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Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition - PubMed

  • ️Sun Jan 01 2017

Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition

Evgenia Shishkova et al. Nat Commun. 2017.

Abstract

Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Global profiling of CARM1 substrates using quantitative mass spectrometry.

(a) Experimental workflow for global detection of CARM1 substrates. Enrichment of ADMA—containing tryptic peptides from three biological replicas of parental and CARM1 KO cell lines—was followed by quantitative mass spectrometry using TMT. (b) Identification and quantification of ADMA-containing peptides and sites. The heat map displays hierarchical clustering using Pearson correlation of mean normalized log2 transformed intensities of ADMA containing peptides in three biological replicas of parental MCF7 and CARM1 KO cells. A TMT reporter region of the spectrum is magnified as an example. The spectrum also illustrates the neutral loss of DMA, characteristic of asymmetric but not symmetric dimethylarginine modification that was used for peptide identification and ADMA site mapping. (c) Identification of putative CARM1 substrates from two breast cancer cell lines using three biological replicas of each sample. Volcano plots illustrate changes in ADMA peptide abundances in MCF7 (left) and MDA-MB-231 cells (right). Greater than twofold reduction on CARM1 loss and maximum P value of 0.01 (two-tailed Student's t-test) were set as the threshold criteria for putative CARM1 substrates. The known CARM1 protein substrates, MED12 and PABP1, were identified among >130 previously unreported ones. (d) Rates of somatic mutations at and nearby CARM1 methylation sites in human cancers. Frequencies of non-synonymous single nucleotide variants at or in the proximity of CARM1-regulated ADMA sites (±5 nucleotides) were over twofold higher than those of non-modified arginines or a randomly selected residue (Fisher's exact test P values of 5.6e-31 and 7.8e-24, respectively), according to the COSMIC. (e) Substrate interaction diagram (STRING 10.0) featuring four biological pathways (Reactome 2016) strongly enriched for the presence of putative CARM1 substrates (combined score >5). Thickness of the lines radiating from CARM1 correlates to the pathway enrichment score (Supplementary Table 3). A black frame around a substrate indicates its causal implementation in cancer (COSMIC).

Figure 2
Figure 2. Discovery of proline-rich CARM1 methylation motifs and high-throughput validation of the detected substrates using peptide arrays.

(a) Amino-acid abundance analysis of CARM1 methylation sites. Frequencies of amino acids in the vicinity of CARM1-regulated methylation sites (±5 nucleotides) were compared to those in the human proteome, revealing pronounced enrichment of proline residues. Glycine, methionine and phenylalanine residues were detected at frequencies typical of the human proteome (P value >0.01; two-tailed Student's t-test). (b) Sequence logos of CARM1 methylation motifs. Various proline-containing motifs were enriched in the proximity of the detected CARM1 methylation sites. (c) Western blot with ADMA antibodies of FLAG-tagged TET2 in wild type and CARM1 KO HEK293T cells showing the reduction of TET2 dimethylation in CARM1 KO. (d) Autoradiography of 192-spot peptide arrays following the in vitro methylation assay using 3H-labelled SAM and indicated purified mammalian PRMTs. The peptide arrays consisted of ∼15 amino-acid sequences centred on substrate arginines. Designated by the blue bracket are substrates of CARM1 identified by IP-MS; the red bracket indicates substrates predicted based on the extracted motifs. Positive control (BAF155 peptide) is denoted with a blue circle; negative control (no peptide) is indicated with a black circle. (e) Autoradiography of 96-spot peptide arrays following the in vitro methylation assay using 3H-labelled SAM and indicated purified mammalian PRMTs. The design of peptide array is depicted at the bottom. Light blue colour designates the location of novel, cancer-related substrates of CARM1 identified by IP-MS; blue—additional substrates and their naturally occurring mutations in human cancer; dark-blue—point mutations of the recognition sequence adjacent to the CARM1 methylation site on BAF155; grey—known substrates of PRTM1; purple—known substrates of PRMT5; pink—known substrates of PRMT6; white—negative control (no peptide).

Figure 3
Figure 3. Requirement of the N-terminal domain for substrate recognition by CARM1.

