Abstract
SUMO conjugation to proteins is involved in the regulation of diverse cellular functions. We have identified a protein, RWD-containing sumoylation enhancer (RSUME), that enhances overall SUMO-1, -2, and -3 conjugation by interacting with the SUMO conjugase Ubc9. RSUME increases noncovalent binding of SUMO-1 to Ubc9 and enhances Ubc9 thioester formation and SUMO polymerization. RSUME enhances the sumoylation of IkB in vitro and in cultured cells, leading to an inhibition of NF-kB transcriptional activity. RSUME is induced by hypoxia and enhances the sumoylation of HIF-1alpha, promoting its stabilization and transcriptional activity during hypoxia. Disruption of the RWD domain structure of RSUME demonstrates that this domain is critical for RSUME action. Together, these findings point to a central role of RSUME in the regulation of sumoylation and, hence, several critical regulatory pathways in mammalian cells.
Highlights
Small ubiquitin-related modifiers (SUMOs) are small (12 kDa) proteins with low sequence identity to ubiquitin but very similar 3D structure
Given that RWD-containing sumoylation enhancer (RSUME) is induced by hypoxia (Figures 2B and 2C) and plays a role in sumoylation and that sumoylation of some proteins with high molecular weight is increased by hypoxia (Figure S2), we hypothesized that RSUME might play a role in the cell’s response to hypoxic stress
Reaction mixes were subjected to Western blot (WB) with anti-IkB. (F) Ubc9-SUMO1 thioester formation reactions were performed in vitro in the presence or absence of 0.3 mg of recombinant RSUME protein for the indicated times
Summary
Small ubiquitin-related modifiers (SUMOs) are small (12 kDa) proteins with low sequence identity to ubiquitin but very similar 3D structure. Unlike ubiquitination, which requires an E3 ligase to complete the final transfer from the E2 to the substrate protein, sumoylation can occur without E3 ligases in vitro, somewhat inefficiently (Hay, 2005). Several SUMO E3 ligases, such as RanBP2 (Pichler et al, 2002), whose mechanism of action has been described in detail (Pichler et al, 2002; Reverter and Lima, 2005), and the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family (Johnson and Gupta, 2001; Hochstrasser, 2001) interact with Ubc and are essential for the correct sumoylation of proteins in vivo (Hay, 2005). SUMO is removed from substrates by specific cysteine proteases (SUMO isopeptidases) called SENPs (Melchior et al, 2003)
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