Expanding the Repertoire of an ERK2 Recruitment Site:  Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Abramczyk, Olga; Rainey, Mark A.; Barnes, Richard H.; Martin, Lance; Dalby, Kevin N.

doi:10.1021/bi7002058

Cited by 35 publications

(53 citation statements)

References 38 publications

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“…An ERK2 docking site is located on the Ets1 and Ets2 PNT domains (11,12). The conformational flexibility of helix H0 may contribute to the accessibility of Thr38 and Ser41 to the catalytic site of ERK2 while the PNT domain is docked at an ancillary site on the enzyme (30). We conclude that the conservation in spacing between the core PNT domain and phosphoacceptors sites is likely explained by the role of helix H0 in both CBP binding and potentially ERK2 docking.…”

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

confidence: 86%

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

confidence: 86%

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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“…These spin-labeled species were used together with an EtsΔ138 construct that lacks all four native cysteines to obtain PREs. These various mutations lead to minimal changes in the enzymatic parameters (32), confirming that native interactions between ERK2 and EtsΔ138 are preserved. The spin-label at position 240 produced a number of significant PREs (indicative of shorter distances); a smaller effect was seen for the spin-label at position 252.…”

Section: Resultsmentioning

confidence: 62%

“…We suspect that this interaction, although present, occurs only transiently. This scenario is supported by the fact that cysteine footprinting studies show no significant protection of Asp316 in the presence of EtsΔ138 and only a ∼1.7-fold increase in K M for the phosphorylation of EtsΔ138 by an ERK2 construct carrying a Asp316Ala/Asp319Ala double mutation (32).…”

Section: Resultsmentioning

confidence: 92%

Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

Piserchio

Warthaka

Kaoud

et al. 2017

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

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Mitogen-activated protein (MAP) kinase substrates are believed to require consensus docking motifs (D-site, F-site) to engage and facilitate efficient site-specific phosphorylation at specific serine/ threonine-proline sequences by their cognate kinases. In contrast to other MAP kinase substrates, the transcription factor Ets-1 has no canonical docking motifs, yet it is efficiently phosphorylated by the MAP kinase ERK2 at a consensus threonine site (T38). Using NMR methodology, we demonstrate that this phosphorylation is enabled by a unique bipartite mode of ERK2 engagement by Ets-1 and involves two suboptimal noncanonical docking interactions instead of a single canonical docking motif. The N terminus of Ets-1 interacts with a part of the ERK2 D-recruitment site that normally accommodates the hydrophobic sidechains of a canonical D-site, retaining a significant degree of disorder in its ERK2-bound state. In contrast, the C-terminal region of Ets-1, including its Pointed (PNT) domain, engages in a largely rigid body interaction with a section of the ERK2 F-recruitment site through a binding mode that deviates significantly from that of a canonical F-site. This latter interaction is notable for the destabilization of a flexible helix that bridges the phospho-acceptor site to the rigid PNT domain. These two spatially distinct, individually weak docking interactions facilitate the highly specific recognition of ERK2 by Ets-1, and enable the optimal localization of its dynamic phospho-acceptor T38 at the kinase active site to enable efficient phosphorylation.MAP kinase | transcription factor | proximity-mediated catalysis | solution NMR T he mitogen activated protein (MAP) kinase ERK2 (extracellular signal-regulated kinase 2) lies at the terminus of a three-tiered phosphorylation-based response to a wide range of extracellular cues that include cytokines, hormones, and growth factors (1-4). ERK2 phosphorylates numerous substrates in both the nucleus and the cytoplasm, including a variety of transcription factors, regulatory kinases, phosphatases, proteins of the nuclear pore complex, and cytoskeleton proteins, among others (3, 4). ERK2-mediated phosphorylation occurs on specific serine/threonine residues that immediately precede a proline; however, the (S/T)P motif alone does not provide sufficient substrate affinity or selectivity to distinguish it from other proline-directed kinases, such as CDK2 (5). To attain high specificity toward native substrates, ERK2 and other MAP kinases use one of two so-called "docking sites," the D-recruitment site (DRS) or the F-recruitment site (FRS) (SI Appendix, Fig.

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“…ERK2-substrate model-In recent work, we have studied the ERK2 recruitment sites in detail utilizing a range of biochemical approaches [4,10,[76][77][78][79]. ERK2 is known to have two recruitment sites called the D-recruitment site (DRS) and the F-recruitment site (FRS) (Figures 2 and 6), which bind the D-site and F-site of protein substrates, respectively [79,80].…”

Section: Extracellular Signal-regulated Kinasesmentioning

confidence: 99%

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

Schnieders¹,

Kaoud²,

Cheng³

et al. 2012

curr drug metab

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Due to their role in cellular signaling mitogen activated protein (MAP) kinases represent targets of pharmaceutical interest. However, the majority of known MAP kinase inhibitors compete with cellular ATP and target an ATP binding pocket that is highly conserved in the 500 plus representatives of the human protein kinase family. Here we review progress toward the development of non-ATP competitive MAP kinase inhibitors for the extracellular signal regulated kinases (ERK1/2), the c-jun N-terminal kinases (JNK1/2/3) and the p38 MAPKs (α, β, γ, and δ). Special emphasis is placed on the role of computational methods in the drug discovery process for MAP kinases. Topics include recent advances in X-ray crystallography theory that improve the MAP kinase structures essential to structure-based drug discovery, the use of molecular dynamics to understand the conformational heterogeneity of the activation loop and inhibitors discovered by virtual screening. The impact of an advanced polarizable force field such as AMOEBA used in conjunction with sophisticated kinetic and thermodynamic simulation methods is also discussed.

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Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

Cited by 35 publications

References 38 publications

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

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

Local destabilization, rigid body, and fuzzy docking facilitate the phosphorylation of the transcription factor Ets-1 by the mitogen-activated protein kinase ERK2

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

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