Beads-on-a-String, Characterization of Ets-1 Sumoylated within Its Flexible N-terminal Sequence

Abstract

Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, we have characterized the conjugation of Ets-1 with SUMO-1. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, we demonstrate that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 (K d ϳ400 M) and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as "beads-on-a-string" with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. We also hypothesize that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors.The regulation of gene expression requires the spatial and temporal orchestration of a complex network of protein-protein and protein-DNA interactions. Central to the control of these interactions are a myriad of post-translational modifications. One such modification involves the reversible, covalent attachment of the ubiquitin-like protein SUMO.4 In addition to serving as an antagonist against ubiquitinmediated degradation, sumoylation affects a growing number of recognized biological processes, including nuclear transport, signal transduction, transcription, and DNA repair (recent reviews Refs. 1-5). However, despite extensive studies, the molecular mechanisms for this regulation, which likely arise through SUMO-dependent modulation of target protein interactions with other macromolecules, remain largely undefined (2, 6 -8).In contrast, the pathways for the sumoylation and desumoylation of target proteins are well established. After proteolytic maturation and ATP-dependent activation, SUMO family members are transferred from the heterodimeric E1-activating enzyme to Cys-93 in the single E2-conjugating enzyme, UBC9. Although E3-ligating enzymes, which facilitate the specificity and efficiency of sumoylation, have been identified, UBC9 is sufficient for conjugating SUMO to many proteins, at least in vitro (9). The final product is an isopeptide bond joining the C-terminal glycine carboxyl of SUMO with the side...

Decreased Recognition of SUMO-Sensitive Target Genes following Modification of SF-1 (NR5A1)

“…The only observed changes in SUMO1 were in Gly97, the site of isopep- tide bond formation with Lys194 in SF-1, and in the neighboring residue Gly96. These data are similar to the NMR structure reported for sumoylated Ets-1 and RanGAP1 (37,38). The complementary experiment using 13 C ILV methyl-labeled SF-1 LBD also revealed no change in the HSQC spectrum of 13 C ILV methyl-SF-1 LBD after conjugation to SUMO1 (see Fig.…”

“…For instance, sumoylation of thymine-DNA glycosylase (TGD) was shown to alter its conformation and promote dissociation from DNA (3,49), and several other studies suggest that transcription factor DNA binding is completely abrogated after sumoylation (2,10,52). On the other hand, structural analysis of sumoylated Ets-1 revealed a "beads-on-a-string" conformation, where SUMO1 and Ets-1 behaved as two independent domains (38). More recently, it has been suggested that SUMO-dependent repression of the glucocorticoid receptor actually requires DNA binding to multiple GREs (25).…”

Regulation of the Ets-1 transcription factor by sumoylation and ubiquitinylation

“…Indeed, the sumoylation profile of Ets-1 consisted in two major sumoylated Ets-1 bands having a molecular mass of B75 and B90 kDa and a minor band at B110 kDa and all these bands were abolished using the double Ets-1 K15R/K277R mutant. A recent structural study adds to the knowledge of recognition of SUMO by Ets-1 (Macauley et al, 2005). These authors also identified K15 as being a SUMO-1 acceptor site in Ets-1.…”

Fli1 and Ets1 Have Distinct Roles in Connective Tissue Growth Factor/CCN2 Gene Regulation and Induction of the Profibrotic Gene Program

“…Interestingly, increased Ets1 acetylation correlated with the dissociation of Ets1 from p300 (9). In addition, recent studies have also demonstrated that Ets1 is modified by sumoylation that involves Lys 15 within the N-terminal segment of Ets1 (10). In contrast to Ets1 phosphorylation, the functional significance of other post-translational modifications are not known.…”