Proteomic profiling of VCP substrates links VCP to K6‐linked ubiquitylation and c‐Myc function

• Published in: EMBO reports Vol. 19; no. 4
• Main Authors: Heidelberger, Jan B, Voigt, Andrea, Borisova, Marina E, Petrosino, Giuseppe, Ruf, Stefanie, Wagner, Sebastian A, Beli, Petra
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.04.2018; John Wiley and Sons Inc
• Subjects: Adenosine triphosphatase; Cell cycle; Cell Line; Cell Nucleus - metabolism; c‐Myc; Deoxyribonucleic acid; DNA; DNA repair; Gene expression; Homeostasis; Humans; HUWE1; Huwe1 protein; Inhibition; K6‐linked ubiquitylation; Mass spectrometry; Mass spectroscopy; Models, Biological; Myc protein; Proteasome Endopeptidase Complex - metabolism; Proteasomes; Protein Binding; Protein Interaction Mapping; Protein Interaction Maps; Protein Transport; Proteins; Proteolysis; Proteome; Proteomics; Proteomics - methods; Proto-Oncogene Proteins c-myc - metabolism; Scientific imaging; Spectroscopy; Substrates; Transcription factors; Ubiquitin; ubiquitin remnant profiling; Ubiquitin-protein ligase; Ubiquitination; Valosin Containing Protein - metabolism; VCP; K6‐linked ubiquitylation; HUWE1; VCP; ubiquitin remnant profiling; c‐Myc
• ISSN: 1469-221X; 1469-3178
• DOI: 10.15252/embr.201744754

Valosin‐containing protein (VCP) is an evolutionarily conserved ubiquitin‐dependent ATPase that mediates the degradation of proteins through the ubiquitin–proteasome pathway. Despite the central role of VCP in the regulation of protein homeostasis, identity and nature of its cellular substrates remain poorly defined. Here, we combined chemical inhibition of VCP and quantitative ubiquitin remnant profiling to assess the effect of VCP inhibition on the ubiquitin‐modified proteome and to probe the substrate spectrum of VCP in human cells. We demonstrate that inhibition of VCP perturbs cellular ubiquitylation and increases ubiquitylation of a different subset of proteins compared to proteasome inhibition. VCP inhibition globally upregulates K6‐linked ubiquitylation that is dependent on the HECT‐type ubiquitin E3 ligase HUWE1. We report ~450 putative VCP substrates, many of which function in nuclear processes, including gene expression, DNA repair and cell cycle. Moreover, we identify that VCP regulates the level and activity of the transcription factor c‐Myc. Synopsis Mass spectrometry‐based proteomics identifies putative substrates of the ubiquitin‐dependent remodeler VCP in human cells and reveals its role in the regulation of the c‐Myc level and activity. Chemical inhibition of VCP increases cellular K6‐linked ubiquitylation that is dependent on the HECT‐type ubiquitin ligase HUWE1. Ubiquitin remnant profiling after chemical inhibition of VCP identifies its putative substrates in human cells. VCP interacts with and regulates the level and activity of the transcription factor c‐Myc. Chemical inhibition of VCP increases cellular K6‐linked ubiquitylation that is dependent on the ubiquitin E3 ligase HUWE1. Proteins modified with K6‐linked ubiquitylation are identified by K6‐linked ubiquitin‐specific affimer and quantitative mass spectrometry. Mass spectrometry‐based proteomics identifies putative substrates of the ubiquitin‐dependent remodeler VCP in human cells and reveals its role in the regulation of the c‐Myc level and activity. Chemical inhibition of VCP increases cellular K6‐linked ubiquitylation that is dependent on the HECT‐type ubiquitin ligase HUWE1.