The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity

Emrick, Michelle A.; Lee, Thomas; Starkey, Paul J.; Mumby, Marc C.; Resing, Katheryn A.; Ahn, Natalie G.

doi:10.1073/pnas.0608849103

Cited by 99 publications

(133 citation statements)

References 33 publications

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“…A number of drug-resistant kinase mutants with altered gatekeeper residues have been identified in multiple kinases that have been effectively targeted clinically: BCR-Abl (T 315I ), EGFR (T 790M ), KIT (T 670I ), and ALK (L 1196M ). Mutation of the gatekeeper residue in rat ERK2 (ERK2 Q103 ) to glycine or alanine has been shown to increase autoactivation by enhancing autophosphorylation of the Thr 183 and Tyr 185 in the activation loop (32). Exploring this further, we found that mutation of human ERK2 glutamine 105 to glutamic acid (Q 105E ) was sufficient to mediate resistance in the presence of an ERK inhibitor through mechanisms that are not fully defined (data not shown).…”

Section: Discussionmentioning

confidence: 74%

Dissecting Therapeutic Resistance to ERK Inhibition

Jha¹,

Morris²,

Hruza³

et al. 2016

Molecular Cancer Therapeutics

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The MAPK pathway is frequently activated in many human cancers, particularly melanomas. A single-nucleotide mutation in BRAF resulting in the substitution of glutamic acid for valine (V 600E ) causes constitutive activation of the downstream MAPK pathway. Selective BRAF and MEK inhibitor therapies have demonstrated remarkable antitumor responses in BRAF V600E-mutant melanoma patients. However, initial tumor shrinkage is transient and the vast majority of patients develop resistance. We previously reported that SCH772984, an ERK 1/2 inhibitor, effectively suppressed MAPK pathway signaling and cell proliferation in BRAF, MEK, and concurrent BRAF/MEK inhibitor-resistant tumor models.ERK inhibitors are currently being evaluated in clinical trials and, in anticipation of the likelihood of clinical resistance, we sought to prospectively model acquired resistance to SCH772984. Our data show that long-term exposure of cells to SCH772984 leads to acquired resistance, attributable to a mutation of glycine to aspartic acid (G 186D

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Section: Discussionmentioning

confidence: 74%

Dissecting Therapeutic Resistance to ERK Inhibition

Jha¹,

Morris²,

Hruza³

et al. 2016

Molecular Cancer Therapeutics

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“…The buffer used for ERK2 in NMR experiments contained 50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM MgSO 4 , 0.1 mM EDTA, 5 mM DTT, 100% D 2 O, and 2.5% (vol/vol) glycerol. Kinase activities of 0P-ERK2, ME/GG-ERK2, and 2P-ERK2 were measured as described in a previous study (36). Nucleotide binding experiments were performed by titrating AMP-PNP (Roche Applied Science) into the NMR samples of 0P-ERK2 and 2P-ERK2, to final AMP-PNP concentrations of 1.35 mM and 1.15 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation releases constraints to domain motion in ERK2

Xiao¹,

Lee

Latham³

et al. 2014

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

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143

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Protein motions control enzyme catalysis through mechanisms that are incompletely understood. Here NMR 13 C relaxation dispersion experiments were used to monitor changes in side-chain motions that occur in response to activation by phosphorylation of the MAP kinase ERK2. NMR data for the methyl side chains on Ile, Leu, and Val residues showed changes in conformational exchange dynamics in the microsecond-to-millisecond time regime between the different activity states of ERK2. In inactive, unphosphorylated ERK2, localized conformational exchange was observed among methyl side chains, with little evidence for coupling between residues. Upon dual phosphorylation by MAP kinase kinase 1, the dynamics of assigned methyls in ERK2 were altered throughout the conserved kinase core, including many residues in the catalytic pocket. The majority of residues in active ERK2 fit to a single conformational exchange process, with k ex ≈ 300 s −1 (k AB ≈ 240 s −1 /k BA ≈ 60 s −1 ) and p A /p B ≈ 20%/80%, suggesting global domain motions involving interconversion between two states. A mutant of ERK2, engineered to enhance conformational mobility at the hinge region linking the N-and C-terminal domains, also induced two-state conformational exchange throughout the kinase core, with exchange properties of k ex ≈ 500 s −1 (k AB ≈ 15 s −1 /k BA ≈ 485 s −1 ) and p A /p B ≈ 97%/3%. Thus, phosphorylation and activation of ERK2 lead to a dramatic shift in conformational exchange dynamics, likely through release of constraints at the hinge.T he MAP kinase, extracellular signal-regulated kinase 2 (ERK2), is a key regulator of cell signaling and a model for protein kinase activation mechanisms (1). ERK2 can be activated by MAP kinase kinases 1 and 2 (MKK1 and 2) through dual phosphorylation of Thr and Tyr residues located at the activation loop (Thr183 and Tyr185, numbered in rat ERK2) (1, 2). Phosphorylation at both sites is required for kinase activation, resulting in increased phosphoryl transfer rate and enhanced affinity for ATP and substrate (3).Conformational changes accompanying the activation of ERK2 have been documented by X-ray structures of the inactive, unphosphorylated (0P-ERK2) and the active, dual-phosphorylated (2P-ERK2) forms (4, 5). Phosphorylation rearranges the activation loop, leading to new ion-pair interactions between phosphoThr and phospho-Tyr residues and basic residues in the N-and C-terminal domains of the kinase core structure. This leads to a repositioning of active site residues surrounding the catalytic base, enabling recognition of the Ser/Thr-Pro sequence motif at phosphorylation sites and exposing a recognition site for interactions with docking sequences in substrates and scaffolds (6).Less is known about how changes in internal motions contribute to kinase activation. Previous studies using hydrogenexchange mass spectrometry (HX-MS) and electron paramagnetic resonance spectroscopy (7-9) led to a model where conformational mobility at the hinge linking the N-and C-terminal domains is increased by phosph...

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“…Hydrogen-deuterium exchange coupled to mass spectrometric (HDX-MS) analysis provides an alternative approach to determining ligand-linked structural and/or dynamic changes in the BirA monomer (19,20). This method has proven powerful in localizing changes in the levels and rates of peptide backbone H-D exchange in proteins as they form complexes and it has been successfully applied to analyzing the allosteric response in several protein systems (21)(22)(23)(24). Its major advantage over other techniques for analyzing the allosteric response in BirA is that the exchange can be performed at concentrations at which even the liganded forms of BirA are predominantly monomeric.…”

Section: The Biotin Repressor: An Allosteric Site-specific Dna Bindinmentioning

confidence: 99%

Allosteric Signaling in the Biotin Repressor Occurs via Local Folding Coupled to Global Dampening of Protein Dynamics

Laine

Streaker

Nabavi

et al. 2008

Journal of Molecular Biology

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SummaryThe biotin repressor is an allosterically regulated site-specific DNA binding protein. Binding of the small ligand, bio-5'-AMP, activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand binding site concomitant with effector binding to the repressor monomer. In this work the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using HydrogenDeuterium exchange coupled to Mass Spectrometry. Measurements were performed on the apoprotein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from H-D exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with magnitude of the functional response to effector binding.

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The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity

Cited by 99 publications

References 33 publications

Dissecting Therapeutic Resistance to ERK Inhibition

Dissecting Therapeutic Resistance to ERK Inhibition

Phosphorylation releases constraints to domain motion in ERK2

Allosteric Signaling in the Biotin Repressor Occurs via Local Folding Coupled to Global Dampening of Protein Dynamics

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