Abstract #5351: The noncovalent binding of SUMO to the Ubc9 conjugating enzyme regulates the formation and lysine linkages of polymeric SUMO chains

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Posttranslational attachment of the ubiquitin-like modifier SUMO (yeast Smt3) to lysine residues in target proteins impacts protein stability, localization, interaction and activity. Dysregulation of SUMO conjugation has been linked with a variety of human diseases, including cancer, with specific alterations in cell-cycle regulation, DNA damage responses and maintenance of genome integrity. Protein SUMOylation is a transient and dynamic process, representing a balance between the E2 conjugating enzyme Ubc9, which acts alone or in concert with E3 ligases, and the deSUMOylating activity of specific isopeptidases. Analogous to ubiquitin, SUMO can also form polymeric chains, which may also dictate protein interactions by binding to SUMO interaction motifs (SIMs) in protein partners. In yeast, the toxic effects of dysregulated chain formation are suppressed, in part, by the Ulp2 isopeptidase. Yet, relatively little is known about the mechanisms regulating SUMO chain formation and, as in the case of ubiquitin, if alternative lysine (K) linkages have any biological importance. To address these questions, we considered the noncovalent binding of SUMO (yeast Smt3) to Ubc9, which involves hydrophobic and electrostatic contacts between Ubc9 residues Phe22, Gly23, and Tyr25 and select Smt3 backbone carbon atoms. The Ubc9F22A,G23Q,Y25S (Ubc9FGY) mutant is unable to interact noncovalently with Smt3 in vitro or to maintain the viability of yeast cells lacking wild-type Ubc9. Yet, the Ubc9FGY and single Ubc9F22A (Ubc9FA) mutant enzymes are active in in vitro SUMOylation assays, where Ubc9 and Smt3 serve as substrates. However, these mutants exhibited specific alterations in the extent and the lysine linkages when forming SUMO chains. For instance, the majority of high molecular weight Ubc9-Smt3 conjugates formed by Ubc9FGY, but not wild-type Ubc9, involved labile thioester linkages. Also, di-Smt3 conjugates were enhanced in the presence of Ubc9FGY and Ubc9FA, while wild-type Ubc9 preferentially formed Ubc9-Smt3 conjugates. These data suggest that the non-covalent interactions of Smt3 and Ubc9 are necessary to regulate the kinetics of higher-order chain formation. Mutational analysis of Smt3 lysine residues revealed efficient formation of K11, K15, K19 and K54 linkages in vitro. However, K54 linkages were preferentially formed by Ubc9FA, while K19 suppressed di-Smt3 assembly at K11 and K15 by Ubc9FGY and Ubc9FA, but not Ubc9. The biological consequences of mutant induced alterations in the extent and character of SUMO chains are being investigated in yeast strains engineered to express the ubc9 and smt3 mutant alleles. Taken together, our findings indicate that the noncovalent interaction of Smt3 and Ubc9 suppress the inappropriate formation of thioester linkages, and is necessary for appropriate polymeric Smt3 chain formation. Supported by NIH grant CA111542 and ALSAC. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5351.