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

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

“…28 Based on this analysis, PntP2 142-252 contains six helical regions that closely match those identified in Ets1 [ Figs. 2(A,B)], as well as Ets2 69-172 (data not shown 4 ). This strongly suggests that the PNT domains of Pointed-P2, ETS1, and ETS2 also share a common tertiary structure of a SAM core (helices H2-H5) with appended N-terminal helices H0/H1.…”

“…This in turn contributes to enhanced electrostatically driven interactions with the TAZ1 domain of the general transcriptional coactivator acetyltransferase CBP, and ultimately, increased expression of Ras-responsive ETS1 target genes. 4,17 Pointed-P2, the Drosophila ortholog of ETS1, plays a similar role in the well-characterized EGF and Sevenless receptor tyrosine kinase/Ras-mediated signal transduction pathway to control fly eye development. [18][19][20][21] Monomeric Pointed-P2 is a transcriptional activator and antagonist to Yan, an ETS family transcriptional repressor that polymerizes via head-to-tail self-association of its PNT/SAM domain.…”

“…The PNT domains of Yan, ETV6 (or Tel), ERG, ELF3, and FLI1 contain only the minimal helical bundle, whereas those of GABPA and SPDEF have one additional N-terminal helix 3 and those of ETS1 and ETS2 have two. 4 Furthermore, owing to differing surface features, 5 the PNT domains of Yan and ETV6 self-associate, 6,7 whereas the remainder are monomeric in isolation. Also, in disordered regions immediately preceding the PNT domains of closely related mammalian ETS1/ETS2 and Drosophila melanogaster Pointed-P2 are phosphoacceptors for the orthologous Ras-activated MAP kinases ERK2 and Rolled, respectively.…”

“…16 Indeed, NMR relaxation and amide hydrogen exchange (HX) measurement have shown that this helix is only marginally stable and structurally flexible. 4 Furthermore, upon phosphorylation, this helix H0 remains folded, but adopts a broad distribution of conformations displaced from the core helical bundle (H2-H5). This in turn contributes to enhanced electrostatically driven interactions with the TAZ1 domain of the general transcriptional coactivator acetyltransferase CBP, and ultimately, increased expression of Ras-responsive ETS1 target genes.…”

“…Also, in disordered regions immediately preceding the PNT domains of closely related mammalian ETS1/ETS2 and Drosophila melanogaster Pointed-P2 are phosphoacceptors for the orthologous Ras-activated MAP kinases ERK2 and Rolled, respectively. 4,8,9 The role of the ETS1 PNT domain in Ras/MAP kinase signaling has been investigated extensively using combination of cell-based and biophysical measurements. The monomeric PNT domain is a docking module for ERK2, enhancing the efficiency of phosphorylating Thr38 and Ser41 in a disordered region preceding this structured domain.…”