The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition

Lee, Gregory M.; Donaldson, Logan W.; Pufall, Miles A.; Kang, Hyun-Seo; Pot, Isabelle; Graves, Barbara J.; McIntosh, Lawrence P.

doi:10.1074/jbc.m410722200

Cited by 96 publications

(173 citation statements)

References 70 publications

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“…A, summary of the domain structure and consensus sumoylation sites in murine Ets-1. The PNT domain (black) and DNA-binding ETS domain (gray) are indicated (38,63). B, full-length Ets-1 was sumoylated in vivo, and mutation of Lys-15 significantly reduced this modification.…”

Section: Ets-1 Is Sumoylated In Vivo and Inmentioning

confidence: 99%

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

Macauley

Errington

Schärpf

et al. 2006

Journal of Biological Chemistry

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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...

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Section: Ets-1 Is Sumoylated In Vivo and Inmentioning

confidence: 99%

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

Macauley

Errington

Schärpf

et al. 2006

Journal of Biological Chemistry

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“…1A) (5). Helix HI-1 is marginally stable and unfolds on DNA binding, thus implicating an allosteric mechanism of inhibition (6,7). The modest twofold repression afforded by the IM is increased to ∼20-fold by an intrinsically disordered serine-rich region (SRR) (8,9).…”

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confidence: 99%

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor

Desjardins

Meeker

Bhachech

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The E26 transformation-specific (Ets-1) transcription factor is autoinhibited by a conformationally disordered serine-rich region (SRR) that transiently interacts with its DNA-binding ETS domain. In response to calcium signaling, autoinhibition is reinforced by calmodulin-dependent kinase II phosphorylation of serines within the SRR. Using mutagenesis and quantitative DNA-binding measurements, we demonstrate that phosphorylation-enhanced autoinhibition requires the presence of phenylalanine or tyrosine (ϕ) residues adjacent to the SRR phosphoacceptor serines. The introduction of additional phosphorylated Ser-ϕ-Asp, but not Ser-Ala-Asp, repeats within the SRR dramatically reinforces autoinhibition. NMR spectroscopic studies of phosphorylated and mutated SRR variants, both within their native context and as separate trans-acting peptides, confirmed that the aromatic residues and phosphoserines contribute to the formation of a dynamic complex with the ETS domain. Complementary NMR studies also identified the SRR-interacting surface of the ETS domain, which encompasses its positively charged DNArecognition interface and an adjacent region of neutral polar and nonpolar residues. Collectively, these studies highlight the role of aromatic residues and their synergy with phosphoserines in an intrinsically disordered regulatory sequence that integrates cellular signaling and gene expression.transcription factor regulation | protein dynamics | intrinsically disordered region | fuzz complex I ntrinsically disordered protein regions (IDRs) are increasingly recognized for their prevalence in the eukaryotic proteome and their roles in normal biological processes, as well as in disease (1-3). IDRs serve as flexible linker sequences between modular domains and as key components of complex protein-interaction networks. These sequences are often sites of posttranslational modifications, and their plasticity enables accessible and reversible interactions necessary for the integration of cellular signals.The autoinhibition of the E26 transformation-specific (Ets-1) transcription factor provides an illuminating example of how a flexible, disordered region can modulate the regulatable DNAbinding ETS domain and thereby tune it for biological control by calcium-dependent phosphorylation and cooperative protein partnerships (4). The inhibitory module (IM) of Ets-1 is composed of four α-helices (HI-1, HI-2, H4, and H5) that pack onto the ETS domain distal from the DNA interface (Fig. 1A) (5). Helix HI-1 is marginally stable and unfolds on DNA binding, thus implicating an allosteric mechanism of inhibition (6, 7). The modest twofold repression afforded by the IM is increased to ∼20-fold by an intrinsically disordered serine-rich region (SRR) (8, 9). The dynamic SRR interacts transiently with both the ETS domain DNA-binding interface and the IM, and thus plays steric and allosteric inhibitory roles (10). Phosphorylation of five SRR serines by calmodulindependent kinase II (CaMKII) leads to a dramatic ∼500-fold autoinhibition (11). In paral...

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“…Indeed, Ets-1 is autoinhibited for DNA binding due to the presence of two inhibitory domains surrounding and interacting with its DBD (Lee et al, 2005). To counteract autoinhibition and improve DNA binding, Ets-1 interacts with partners, enabling their cooperative binding to adjacent DNA elements (Pufall and Graves, 2002).…”

Section: Introductionmentioning

confidence: 99%

Ets-1 p27: a novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51

et al. 2009

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The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1D(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.

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The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition

Cited by 96 publications

References 70 publications

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

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

Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor

Ets-1 p27: a novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51

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