Use of docking peptides to design modular substrates with high efficiency for mitogen-activated protein kinase extracellular signal-regulated kinase

Abstract

The mitogen-activated protein kinase extracellular regulated kinase (ERK) plays a key role in the regulation of cellular proliferation. Mutations in the ERK cascade occur in 30% of malignant tumors. Thus understanding how the kinase identifies its cognate substrates as well as monitoring the activity of ERK is central to cancer research and therapeutic development. ERK binds to its protein targets, both downstream substrates and upstream activators, via a binding site distinct from the catalytic site of ERK. The substrate sequences that bind, or dock, to these sites on ERK influence the efficiency of phosphorylation. For this reason, simple peptide substrates containing only phosphorylation sequences typically possess low efficiencies for ERK. Appending short docking peptides derived from full-length protein substrates and activators of ERK to a phosphorylation sequence increased the affinity of ERK for the phosphorylation sequence by as much as 200-fold while only slightly diminishing the maximal velocity of the reaction. The efficiency of the phosphorylation reaction was increased by up to 150-fold, while the specificity of the substrate for ERK was preserved. Simple modular peptide substrates, which can be easily tailored to possess high phosphorylation efficiencies, will enhance our understanding of the regulation of ERK and provide a tool for the development of new kinase assays.

Figure 1

A linker is required to connect the docking peptide to the substrate sequence. a) Structure of ERK2 (PDB 2FYS) indicating the residues interacting with the substrate D domain (green), substrate sequence (blue), and ATP (red). The image was created with Pymol (DeLano Scientific LLC). b) Shown is the linker AOO₃ with a glycine at both termini (Gly-(AOO)₃-Gly).

Figure 1

A linker is required to connect the docking peptide to the substrate sequence. a) Structure of ERK2 (PDB 2FYS) indicating the residues interacting with the substrate D domain (green), substrate sequence (blue), and ATP (red). The image was created with Pymol (DeLano Scientific LLC). b) Shown is the linker AOO₃ with a glycine at both termini (Gly-(AOO)₃-Gly).

Figure 2

Location of the docking peptide and substrate peptide when bound to the kinase. a) Model of ERK2 (PDB 2FYS) with a docking peptide (green) depicted in a stick form. The docking peptide GIMLRRLQKGNLPVRAL is derived from the D-domain of MAP kinase phosphatase 3 (18). The N terminus is marked with an “N”. b) Model of CDK2 (PDB 1QMZ) with a substrate peptide (HHASPRK) depicted in a stick form (blue). The C terminus of the substrate peptide is labeled. In a) and b), the ATP-interacting residues of the kinase are highlighted in red. All images were generated with Pymol.

Figure 3

Phosphorylation of the designed substrates by ERK1. The designed substrates (1μM) were incubated with ERK1 in the presence of ATP and Mg²⁺. Aliquots of the reaction mixture were removed at varying time points and the amount of phosphorylated peptide was measured. The data points represent the average of three measurements and the error bars indicate their standard deviation.

Figure 4

The rates of phosphorylation of the designed substrates fit to an equation of the form v = V_max^app*[D-S]/(KM^app + [D-S]) where [D-S] is the concentration of the docked substrate peptide. a) Shown is the v vs substrate concentration ([S]) curve for ERKSub. b) and c), Shown is the v vs docked substrate concentration ([D-S]) curve for ERKMEK1 (B) and MEK1ERK (C). The solid lines represent the fits to the Michaelis-Menten equation (a) or the equation above (b and c). The initial velocity v is reported per μmole of enzyme used.

Figure 5

Time course of ERKSub phosphorylation by ERK in the presence and absence of the free MEK1 docking peptide. The initial concentration of ERKSub was 150 μM. The data points represent the average of three measurements while the error bars represent the standard deviation.