%0 Journal Article
%A Hans, Friederike
%A Eckert, Marita
%A von Zweydorf, Felix
%A Gloeckner, Christian Johannes
%A Kahle, Philipp J
%T Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43).
%J The journal of biological chemistry
%V 293
%N 41
%@ 0021-9258
%C Bethesda, Md.
%I Soc.60645
%M DZNE-2020-06592
%P 16083-16099
%D 2018
%X TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193-414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.
%K Active Transport, Cell Nucleus
%K Cell Nucleus: metabolism
%K DNA-Binding Proteins: chemistry
%K DNA-Binding Proteins: genetics
%K DNA-Binding Proteins: metabolism
%K HEK293 Cells
%K Humans
%K Lysine: chemistry
%K Mutagenesis, Site-Directed
%K Mutation
%K Phosphorylation
%K Protein Processing, Post-Translational
%K Solubility
%K Ubiquitin: metabolism
%K DNA-Binding Proteins (NLM Chemicals)
%K TDP-43 protein, human (NLM Chemicals)
%K Ubiquitin (NLM Chemicals)
%K Lysine (NLM Chemicals)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:30120199
%2 pmc:PMC6187624
%R 10.1074/jbc.RA118.003440
%U https://pub.dzne.de/record/140270