was immunoprecipitated from HeLa cell lysate and treated with or without λ-phosphatase (λ ppase). Following extensive washing, precipitated CARM1 was used for in vitro methylation assay. Total CARM1 protein was determined by Western blot analysis.

Mutation of CARM1 Ser²¹⁷ phosphorylation site diminishes its coactivator function. A, CARM1 Ser²¹⁷ mutants showed little coactivator activity for ERα-mediated transcription. CV-1 cells were transiently transfected with 200 ng of ERE-Luc reporter, 6 ng of pCR3.1-ERα, 50 ng of pSG5-FLAG-SRC-3, and 100 ng of pSG5-HA-CARM1 wild type (WT) or indicated mutant in each well of a 12-well plate. 10 nm estradiol (E2) was added 24 h after transfection, and luciferase activity was measured at 48 h post-transfection. Luciferase activity was normalized against total cell lysate protein. B, CARM1 Ser²¹⁷ phosphorylation mutants lost coactivator activity on expression of an endogenous ER targeting gene. MCF-7 cells in 6-well plates were transiently transfected with 2 μg of pSG5-HA-CARM1 wild type or indicated mutant. 48 h after transfection, cells were treated with 10 nm estradiol for 18 h to induce endogenous ER target gene expression. The mRNA level of pS2 was determined by real-time reverse transcription-PCR, and the relative amount is shown. The 18 S rRNA was used as an internal control. +, present; −, absent. The exogenously expressed CARM1 proteins were examined by Western blot analysis, as shown in the right panel. vec, vector.

Ser²¹⁷ phosphorylation promotes CARM1 cytoplasmic localization and occurs at cell mitosis. A, Ser²¹⁷ phosphorylation of CARM1 changes its subcellular localization. HA-tagged CARM1 Ser²¹⁷ mutants and wild-type (WT) protein were exogenously expressed in HeLa cells. HA staining shows the CARM1 protein, and 4′,6-diamidino-2-phenylindole (DAPI) shows the DNA. 200 positively stained cells were counted for each sample, and the percentage of cells showing stronger cytoplasmic signal is shown. B, CARM1 Ser²¹⁷ phosphorylation level greatly increases during cell mitosis. HeLa cells were cycle-synchronized at M phase using thymidine/nocodazole treatment. Ser²¹⁷ phosphorylation level was determined by direct immunoprecipitation with pSer²¹⁷ antibody followed by Western blot analysis against CARM1. CARM1 and β-actin levels shown by Western blot analysis. Histones were prepared by acid extraction method, and the amount of Ser¹⁰ phosphorylation and Arg¹⁷ methylation of histone H3 in different samples was determined by Western blot analysis. IgG heavy chain is labeled with an asterisk. C, Ser²¹⁷ phosphorylation of CARM1 is a mitotic event. HeLa cells were synchronized at the G₁/S boundary by double thymidine treatment. After being released into fresh medium, cells were collected every 2 h, and the level of Ser²¹⁷ phosphorylation (pSer217) of CARM1 was determined by immunoprecipitation-Western blot analysis, as described for B. Direct Western blot analysis also shows the amount of CARM1, β-actin, pSer¹⁰, and total histone H3 in different samples. IgG heavy chain is labeled with an asterisk. D, characterization of CARM1 antibody in immunofluorescent staining. CARM1^+/+ and CARM1^−/− mouse embryonic fibroblast cells were used for immunofluorescent staining by CARM1 antibody. CARM1 protein is shown in red, whereas DNA is shown in blue (DAPI). E, CARM1 localizes in the cytoplasm during M and early G₁ phases. HeLa cells were synchronized at the G₁/S boundary by double thymidine block. After being released into regular medium, immunofluorescent staining was performed at 0, 3, 7, 10, and 12 h to show CARM1 cellular localization at late G₁, S, G₂, M, and early G₁ phases, respectively. CARM1 is labeled in red, and DNA is shown in blue (DAPI). Typical cells at the designated cell cycle stage are indicated out by white arrows.