Substrate discrimination among mitogen-activated protein kinases through distinct docking sequence motifs

Abstract

Mitogen-activated protein kinases (MAPKs) mediate cellular responses to a wide variety of extracellular stimuli. MAPK signal transduction cascades are tightly regulated, and individual MAPKs display exquisite specificity in recognition of their target substrates. All MAPK family members share a common phosphorylation site motif, raising questions as to how substrate specificity is achieved. Here we describe a peptide library screen to identify sequence requirements of the DEF site (docking site for ERK FXF), a docking motif separate from the phosphorylation site. We show that MAPK isoforms recognize DEF sites with unique sequences and identify two key residues on the MAPK that largely dictate sequence specificity. Based on these observations and computational docking studies, we propose a revised model for MAPK interaction with substrates containing DEF sites. Variations in DEF site sequence requirements provide one possible mechanism for encoding complex target specificity among MAPK isoforms.

FIGURE 1.

MAPKs share a common consensus phosphorylation site specificity. Preferred amino acids flanking the Ser or Thr phosphoacceptor were identified for four representative MAPKs by measuring the relative rate of phosphorylation of 198 members within a positional scanning peptide library (sequence shown at top). Kinase reactions were performed in parallel in solution in the presence of [γ-³³P]ATP. Biotin-labeled peptides were then captured on a streptavidin membrane, and the relative rate of phosphorylation was assessed by phosphorimaging. ERK2, p38α, p38δ, and JNK2 share a common active site motif with a requirement for Pro at the +1 position and a preference for Pro at -2. pT, phosphothreonine; pY, phosphotyrosine.

FIGURE 2.

Optimization of peptide substrates containing DEF sites. A, pairs of DEF site peptides based on amino acids 386–399 of human Elk-1 that were evaluated for phosphorylation by ERK2. Peptides contain the endogenous ERK phosphorylation site (Ser-389) and either an intact DEF site (FQFP) or a nonfunctional DEF site with charged residue in position P1 (DQFP). B, extent of phosphorylation was measured by radiolabel incorporation from [γ-³³P]ATP. The increase in phosphorylation rate afforded by an intact DEF site is potentiated by incorporating a suboptimal phosphorylation site sequence (peptide AF) or by separating the phosphorylation site from the DEF site by one additional residue (peptide PKF). Error bars reflect the standard deviation of three separate experiments.

FIGURE 3.

Identification of MAPK DEF site motifs using a novel positional scanning peptide library. A, peptide library incorporates 19 amino acid substitutions (Z) at each of the four positions within the DEF site (P1–P4). B, peptides were subjected to phosphorylation by the MAPK in the presence of [γ-³³P]ATP followed by capture on streptavidin membrane and exposure to a phosphor screen (left panel). p38 MAPK isoforms displayed unique selectivity at the P1 position. The p38α selectivity is similar to ERK2, with a preference for aromatic residues at P1, although p38δ favors aliphatic residues at P1. p38β and p38γ displayed intermediate P1 selectivity with p38β more similar to p38α and p38γ more similar to p38δ. JNK2 did not display a strong sequence preference in the DEF site (<2-fold difference for any substitution). The P4 Tyr peptide was not included because of failed synthesis (space indicated with an asterisk). Histograms depict the quantified radiolabel incorporation for the P1 position. The selectivity value is calculated as the level of phosphorylation of a given peptide divided by the average level of phosphorylation for all 19 peptides. Error bars represent the standard deviation from three separate experiments.

FIGURE 4.

DEF site-dependent phosphorylation of consensus peptide and protein substrates. A, sequences of consensus peptides used in this study. B, phosphorylation rates of consensus peptides by p38α and p38δ were assayed at concentrations ranging from 2.5 to 120 μm. The two p38 isoforms phosphorylate their respective consensus peptides at a faster rate at all concentrations tested. A peptide with Ala substitutions in positions P1 and P3 in the DEF site (DEFless) was phosphorylated inefficiently by both kinases. C, rate of phosphorylation of ALPHAtide or DELTAtide at 5 μm concentration by all four p38 isoforms is shown (relative to their phosphorylation rate of MAPKtide at 100 μm). Selectivity for the two consensus peptides reflects their specificity at the P1 position observed in the DEF peptide library, with p38β and p38γ having intermediate selectivity between p38α and p38δ (cf. Fig. 3B). Error bars reflect the standard deviation of three separate experiments. D, fragment of human Elk-1 containing its MAPK phosphorylation and docking sites (residues 307–428, WT), and the same fragment with its DEF sequence mutated from FQF to either AAA or IVW as indicated, was phosphorylated in vitro with ERK2. Site-specific phosphorylation was determined by immunoblotting with a phosphospecific antibody against phospho-Ser-383, and total phosphorylation was determined by autoradiography. E, NIH3T3 cells were transfected with either an empty vector or plasmids encoding the same fragment of Elk-1 used in D and the indicated mutants. Cells were serum-starved overnight and either harvested without further treatment or treated with media containing 10% calf serum to stimulate ERK activity. Cells were pretreated where indicated with the MEK1/2 inhibitor U0126 (5 μm) to block ERK activation. Shown are immunoblots of cell lysates using antibodies to detect phosphorylated and total Elk-1 and ERK1/2.

FIGURE 5.

Mutagenesis of the DEF site interaction pocket. A, primary sequence alignment of the residues surrounding the DEF site interaction pocket (highlighted residues) of ERK2, p38 isoforms, and JNK2 reveals a high degree of sequence conservation. Amino acids that were mutated in this study are indicated with an asterisk. B, single amino acid substitutions to Ala were made in p38α at Leu-195 and Tyr-258 and in p38δ at Leu-195, Leu-232, and Tyr-258. With the exception of p38δ L195A, the phosphorylation rates of DEF site consensus peptides were attenuated ∼4-fold relative to the WT kinase. Results from screening the DEF peptide library with these mutants are shown in supplemental Fig. 2. C, specificity transposition mutants (p38α I193V/M194I and p38δ V193I/I194M) were screened against the DEF peptide library. Mutation of these residues interchanges the selectivity for aromatic versus aliphatic side chains at the P1 position between p38α and p38δ. D, relative rates of phosphorylation of ALPHAtide and DELTAtide consensus peptides by the transposition mutants mirror those for the WT p38 isoforms. Error bars reflect the standard deviation of three separate experiments.

FIGURE 6.

Proposed model for DEF site-MAPK interactions. Docking of a capped pentapeptide ligand representing an aromatic-X-aromatic DEF sequence (acetyl-SFQFP-amide) to the active form of ERK2 (Protein Data Bank code 2ERK) was modeled using AutoDock. A, docking of the peptide ligand (orange) was restricted to the previously identified hydrophobic pocket on the ERK2 surface (green). Residues constituting the P +1 selectivity pocket are highlighted in blue. B, close up view of the docking model (rotated 90° counterclockwise from A). Consistent with peptide screening data showing strongest selectivity at the P1 and P3 positions, the two Phe residues extend deeply into the ERK2 hydrophobic pocket, whereas the Pro residue at P4 makes less intimate contact. C, removal of the surface detail from B. Residues lining the hydrophobic pocket are shown in stick representation. The P1 Phe residue is in closest proximity to Met-197, consistent with the change in specificity observed when the analogous residue in p38α is mutated to Ile.