Extracellular 5'-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins

Abstract

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Keywords: DNA-binding protein; metabolism; methionine; protein methylation; proteomics.

Figure 1

sDMA decreased in MTAP− cells.A, isogenic HTM+ and HTM− cells probed with sDMA-recognizing antibodies from various commercial and academic sources (Fig. S1 for additional information on each antiserum). B, Cell-Signaling sDMA antibodies (catalog no.: 13222) probed against HTM+ and HTM− cell lysates in the presence or the absence of indicated competitor peptides. Fivefold molar excess of competitor peptide was added. C, Western blot of MCM cell lysates from cells exposed to doxycycline (Dox) at various times or concentrations probed with either α-MTAP or α-actin. D, extracts from MCM cells grown in either the presence (+D) or the absence (−D) of Dox probed with two different sDMA-recognizing antiserum. E, first two lanes same as D, third lane contains induced MCM cells treated with MTAP-specific inhibitor MT-DADMe-ImmA for 48 h. Lane 4 has HTM+ cell extract as positive control. F, MCM cells with/without Dox probed with antibodies recognizing either aDMA or MMA. Loading controls are shown below. aDMA, asymmetric dimethylarginine; MTAP, methylthioadenosine phosphorylase; sDMA, symmetric dimethylarginine.

Figure 2

Assessment of methylated arginine in hydrolyzed protein lysates. Lysates from HTM−, HTM+, and HTM+ cells treated with 100 mM MTA (48 h) were prepared and then hydrolyzed by XXXYYY. Resulting material was then analyzed using an amino acid analyzer for aDMA, sDMA, and MMA. Note that we were unable to resolve the arginine from the MMA peak, so they are quantitated together. aDMA, asymmetric dimethylarginine; MTA, 5′-deoxy-5′-methylthioadenosine; sDMA, symmetric dimethylarginine.

Figure 3

Inhibition of sDMAylation by extracellular MTA.A, cells of indicated genotype were treated for 48 h with indicated amounts of MTA. Extracts were probed with Millipore 07-412 (sDMA, Sym10). B, MTA concentrations in extracts from same samples. bd, below detection. C, Western blot of HTM− and HTM+ cells treated with either 80 μM MTA or 1.5 μM EPZ015666 for 48 h probed with CS13222 anti-sDMA. D, Western blot of MCM cells with and without 1 μg/ml doxycycline (+Dox) and/or indicated amount of MTA probed with CS13222 anti-sDMA. E, MTA concentrations in same extracts. MTA, 5′-deoxy-5′-methylthioadenosine; sDMA, symmetric dimethylarginine.

Figure 4

Time course of sDMA inhibition and reactivation.A, Western blot showing effect of addition of 100 μM MTA over the course of 48 h. B, intracellular MTA as measured in same lysates. C, MTA in media at same time points. D, cells were exposed to 50 μM MTA for 72 h. Cells were then washed and replated in media lacking MTA for the period shown. Western blot shows sDMA levels. E, intracellular MTA as measured in same lysates as D. F, extracellular MTA measured at time of harvest. MTA, 5′-deoxy-5′-methylthioadenosine; sDMA, symmetric dimethylarginine.

Figure 5

Mixing experiment.A, Western blot showing protein sDMAylation in HTM−, HTM+, and a 50:50 mixture of both cells plated together. Cells were incubated for 48 h. Experiment was performed in triplicate. B, quantitation of bands shown with arrows in A. C, intracellular MTA in same lysates. D, MTA present in the media at time of harvest. MTA, 5′-deoxy-5′-methylthioadenosine; sDMA, symmetric dimethylarginine.

Figure 6

Immunoprecipitation (IP) of sDMAylated proteins.A, Western blot showing levels of FUBP1, FUBP3, PABN1, PSPC1, and EIF4H in HTM+ (M+) and HTM− (M−) cell lysates. B, IP of indicated extracts using CS13222 antibodies to pull down sDMAylated proteins. Precipitated material was then subjected to Western analysis using indicated antibody. For each IP, a beads-only negative control is also shown. Note that for FUBP1, only lower band shows differential pulldown. C, same as B, but MTM cell extracts are used. sDMA, symmetric dimethylarginine.

Figure 7

FACS analysis of FUSE site function.A, cells transfected with reporter plasmid either containing or missing FUSE site were treated with zinc to induce the MT-I protomer driving a GFP promoter in either the presence or the absence of 100 μM MTA. Horizontal bars show windows used to assess parent induced versus uninduced cells. B, comparison of histograms with and without MTA treatment. FUSE, far upstream binding sequence element; MTA, 5′-deoxy-5′-methylthioadenosine.