Rhes, a Physiologic Regulator of Sumoylation, Enhances Cross-sumoylation between the Basic Sumoylation Enzymes E1 and Ubc9

Abstract

We recently reported that the small G-protein Rhes has the properties of a SUMO-E3 ligase and mediates mutant huntingtin (mHtt) cytotoxicity. We now demonstrate that Rhes is a physiologic regulator of sumoylation, which is markedly reduced in the corpus striatum of Rhes-deleted mice. Sumoylation involves activation and transfer of small ubiquitin-like modifier (SUMO) from the thioester of E1 to the thioester of Ubc9 (E2) and final transfer to lysines on target proteins, which is enhanced by E3s. We show that E1 transfers SUMO from its thioester directly to lysine residues on Ubc9, forming isopeptide linkages. Conversely, sumoylation on E1 requires transfer of SUMO from the thioester of Ubc9. Thus, the process regarded as "autosumoylation" reflects intermolecular transfer between E1 and Ubc9, which we designate "cross-sumoylation." Rhes binds directly to both E1 and Ubc9, enhancing cross-sumoylation as well as thioester transfer from E1 to Ubc9. Huntington disease (HD)3 is an autosomal dominant disorder caused by an expansion of glutamine residues in the gene encoding the protein huntingtin (Htt) (1). Htt and mutant Htt (mHtt) are ubiquitously expressed throughout the brain and peripheral tissues, yet HD is associated with highly selective degradation of the striatum with no notable alterations in peripheral tissues. Rhes, a member of the Ras family of small G-proteins, was identified on the basis of its selective localization to the corpus striatum of the brain, hence its designation as Ras homolog enriched in striatum (2). We recently reported that Rhes binds mHtt selectively and with high avidity to enhance mHtt sumoylation, with Rhes acting as an apparent E3 ligase for mHtt as well as other substrates such as RanGAP and SP100 (3). Sumoylation of mHtt leads to its disaggregation and augmented neurotoxicity (3, 4). Thus, selective striatal neuronal loss in HD may reflect the tissue-specific influence of Rhes on mHtt sumoylation and cytotoxicity.Sumoylation involves a multistep enzymatic cascade analogous to ubiquitination, beginning with the activation and attachment of SUMO as a thioester bond to E1, the Aos1/Uba2 heterodimer. The SUMO group is then transferred from E1 to Ubc9, the E2-conjugating enzyme, as a thioester bond. In the final step, SUMO is transferred to lysine residues of target proteins forming an isopeptide bond, a process enhanced by E3 ligases (5).Sumoylation can regulate protein function through changes in localization, stability, and activity. Because the sumoylation cascade employs only a single E1 (Aos1/Uba2) and a single E2 (Ubc9), regulation of these enzymes could have substantial effects upon sumoylation. SUMO E1 and Ubc9 are themselves sumoylated in yeast and mammalian cells (6, 7). Recently, it was reported that attachment of SUMO at lysine 14 on Ubc9 acts as a molecular switch to modulate sumoylation of target proteins (8). Molecular mechanisms underlying "autosumoylation" of SUMO-E1 and Ubc9 have not hitherto been elucidated.We now report that mice with deletion of Rhes manifest a p...

UBC9 Autosumoylation Negatively Regulates Sumoylation of Septins in Saccharomyces cerevisiae

“…Lys-153 appears to be the major SUMO conjugation site, and SUMO conjugation to Lys-157 inhibits SUMO conjugation to Lys-153. Interestingly, a similar self-inhibitory effect was observed for the three human Ubc9 autosumoylation lysine residues, namely Lys-14, Lys-49, and Lys-153 (43), and mutation of Lys-153 also increased human Ubc9 autosumoylation (26). Despite the different lysine residues involved in Ubc9 autosumoylation, this self-modification process is conserved from yeast to humans and functions to regulate both Ubc9 autosumoylation and SUMO conjugating activities.…”

“…However, Ubc9 may also interact with its substrates at multiple positions and recognize alternative sites to modify nonconsensus sequences. A number of nonconsensus SUMO conjugations were reported including, for instance, E2-25K (40), Cdc3 (14), proliferating cell nuclear antigen (41), human PML (42), and human Ubc9 (26,43). As revealed in our study, yeast Ubc9 autosumoylation at the C-terminal amino acid residues Lys-153 and Lys-157 appears to follow the pattern of nonconsensus SUMO conjugation, and the resulting modification regulates the function of Ubc9.…”

Rhes, a Striatal-selective Protein Implicated in Huntington Disease, Binds Beclin-1 and Activates Autophagy

“…Rhes binds mHtt and acts as a SUMO (Small Ubiquitin-like MOdifier) E3 ligase to stimulate sumoylation of mHtt, a post-translation modification known to augment mHtt toxicity (4,6). Rhes also physiologically regulates sumoylation and enhances a process we have termed "cross-sumoylation" (7). Independent work by other groups have confirmed the importance of Rhes in mHtt cytotoxicty using primary neuron and stem cell models of HD (8,9).…”

“…Rhes activates autophagy by competitively displacing the inhibitory binding of Bcl-2 to Beclin-1. Rhes enhances cross-sumoylation and regulates sumoylation in the striatum (7). Mice lacking Rhes have decreased L-DOPA-induced dyskinesia, demonstrating that Rhes physiologically activates mTOR in the striatum (24).…”

Sumoylation of SAE2 C Terminus Regulates SAE Nuclear Localization

“…Briefly, Ubc9 catalyzed SUMOylation of SAE2 was obtained by incubation of E1 and E2 with SUMO, ATP, and Mg 2ϩ , as previously described (27,28) (Fig. 1A, left panel).…”