(a) Schematic diagram of FL and N-terminal truncated CARM1 derivatives. CARM1 FL protein contained 608 residues. CARM1 28–608 lacks the first, unstructured 28 residues denoted in grey. CARM1 140–608 lacks the first 140 residues encompassing the EVH1 domain denoted in green. (b) Western blot analyses of co-immunoprecipitated BAF155, MED12, PABP1, NCOA3 and TET2 with FLAG-tagged CARM1, transiently transfected into HEK293T CARM1 KO cells. CARM1 was immunoprecipiated with the anti-FLAG antibody, and the presence of BAF155, MED12, PABP1, NCOA3 and TET2 in the immunoprecipitates was detected with western blots using the respective antibodies. The loading controls are depicted below the corresponding western blot results, separately for BAF155, MED12 and PABP1, and the other two proteins. In all cases the amount of co-precipitated protein was strongly reduced in cell lines expressing N-terminus truncated CARM1 140–608. (c) Western blot analyses of total ADMA-containing proteins co-precipitated with CARM1. The FLAG-tagged CARM1 immunoprecipitates in b were probed with ADMA antibodies. The strong reduction in the levels of ADMA—containing proteins in cells expressing CARM1 140–680 was evident on both short (left) and long exposure (right). The corresponding loading control was shared between the experiments in b (BAF155, MED12 and PABP1) and c and is depicted in b labelled with IB: FLAG. (d) FP assay using purified recombinant 6xHis-CARM1 28–140 and fluorescein-labelled BAF155 peptide. Pronounced increase in FP was observed at high concentrations of recombinant CARM1, but not with the BSA control, demonstrating that the EVH1 domain of CARM1 directly interacts with the enzyme's substrate at low affinity. (e) Coomassie Blue staining of highly purified recombinant 6xHis-CARM1 28–140 used in the FP assay (d).

Figure 4
Figure 4. In vitro and in vivo requirement of the N-terminal domain for substrate methylation by CARM1.

(a) In vitro methylation assays using recombinant CARM1 proteins and 3H-SAM to test methylation of FLAG-BAF155 protein. Coomassie Brilliant Blue staining of purified BSA (control), FL CARM1, the N-terminally truncated CARM1 28–608 and 140–608, and FLAG-BAF155 are shown in the top two panels. Radioactive labelling of BAF155 is visualized using autoradiography after in vitro methylation assays. (b) In vitro methylation assay using recombinant CARM1 proteins, 3H-SAM and total cell lysates derived from MCF7 CARM1 KO cells. Coomassie Brilliant Blue staining (left panel) and autoradiograph (right panel) of total cell lysates after in vitro methylation assays are shown. (c) Western blot analyses of in vivo methylated BAF155 in parental MCF7 or MCF7 CARM1 KO cells stably expressing GFP control, CARM1 FL, CARM1 28–608 or CARM1 140–608. Expression of CARM1 in total cell lysates was detected by anti-FLAG antibody (bottom panel) and the levels of me-BAF155 (top panel) and total BAF155 (middle panel) were detected using corresponding antibodies. (d) Western blot analyses of ADMA-containing proteins in the total cell lysates as described in c. While Ponceau S staining (left panel) confirms equal loading, Western blot (right panel) demonstrates the reduced levels of ADMA in GFP control and CARM1 140–608 expressing cells, as compared with those in parental cells and cells expressing CARM1 FL and CARM1 28–608. (e) Heat map displaying hierarchical clustering of log2 transformed ADMA peptide intensities in complete CARM1 KO MCF7 cells and CARM1 KO, CARM1 140–608 expressing MCF7 cells, using three biological replicas of each. The intensities were normalized to their average respective levels in CARM1 KO, CARM1 28–608 expressing MCF7 cells. The ADMA peptides were prepared and quantified via nanoLC-MS/MS analysis, as described in Fig. 1a. (f) Comparison of the abundance of ADMA-containing peptides in CARM1 KO MCF7 cells and CARM1 KO MCF7 cells expressing CARM1 140–608. A close Pearson correlation in the levels of ADMA-containing peptides was detected in two cell lines (R2 of 0.82).

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