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.MAP kinase | protein structure/dynamics | transcriptional regulation | protein-protein interaction | Ets2

DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.

DAXX is a scaffold protein with diverse roles that often depend upon binding SUMO via its N-and/or C-terminal SUMO-interacting motifs (SIM-N and SIM-C). Using NMR spectroscopy, we characterized the in vitro binding properties of peptide models of SIM-N and SIM-C to SUMO-1 and SUMO-2. In each case, binding was mediated by hydrophobic and electrostatic interactions and weakened with increasing ionic strength. Neither isolated SIM showed any significant paralog specificity, and the measured M range K D values of SIM-N toward both SUMO-1 and SUMO-2 were ϳ4-fold lower than those of SIM-C. Furthermore, SIM-N bound SUMO-1 predominantly in a parallel orientation, whereas SIM-C interconverted between parallel and antiparallel binding modes on an ms to s time scale. The differences in affinities and binding modes are attributed to the differences in charged residues that flank the otherwise identical hydrophobic core sequences of the two SIMs. In addition, within its native context, SIM-N bound intramolecularly to the adjacent N-terminal helical bundle domain of DAXX, thus reducing its apparent affinity for SUMO. This behavior suggests a possible autoregulatory mechanism for DAXX. The interaction of a C-terminal fragment of DAXX with an N-terminal fragment of the sumoylated Ets1 transcription factor was mediated by SIM-C. Importantly, this interaction did not involve any direct contacts between DAXX and Ets1, but rather was derived from the non-covalent binding of SIM-C to SUMO-1, which in turn was covalently linked to the unstructured N-terminal segment of Ets1. These results provide insights into the binding mechanisms and hence biological roles of the DAXX SUMOinteracting motifs.DAXX is an enigmatic protein with diverse and often controversial roles. Although first discovered through a screen for association with the cytoplasmic Fas death domain (1), DAXX is now recognized to function primarily in the nucleus and to often be localized within subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs) 3 (2, 3). Perhaps the best established role of DAXX lies with the regulation of transcription factors. Although some of these factors may be activated by DAXX (4, 5), in most cases, DAXX is linked with transcriptional repression (3). DAXX also associates with histone deacetylases as well as other chromatin-remodeling systems, thereby providing plausible routes to the repression of gene expression (6 -10). The key to the localization and function of DAXX is its ability to bind the ubiquitin-like protein SUMO (3, 11).The human genome encodes four SUMO paralogs (for reviews, see Refs. 12-16). SUMO-2 and SUMO-3 are virtually identical and are generally referred to as SUMO-2/3. SUMO-1 and SUMO-2/3 have ϳ50% sequence identity and are expressed in all cells, whereas the function of SUMO-4 remains unclear. Using a common enzymatic pathway, many substrates can be covalently modified by both SUMO-1 and SUMO-2/3. However, the latter can also form chains on substrate proteins, and the levels of SUMO-2/3 conjugation ...