Abstract
The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)2-3-X2-6-ΦA-X-ΦB, where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus 7RK-X2-ΦA-X-ΦB 13. This results in a 2-fold increase in k cat/K m for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k cat/K m by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k cat/K m, with little apparent change in k cat. A peptide that binds to the DRS of ERK2 affects K m, but not k cat. Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.
Highlights
Mitogen-activated protein kinases (MAPKs) are cell-signaling enzymes that regulate an extraordinarily diverse range of biological processes in eukaryotic organisms [1,2]
We briefly describe the approach used to model the structures of three peptide sequences onto the surface of activated ERK2: a) For sequence FQRKTLQ-RRNLKGLNLNL (Lig-D), coordinates were first obtained for the fragment RRNLKGLNLNL using the known structure of this fragment bound to the MAPK FUS3 (PDB ID: 2B9H) [18]
Modeling the ERK2NEts-1 complex A considerable amount of experimental data suggests that activated ERK2, is a monomer in vitro, and forms a 1:1 complex with residues 1–138 of Ets-1 1-138 (Ets)-1 where two docking interactions contribute to the stability of the complex [11] (Fig. 2)
Summary
Mitogen-activated protein kinases (MAPKs) are cell-signaling enzymes that regulate an extraordinarily diverse range of biological processes in eukaryotic organisms [1,2]. Despite this diversity of signaling, MAPKs are characterized by a single pronounced specificity, namely a preference for phosphorylating proteins at a Ser/Thr-Pro motif [3] This specificity comes from their ability to negatively select against many potential substrates by virtue of the activation segment, a loop at the active site that creates a shallow hydrophobic pocket most compatible with the binding of proline [3,4]. Despite this specificity, proteins containing just a Ser/Thr-Pro element are poor MAPK substrates, generally exhibiting large Michaelis-Menton constants when compared to specific protein substrates. Docking interactions underlie the ability of some MAPKs to phosphorylate as many as fifty substrates or more in vivo [6]
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