Chmp1A regulates pancreatic cancer cell growth via SIM-medicated SUMOylation and UIM-mediated ubiquitination, opposed through NLS-mediated signaling activities

Background: Chmp1A is a member of ESCRT-III family that functions in the lysosomal degradation of proteins via the formation of MVBs at the final stage of lysosomal degradation. ESCRT-0, -I, and –II were shown to contain UIM for interaction with ubiquitinated cargo proteins destined for MVBs. However, Chmp1A of ESCRT-III has not previously been reported to contain UIM. The purpose of this study is to examine if Chmp1A contains motifs such as UIM through which it is modified and to examine the effect of these modifications on pancreatic cancer cell growth. Methods: Protein database search was employed to investigate whether Chmp1A contains UIM or similar motif. Immunoprecipitation assays were used for examining posttranslational modifications, WB for protein expression, microscopy for subcellular localization, MTT assays for proliferation, transfection method for overexpression of various Chmp1A constructs in pancreatic cancer cells, and in vitro Ubiquitination assays for ubiquitination of rhChmp1A. Results: We found that Chmp1A contains putative SIMs and a UIM. We observed that Chmp1A is slightly SUMOylated in normal pancreatic epithelial cells, which increases as pancreatic cancer progresses towards malignancy. In pancreatic cancer cells, full-length Chmp1A protein is significantly multi-SUMOylated but slightly mono-ubiquitinated. The NLS-deleted Chmp1A eliminated the PTMs by Sumo and ubiquitin, which were verified by SIM and UIM mutants. When overexpressed, Chmp1A was detected at the nucleus and the cytoplasm of which overlaps with late endosomal protein Lamp1. However, both mutants had no expression in the nucleus. SIM mutant showed spotty cytoplasm expression that overlaps with Lamp1. UIM mutant exhibited uniform cytoplasmic expression with little overlap with Lamp1. Both mutants of Chmp1A inhibited cell growth significantly, demonstrating that PTMs of Chmp1A are required for cancer cell growth. Conclusion: We identified SIMs and a UIM of Chmp1A and verified its PTM by Sumo and ubiquitin. Based on our data, we conclude that SUMOylation and/or ubiquitination is essential for the nuclear signaling mediated cancer inhibitory functions of Chmp1A. We also conclude that the excess PTM of Chmp1A by Sumo and/or ubiquitin could lead to cancer promotion either by inhibiting tumor suppressors such as ATM or p53 or by activating oncogenic activities.