Diversity in Structure and Function of the Ets Family PNT Domains

Mackereth, Cameron D.; Schärpf, Manuela; Gentile, Lisa; MacIntosh, Scott; Slupsky, Carolyn M.; McIntosh, Lawrence P.

doi:10.1016/j.jmb.2004.07.094

Cited by 65 publications

(71 citation statements)

References 83 publications

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“…Unlabeled and uniformly 15 N-or 15 N∕ 13 C-labeled murine Ets1 constructs were prepared as described previously (10,11,13,29). Ets1 1-138 and Ets1 were phosphorylated in vitro using 50 mM ATP and a 1∶20 molar ratio of ERK2∶Ets1 (8).…”

Section: Methodsmentioning

confidence: 99%

“…Ets1, Ets2, and their Drosophila ortholog Pnt-P2, as well as GABPα and SPDEF share an appended N-terminal helix H1. The highly similar proteins Ets1 and Ets2 also bear the dynamic helix H0 (29). These helices are preceded with conserved spacing by an ERK2 consensus site only in Ets1, Ets2, and Pnt-P2.…”

Section: Role Of Protein Dynamics In Phosphorylation-regulated Ets1/cbpmentioning

confidence: 99%

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

Nelson

Kang

Lee

et al. 2010

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

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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

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Section: Methodsmentioning

confidence: 99%

Section: Role Of Protein Dynamics In Phosphorylation-regulated Ets1/cbpmentioning

confidence: 99%

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

Nelson

Kang

Lee

et al. 2010

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

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“…Based on a nuclear magnetic resonance (NMR) approach, the N-terminal PNT domain, which is conserved in Ets-1, Ets-2, and PNT P2, forms a five-helix globular structure that resembles a more broadly observed protein fold known as the SAM domain (Fig. 1B) (22,29,47). The site of MAPK phosphorylation is located at a conserved distance ϳ20 amino acids N terminal to the PNT domain in Ets-1, Ets-2, and PNT P2 (45).…”

mentioning

confidence: 99%

Ras/Mitogen-Activated Protein Kinase Signaling Activates Ets-1 and Ets-2 by CBP/p300 Recruitment

Foulds

Nelson

Błaszczak

et al. 2004

Molecular and Cellular Biology

174

145

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Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.The connection of cell signaling to changes of gene expression represents a central step in many types of biological regulation. The Ras/mitogen-activated protein kinase (MAPK) pathway exemplifies the relevance of signaling to both normal development and disease. In this pathway, Ras, a GTPase, transmits extracellular signaling from receptor tyrosine kinases to two serine/threonine kinases (Raf and MEK) and, finally, to the activation of MAPKs. Upon nuclear import, MAPKs phosphorylate many different transcription factors, modulating DNA binding affinity, nuclear localization, stability, and interactions with coregulators (20, 58), thereby regulating gene expression. Genetic studies of Drosophila (54) and Caenorhabditis elegans (48) demonstrate that Ras/MAPK signaling plays a role in normal development. Furthermore, ϳ15 to 20% of all human tumors have an activating mutation in one of three ras genes (N-, K-, or H-ras), and oncogenic mutations in the B-raf gene have been identified for a wide variety of cancers, with 66% of all melanomas being affected (6,8). Although many of the components of Ras/MAPK signaling have been characterized, the full array of transcription factors affected is not known. Furthermore, the detailed mechanisms by which phosphorylation modulates transcription factors remain unclear in many cases.Multiple members of the ETS family of transcription factors are phosphorylated upon activation of the Ras/MAPK signaling pathway. For example, Elk-1 phosphorylation results in recruitment of the mediator complex via its Sur-2 (MED23) subunit (49), enhanced interaction with the coactivator p30...

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“…TEL, or ETV6, another Ets protein in the pointed domain subfamily, is closely related to Fli-1 and may also function in megakaryocytopoiesis (101). The pointed domain of TEL is a short N-terminal domain involved in self-oligomerization.…”

Section: Figurementioning

confidence: 99%