Expression and subcellular localization of Ewing sarcoma (EWS) protein is affected by the methylation process.
Ewing sarcoma (EWS) protein contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Recently, we had shown that EWS protein is not only localized in the nucleus and cytosol, but also on the surface of T cells and that its RBD is extensively asymmetrically dimethylated on arginine residues. Here we show that stimulation of T cells with phytohemagglutinin (PHA) caused a time-dependent 10-fold increase in expression of methylated EWS protein on the cell surface and a sixfold increase in the nuclei of peripheral blood mononuclear cells (PBMC). Mitogenic stimulation of malignant T cell lines, however, did not increase their inherently high expression of EWS protein. This expression seemed to correlate with methionine adenosyltransferase activity and S-adenosyl-L-methionine (AdoMet) utilization in PBMC and tumor cells and thus indicates dependence on the methylation process. Inhibition of methylation in normal and malignant cells with the methylation inhibitor adenosine dialdehyde (AdOx) resulted in a three to fivefold decreased expression of EWS protein not only in the nucleus but also on the cell surface. The inhibitory effect of AdOx was compensated and negligible in PBMC, but not in tumor cells if they were treated simultaneously with mitogenic PHA concentrations. The present findings indicate that expression of EWS protein in the various subcellular compartments is affected by the methylation process, in particular by the availability of intracellular AdoMet.
Belyanskaya
Larisa L
LL
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Delattre
Olivier
O
Gehring
Heinz
H
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D002458:Cell Fractionation; D006801:Humans; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008745:Methylation; D010835:Phytohemagglutinins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D034802:RNA-Binding Protein EWS / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D010835:Phytohemagglutinins; D034802:RNA-Binding Protein EWS; C027579:periodate-oxidized adenosine; D000241:Adenosine