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

ABSTRACT: 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.

SUBMITTER: Piserchio A

PROVIDER: S-EPMC5547609 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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

Piserchio Andrea A Warthaka Mangalika M Kaoud Tamer S TS Callaway Kari K Dalby Kevin N KN Ghose Ranajeet R

Proceedings of the National Academy of Sciences of the United States of America 20170717 31

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 tha ...[more]

PMID: 28716922

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Project description:ERK2, a major effector of the BRAF oncogene, is a promiscuous protein kinase that has a strong preference for phosphorylating substrates on Ser-Pro or Thr-Pro motifs. As part of a program to understand the fundamental basis for ERK2 substrate recognition and catalysis, we have studied the mechanism by which ERK2 phosphorylates the transcription factor Ets-1 at Thr-38. A feature of the mechanism in the forward direction is a partially rate-limiting product release step (koff = 59 +/- 6 s(-1)), which is significant because to approach maximum efficiency substrates for ERK2 may evolve to ensure that ADP dissociation is rate-limiting. To improve our understanding of the mechanism of product release, the binding of the products to ERK2 was assessed and the reaction was examined in the reverse direction. These studies demonstrated that phospho-Ets-1 (p-Ets) binds >20-fold more tightly to ERK2 than ADP (Kd = 7.3 and 165 microM, respectively) and revealed that the products exhibit little interaction energetically while bound to ERK2 and that they can dissociate ERK2 in a random order. The overall equilibrium for the reaction in solution (Keq = 250 M(-1)) was found to be similar to that with the substrate bound to the enzyme (Kint = 525 M(-1)). To determine what limits koff, several pre-steady-state experiments were performed. A catalytic trapping approach furnished a rate constant (k-ADPa) of 61 +/- 12 s(-1) for the dissociation of ADP from the abortive ternary complex, ERK2.Ets.ADP. To examine p-Ets dissociation, the binding of a fluorescent derivative (p-Ets-F), which binds ERK2 with an affinity similar to that of p-Ets, was examined by stopped-flow kinetics. Using this approach, p-Ets-F was found to bind through a single-step mechanism, with the following parameters: k-p-Ets-F = 121 +/- 3.8 s(-1), and kp-Ets-F = (9.4 +/- 0.3) X 10(6) M(-1) s(-1). Similar results were found in the presence of a saturating ADP concentration. These data suggest that koff may be limited by the dissociation of both products and are consistent with the notion that Ets-1 has evolved to be an efficient substrate for ERK2, where ADP release is, at least, partially rate-limiting. A molecular mechanics model of the complex formed between ERK2 and residues 28-138 of Ets-1 provides insight into the role of substrate docking interactions.

Project description:ERK2 primarily recognizes substrates through two recruitment sites, which lie outside the active site cleft of the kinase. These recruitment sites bind modular-docking sequences called docking sites and are potentially attractive sites for the development of non-ATP competitive inhibitors. The D-recruitment site (DRS) and the F-recruitment site (FRS) bind D-sites and F-sites, respectively. For example, peptides that target the FRS have been proposed to inhibit all ERK2 activity (Galanis, A., Yang, S. H., and Sharrocks, A. D. (2001) J. Biol. Chem. 276, 965-973); however, it has not been established whether this inhibition is steric or allosteric in origin. To facilitate inhibitor design and to examine potential coupling of recruitment sites to other ligand recognition sites within ERK2, energetic coupling within ERK2 was investigated using two new modular peptide substrates for ERK2. Modeling shows that one peptide (Sub-D) recognizes the DRS, while the other peptide (Sub-F) binds the FRS. A steady-state kinetic analysis reveals little evidence of thermodynamic linkage between the peptide substrate and ATP. Both peptides are phosphorylated through a random-order sequential mechanism with a k(cat)/K(m) comparable to Ets-1, a bona fide ERK2 substrate. Occupancy of the FRS with a peptide containing a modular docking sequence has no effect on the intrinsic ability of ERK2 to phosphorylate Sub-D. Occupancy of the DRS with a peptide containing a modular docking sequence has a slight effect (1.3 ± 0.1-fold increase in k(cat)) on the intrinsic ability of ERK2 to phosphorylate Sub-F. These data suggest that while docking interactions at the DRS and the FRS are energetically uncoupled, the DRS can exhibit weak communication to the active site. In addition, they suggest that peptides bound to the FRS inhibit the phosphorylation of protein substrates through a steric mechanism. The modeling and kinetic data suggest that the recruitment of ERK2 to cellular locations via its DRS may facilitate the formation of F-site selective ERK2 signaling complexes, while recruitment via the FRS will likely inhibit ERK2 through a steric mechanism of inhibition. Such recruitment may serve as an additional level of ERK2 regulation.

Dataset Information

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

Publications

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

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