Pahlich Steffen, Zakaryan Rouzanna P, Gehring Heinz
Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.
Proteins. 2008 Sep;72(4):1125-37. doi: 10.1002/prot.22004.
Protein arginine methylation is a eukaryotic posttranslational modification that plays a role in transcription, mRNA splicing and transport, in protein-protein interaction, and cell signaling. The type I protein arginine methyltransferase (PRMT) 8 is the only member of the human PRMT family that is localized at the cell membrane and its endogenous substrates have remained unknown as yet. Although PRMT8 was supposed to be expressed only in brain tissue, its presence in HEK 293 (T) cells could be demonstrated. We identified more than 20 PRMT8-binding partners in pull-down experiments using recombinant PRMT8 as bait followed by mass spectrometric identification of the bound proteins. Among the extracted proteins were several heterogeneous nuclear ribonucleoproteins (hnRNP), RNA-helicases (DEAD box proteins), the TET-family proteins TLS, Ewing's sarcoma (EWS), and TAF(II)68, and caprin, which all contain RGG methylation motifs and are potential substrates of PRMT8. Additionally, actin, tubulin, and heat shock proteins belong to the identified proteins. The interaction between PRMT8 and the EWS protein was characterized in more detail. Although binding of endogenous and recombinant EWS protein to PRMT8 as well as colocalization in HEK cells was observed, in vitro methylation assays revealed a rather poor methyltransferase activity of PRMT8 towards the EWS protein and a synthetic RGG-rich reference peptide (K(m), 1.3 microM; k(cat)/K(m), 2.8 x 10(-4) microM(-1) s(-1)) in comparison to PRMT1 (K(m), 0.8 microM; k(cat)/K(m), 8.1 x 10(-3) microM(-1) s(-1)). In contrast, substrate proteins within a cell extract could be methylated by PRMT8 as efficient as by PRMT1. The main interaction site of the EWS protein with PRMT8 was determined to be the C-terminal RGG box 3. Remarkably, complete methylation of the EWS protein did not abrogate the binding to PRMT8, pointing to an adapter role of PRMT8 for nuclear proteins at the cell membrane in addition to its methyltransferase activity.
蛋白质精氨酸甲基化是一种真核生物的翻译后修饰，在转录、mRNA剪接与转运、蛋白质-蛋白质相互作用以及细胞信号传导中发挥作用。I型蛋白质精氨酸甲基转移酶（PRMT）8是人类PRMT家族中唯一定位于细胞膜的成员，其内源底物至今仍不清楚。尽管PRMT8据推测仅在脑组织中表达，但已证实在人胚肾293（T）细胞中也有其存在。我们在以重组PRMT8为诱饵的下拉实验中鉴定出20多个PRMT8结合伙伴，随后通过质谱鉴定结合蛋白。提取的蛋白质中有几种不均一核核糖核蛋白（hnRNP）、RNA解旋酶（DEAD盒蛋白）、TET家族蛋白TLS、尤因肉瘤（EWS）、TAF（II）68和帽蛋白，它们都含有RGG甲基化基序，是PRMT8的潜在底物。此外，肌动蛋白、微管蛋白和热休克蛋白也属于已鉴定的蛋白质。对PRMT8与EWS蛋白之间的相互作用进行了更详细的表征。尽管观察到内源和重组EWS蛋白与PRMT8的结合以及在人胚肾细胞中的共定位，但体外甲基化分析显示，与PRMT1（K m，0.8 μM；k cat/K m，8.1×10−3 μM−1 s−1）相比，PRMT8对EWS蛋白和富含RGG的合成参考肽（K m，1.3 μM；k cat/K m，2.8×10−4 μM−1 s−1）的甲基转移酶活性相当低。相反，细胞提取物中的底物蛋白可被PRMT8甲基化，效率与PRMT1相当。EWS蛋白与PRMT8的主要相互作用位点确定为C端RGG框3。值得注意的是，EWS蛋白的完全甲基化并未消除其与PRMT8的结合，这表明PRMT8除了具有甲基转移酶活性外，在细胞膜上对核蛋白还具有衔接子作用。
