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

Abstract

Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, 15 N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site.

Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition

“…Ets proteins are subclassified by the presence of further domains associated with PPIs (protein–protein interactions) or transcriptional regulation [ 2 , 3 ], including the TCF (containing the B-box [ 6 ]) and PEA3 (containing the unstructured PEA3 transactivation domain [ 7 ]) subfamilies. PNT (pointed) domains are also frequently found N-terminal to the Ets domain, involved in PPI and homodimerization [ 8 , 9 ] ( Figure 1 A).…”

Phosphorylation of an Intrinsically Disordered Segment in Ets1 Shifts Conformational Sampling toward Binding-Competent Substates

“…Although nuclear magnetic resonance (NMR)-derived structural ensembles of the Ets1 PNT domain and adjacent phosphoacceptors in their unmodified and modified forms have been reported, it was not fully established how phosphorylation induces conformational changes that lead to enhanced CBP binding. In particular, the NMR data are consistent with two models: (1) a 0 remains separate from a 1 , adopting a broad conformational distribution that points away from the SAM core; or (2) a 0 and a 1 form one continuous helix that is detached from this core bundle (Lau et al, 2012;Nelson et al, 2010). Furthermore, it is also not clear how the PNT and TAZ1 domains interact.…”

“…Ets1 is a member of the ETS family of transcription factors that are frequently used as downstream effectors in signal transduction cascades to regulate cell proliferation, differentiation, migration, and apoptosis (Hollenhorst et al, 2011;Sharrocks, 2001). Previous studies (Lau et al, 2012;Nelson et al, 2010) revealed that the increased transcriptional activity of Ets1 upon activation of the Ras/MAPK signaling pathway depends on the phosphorylation-enhanced binding of Ets1 to CBP/p300, which are paralog transcriptional coactivators that serve as scaffolds for the assembly of various transcription factors (Kasper et al, 2006). Specifically, the MAPK extracellular signal-regulated kinase 2 (ERK2) phosphorylates T38 and S41 in an ID N-terminal segment of Ets1, immediately adjacent to the folded PNT domain.…”

Lessons from Drosophila Pointed, an ETS family transcription factor and key nuclear effector of the RTK signaling pathway

“…So in addition to the ETS DNA binding domain, the N‐terminal region of PntP2 harbors a SAM domain (also referred to as the Pointed domain). The core structure of the SAM domain of ETS1 and PntP2 is comprised of four α‐helices (H2–H5) and a fifth helix (H1), N‐terminal to this core (Figure b) (Lau, Okon, & McIntosh, ; Slupsky et al, ). The SAM domain of PntP2 and its vertebrate orthologs has been shown to mediate protein–protein interactions.…”