title: Proline-rich sequence recognition: II. Proteomics analysis of Tsg101 ubiquitin-E2-like variant (UEV) interactions.
abstract: The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly "PTAP" interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFkappaB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461-2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.
authors: Protein Engineering Group, Leibniz Institute for Molecular Pharmacology and Freie Universität Berlin, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
authors: Andreas
authors: A
authors: Schlundt
authors: Jana
authors: J
authors: Sticht
authors: Kirill
authors: K
authors: Piotukh
authors: Daniela
authors: D
authors: Kosslick
authors: Nadin
authors: N
authors: Jahnke
authors: Sandro
authors: S
authors: Keller
authors: Michael
authors: M
authors: Schuemann
authors: Eberhard
authors: E
authors: Krause
authors: Christian
authors: C
authors: Freund
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry*; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D019151:Peptide Library; D011392:Proline / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D000099177:Tsg101 Protein
chemical_list: D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D019151:Peptide Library; D011392:Proline; D020543:Proteome; D012333:RNA, Messenger; D014157:Transcription Factors; D044763:Ubiquitin-Conjugating Enzymes; D000099177:Tsg101 Protein