Proximity-Induced Catalysis by the Protein Kinase ERK2

Abstract

Five hundred protein kinases phosphorylate 10 000 proteins in human cells. Frequently, more than one site in a protein is phosphorylated, and often by more than one protein kinase. The mechanistic basis underlying the overlapping specificity of the phospho-proteome is not well understood. We are interested in understanding why ERK2, a proline-directed protein kinase, phosphorylates only a fraction of the (S/T-P) sites found in the surface loops of proteins, at an appreciable rate. To address this fundamental question, we utilized a well-established protein substrate EtsDelta138, which comprises a globular ERK2-recognition domain (pnt domain) and an unstructured peptide-like N-terminal tail. This tail contains T38, the sole ERK2 phosphorylation site. We mutated the TP motif, which is recognized by the active site and found that mutagenesis of the T-38/P-39 motif to TD, TR, TA, TG, and TV has no effect on the stability of the ternary complex but does decrease kcat. We also investigated the effect of perturbing the binding between ERK2 and the pnt domain, which occurs outside the active site, to find that mutation of the pnt domain (F120A) leads to a 10-fold decrease in binding but the kcat remains the same. The data support a mechanism of proximity-mediated catalysis, where the docking of the pnt domain, outside the active site, increases the effective concentration of the TP motif near the active site. The data are consistent with the notion that the interaction between ERK2 and the pnt domain provides uniform binding energy and stabilizes each enzyme intermediate and transition state to an equal extent. While other steps on the reaction pathway contribute towards the specificity of the ERK2 reaction, a docking interaction provides the initial basis for substrate recognition. Those residues within the docked complex, which have the ability to access the active site with an appropriate geometry, can be phosphorylated at an efficient rate if followed by a proline or small hydrophobic amino acid.

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

“…Thus, Ets-1 may contain a novel or cryptic D-site, which resides either in the PNT domain or in its flexible N-terminus. Identifying this site is important to understand the basis for the mechanism of proximity-induced catalysis exhibited by ERK2 (22).…”

“…These steps were attributed to the phosphorylation of bound EtsΔ138, k 2 , and processes associated with product release, k 3 , respectively. Recently, evidence was provided for the existence of two ternary complexes on the reaction pathway, prior to the phosphorylation step (22). It was proposed that one ternary complex is stabilized exclusively by interactions extrinsic to the consensus sequence and that this ternary complex can interconvert to a less stable ternary complex in which the consensus sequence of Ets-1 intimately engages the active site.…”

“…It was proposed that one ternary complex is stabilized exclusively by interactions extrinsic to the consensus sequence and that this ternary complex can interconvert to a less stable ternary complex in which the consensus sequence of Ets-1 intimately engages the active site. This was termed a proximity-induced mechanism of catalysis, highlighting the catalytic role extrinsic docking interactions play in increasing the concentration of the consensus sequence within the proximity of the active site (22).…”

The PNT domain from Drosophila pointed‐P2 contains a dynamic N‐terminal helix preceded by a disordered phosphoacceptor sequence

“…This is attributed to faster 1 N H and 15 N transverse relaxation owing to the formation of a high-molecular-mass complex with the kinase. Based on the kinetic studies and equilibrium-binding measurements, the K m or K d values ERK2 kinases and PNT domains are in the range of 10-100 lM, 10,11,26 and thus partial saturation of PntP2 142-252 is expected under these experimental conditions. More importantly, residues in the phosphoacceptor region, as well as helices H0 and H5, showed substantially greater intensity changes yet no chemical shift perturbations.…”

“…The monomeric PNT domain is a docking module for ERK2, enhancing the efficiency of phosphorylating Thr38 and Ser41 in a disordered region preceding this structured domain. 10,11 The docking interfaces on the PNT domain and the kinase have been mapped coarsely through mutagenesis, NMR spectroscopic, chemical footprinting, and competition studies. 10,[12][13][14][15] To both accommodate the proposed docking mechanism and position the phosphoacceptors in the catalytic site of the kinase, a significant conformational change in the ETS1 PNT domain, such as the unfolding of the appended Nterminal helix H0 (residues Lys42-Thr52), appears to be required.…”