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Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

ABSTRACT: Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2-H5), resembling the SAM domain, appended with two additional helices (H0-H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

SUBMITTER: Nelson ML

PROVIDER: S-EPMC2890480 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

Nelson Mary L ML Kang Hyun-Seo HS Lee Gregory M GM Blaszczak Adam G AG Lau Desmond K W DK McIntosh Lawrence P LP Graves Barbara J BJ

Proceedings of the National Academy of Sciences of the United States of America 20100513 22

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses ...[more]

PMID: 20534573

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Project description:Binding free energies are calculated for the phosphorylated and unphosphorylated complexes between the kinase inducible domain (KID) of the DNA transcriptional activator cAMP response element binding (CREB) protein and the KIX domain of its coactivator, CREB-binding protein (CBP). To our knowledge, this is the first application of a method based on a potential of mean force (PMF) with restraining potentials to compute the binding free energy of protein-protein complexes. The KID:KIX complexes are chosen here because of their biological relevance to the DNA transcription process and their relatively small size (81 residues for the KIX domain of CBP, and 28 residues for KID). The results for pKID:KIX and KID:KIX are -9.55 and -4.96 kcal/mol, respectively, in good agreement with experimental estimates (-8.8 and -5.8 kcal/mol, respectively). A comparison between specific contributions to protein-protein binding for the phosphorylated and unphosphorylated complexes reveals a dual role for the phosphorylation of KID at Ser-133 in effecting a more favorable free energy of the bound system: 1), stabilization of the unbound conformation of phosphorylated KID due to favorable intramolecular interactions of the phosphate group of Ser-133 with the charged groups of an arginine-rich region spanning both ?-helices, which lowers the configurational entropy; and 2), more favorable intermolecular electrostatic interactions between pSer-133 and Arg-131 of KID, and Lys-662, Tyr-658, and Glu-666 of KIX. Charge reduction through ligand phosphorylation emerges as a possible mechanism for controlling the unbound state conformation of KID and, ultimately, gene expression. This work also demonstrates that the PMF-based method with restraining potentials provides an added benefit in that important elements of the binding pathway are evidenced. Furthermore, the practicality of the PMF-based method for larger systems is validated by agreement with experiment. In addition, we provide a somewhat differently structured exposition of the PMF-based method with restraining potentials and outline its generalization to systems in which both protein and ligand may adopt unbound conformations that are different from those of the bound state.

Dataset Information

Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

Publications

Ras signaling requires dynamic properties of Ets1 for phosphorylation-enhanced binding to coactivator CBP.

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