Ubiquitination is a novel post-translational modification of VMP1 in autophagy of human tumor cells

Abstract

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein whose expression in pancreatic cancer stem cells, carrying activated Kirsten rat sarcoma viral oncogene homolog (KRAS), triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis, remaining ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that Cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer Cullin–RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with the Cdt2 overexpression. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor cell therapy resistance.