Residue Ser-228 is identified as the main phosphorylation site on human CARM1. (A) Phosphorylation of CARM1 during mitosis was observed on serine residues, but not on tyrosine or threonine residues. The ³²P-labeled phosphorylated CARM1 was trypsin-digested, acid-treated, and analyzed by phosphoamino acid analysis on a 2D gel. (B) Phosphorylation of human CARM1 is located at Ser-228, which is conserved among human, mouse, rat, zebra fish, fruit fly, and mosquito. The GenBank accession numbers are as follows: Homo sapiens, NP_954592; Mus musculus, NP_067506; Rattus norvegicus, NP_001029259; Danio rerio, XP_701531; Drosophila melanogaster, NP_649963; Anopheles gambiae, XP_318375). (C) The phosphorylation site on CARM1 is not conserved among human PRMT molecules, but next to the conserved asparagine (or aspartate) residue. Notice that hPRMT4 is the alternative name for human CARM1. (D) CARM1 phosphorylation was not detectable with lysates prepared from MEF3−/− cells stably expressing CARM1 S229A mutant (lane 2). Whole-cell lysates from MEF3−/− CARM1 null cells stably expressing either WT or the S229A mutant CARM1 were separated on SDS/PAGE and transferred to nitrocellulose for Western blot. CARM1 was probed with anti-CARM1 antibodies.

The mutant mouse CARM1 S229E shows reduced MTase activity in in vitro methylation assays. (A) In vitro methylation assays. (Top) Western blotting shows the relative amount of WT and mutant (S229A and S229E) mouse CARM1 used in the assay. (Middle) Coomassie staining shows the relative amount of substrate histone used in the assay. (Bottom) Autoradiograph of the methylated ³H-H3. (B) Quantitation of methylated ³H-H3 based on two independent experiments. The ³H-histone H3 bands were excised from the dried gel, immersed in scintillation buffer, and counted for ³H.

S229E mutant CARM1 is impaired in dimerization. (A) Coimmunoprecipitation of HA- and FLAG-tagged CARM1 from MEF20−/− cell extracts transiently transfected with constructs expressing HA- and FLAG-tagged wild-type or mutant CARM1. Cell extracts were directly used in the Western blot experiment (top two blots) to confirm the expression of HA- and FLAG-tagged CARM1. In experiments shown in the lower two blots, HA-CARM1 was first immunoprecipitated from cell extracts with anti-HA antibodies. Then the coimmunoprecipitated FLAG-tagged CARM1 was probed with anti-FLAG antibodies. FLAG-CARM1 was not detected when constructs expressing S229E mutant CARM1 were used (comparing lane 4 with lanes 2 and 3 in the lowest blot). (B) Mouse CARM1 residue S229 lies close to the dimerization interface in the remodeled CARM1 structure. PRMT1 forms homodimers in crystal structure (Right), and the Asn-Asp (N115-D205) interaction was assumed to be critical for stabilizing the dimer status. Residue A114 in PRMT1 (Upper Left) is equivalent to S229A mutant in mouse CARM1 (Lower Left).

CARM1 phosphorylation is observed in vivo. (A) CARM1 phosphorylation was observed during mitosis in HeLa cells. CARM1 was immunoprecipitated from HeLa cells treated with 1 mM hydroxyurea (S-phase arrest) (lanes 1 and 2) or 0.5 μg/ml nocodazole (M-phase arrest) (lanes 3 and 4). The CARM1 immune complex was divided into halves; one half was treated with 2.5 μg of λ-phosphatase (Upstate Biotech) (lanes 2 and 4) in supplied buffer, and the other half was treated with buffer alone (lanes 1 and 3). Samples were loaded on 7% SDS/PAGE and transferred to nitrocellulose membrane. CARM1 was detected by using anti-CARM1 antibody (Upstate Biotech). The slower migrating CARM1 representing phosphorylated-CARM1 is denoted by an *. (B) CARM1 phosphorylation can be detected in SK-BR3 cells but not in MCF7 breast cancer cells. SKBR3 cells were treated with DMSO (lane 1), 50 ng/ml EGF (lane 2), 200 ng/ml phorbol 12-myristate 13-acetate (PMA) (lane 3), and 10 ng/ml NRG-1 (lane 5) for 15 min before harvesting. SB208530, a specific inhibitor of p38 kinase, was preincubated with cells for 1 h before addition of NRG-1 (lane 4). Cells were lysed in RIPA lysis buffer (150 mmol/liter NaCl/1% Nonidet P-40/0.5% deoxycholic acid/0.1% SDS/50 mM Tris·HCl, pH 8.0/2 μg/ml aprotinin/10 μg/ml leupeptin/1 mg/ml sodium orthovanadate/1 mmol/liter PMSF). Equal amounts of total proteins were loaded on 7% SDS/PAGE and transferred to nitrocellulose membrane. CARM1 was detected by using anti-CARM1 antibody (Upstate Biotech).

The mutant mouse CARM1 S229E is defective in binding to substrate S-AdoMet. (A) The recombinant mouse CARM1 (WT and mutants S229E and S229A) were expressed in baculovirus-infected Sf9 cells and purified by affinity chromatography. Samples were run on 7% SDS/PAGE and stained with Coomassie. (B) The chemical structures of AdoMet and Sinefungin. (C) WT and mutant S229A, but not mutant S229E, mouse CARM1 could be cross-linked with AdoMet. Sinefungin, a competitive inhibitor, was preincubated with CARM1 before incubation with ³H-AdoMet in lane 2. Coomassie staining shows the relative amount of protein used in the photo-affinity labeling experiment. ³H count represents the amount of ³H-AdoMet cross-linked with CARM1.

Mouse CARM1 mutant S229E is defective in the activation of ER-dependent transcription. (A) Plasmids for TIF2 (200 ng), Flag-CARM1 (200 ng), and ER (2.5 ng) were transfected into MEF cells from CARM1 knockout mice with TK-(ERE)x3-LUC reporter plasmids (200 ng) and plasmids encoding β-gal as an internal control. (Upper) Relative luciferase activity was normalized to the level of β-gal. (Lower) The cell lysates used for luciferase assays were analyzed for CARM1, TIF2, and Hsp90 expression by Western blotting. The expressed TIF2 and Flag-tagged CARM1 proteins in transfected cells were detected with anti-GRIP1/TIF2 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Flag (Sigma, St. Louis, MO) antibodies, respectively. Hsp90 serves as a control showing the relative amount of loaded samples. (B) Empty vector and CARM1 expression constructs were cotransfected with ERα into CARM1−/− MEF cells. After 4 h of 17β-estradiol treatment, the expression levels of two endogenous ER-target genes, EGFR and EBAG9, were measured with quantitative RT-PCR.