Post‐translational modifications of Chmp1A with ubiquitin or sumo and subsequent function in pancreatic cancer growth

Abstract

Background ESCRT families have been shown to contain ubiquitin-interacting motifs (UIMs) that interact with ubiquitinated membrane proteins for sorting into Multi-vesicular bodies (MVBs). Through an extensive database search, we found that Chmp1A of ESCRT-III contains a putative UIM and SUMO-interacting motifs (SIMs), both which overlap with the nuclear localization signal (NLS) domain. The goal of this project is to test whether Chmp1A binds and undergoes modification with ubiquitin or sumo proteins via its UIM or SIMs, and to examine the effect of these processes on pancreatic cancer development. Methodology Immunoprecipitation, Ubiquitination and SUMOylation assays were carried out to examine the interaction or modification of Chmp1A with ubiquitin or SUMO proteins. NLS- deleted Chmp1A was used to determine the significance of the UIM or SIMs in the interaction of Chmp1A with ubiquitin or SUMOs. New deletion mutants in UIM and SIMs of Chmp1A were generated to corroborate the data obtained from NLS- deleted Chmp1A. With the new deletion mutants, IP assays were performed to examine the interaction between Chmp1A and SUMO proteins. Subcellular localization of these deletion mutants was determined by immunocytochemistry. Results Our data showed that Chmp1A associated with ubiquitin and underwent ubiquitination. Full-length but not the NLS- deleted Chmp1A showed an interaction with ubiquitin, suggesting that the UIM is required for association with ubiquitin. The data showed that the full-length but not the NLS- deleted Chmp1A interacted with SUMO protein. Since two of the three SIMs are contained in the NLS domain, this data suggests the importance of SIMs for the interaction of Chmp1A with SUMO. We detected higher molecular weight bands of SUMO- bound full-length Chmp1A, which suggests modification of Chmp1A with SUMO. This data was confirmed partly with UIM or SIM deletion mutants of Chmp1A. Conclusion We identified UIMs and SIMs of Chmp1A and verified their binding and modification with ubiquitin or SUMO proteins. These motifs overlap with the NLS, which is required for the inhibition of pancreatic tumor growth. Thus, this study provides new insights into yet undiscovered mechanisms of Chmp1A in pancreatic tumor suppression.