A docking site in MKK4 mediates high affinity binding to JNK MAPKs and competes with similar docking sites in JNK substrates

Abstract

Specific docking interactions between MAPKs and their activating MAPK kinases (MKKs or MEKs) are crucial for efficient and accurate signal transmission. Here, we report the identification of a MAPK-docking site, or "D-site," in the N terminus of human MKK4/JNKK1. This docking site conforms to the consensus sequence for known D-sites in other MKKs and contains the first of the two cleavage sites for anthrax lethal factor protease that have been found in the N terminus of MKK4. This docking site was both necessary and sufficient for the high affinity binding of the MAPKs JNK1, JNK2, JNK3, p38 alpha, and p38 beta to MKK4. Mutations that altered conserved residues in this docking site reduced JNK/p38 binding. In addition, a peptide version of this docking site, as well as a peptide version of the JNK-binding site of the JIP-1 scaffold protein, inhibited both MKK4/JNK binding and MKK4-mediated phosphorylation of JNK1. These same peptides also inhibited JNK2-mediated phosphorylation of c-Jun and ATF2, suggesting that transcription factors, MKK4, and the JIP scaffold compete for docking to JNK. Finally, the selectivity of the MKK4, MEK1, and MEK2 D-sites for JNK versus ERK was quantified. The MEK1 and MEK2 D-sites displayed a strong selectivity for their cognate MAPK (ERK2) versus a non-cognate MAPK (JNK). In contrast, the MKK4 D-site exhibited only limited selectivity for JNK versus ERK.

Fig. 1. MKK4 contains a putative D-site

A, diagram of MKK4. The hatched box represents the catalytic domain; the triangular protrusion represents the putative D-site. In the sequence highlighted above, conserved D-site residues are in boldface and underlined; arrows indicate cleavage sites for the anthrax lethal factor protease. Particular amino acid residues are numbered; full-length MKK4 is 399 residues long, including the N-terminal methionine. B, known D-sites in selected human MAPK-binding proteins aligned with the putative D-site of MKK4. Conserved MAPK-docking site residues are in boldface and marked as either basic (+) or hydrophobic (ϕ). Dashes denote gaps inserted to optimize the alignment; spaces are for visual clarity. The corresponding residues of the full-length proteins are given on the right. GenBank^™/EBI accession numbers are as follows: MEK1, NM_002755; MEK2, NM_030662; MKK4, NM_003010; c-Jun, NM_002228; ATF2, NM_001880; JIP-1, NM_005456; and Elk-1, NM_005229.

Fig. 2. The MKK4 D-site is required for JNK binding

A, ³⁵S-labeled MKK4 derivatives were tested for binding to GST-JNK3. B, ³⁵S-radiolabeled full-length MKK4 protein and N-terminal truncations thereof were prepared by in vitro translation and partially purified by ammonium sulfate precipitation, and portions (10% of the amount added in the binding reactions) were resolved on a 10% SDS-polyacrylamide gel (lane 1). Samples (~1 pmol) of the same proteins were incubated with >40 μg of GST (lane 2) or with 10 or 40 μg of GST-JNK3 (lanes 3 and 4, respectively) bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were isolated by sedimentation and resolved by 10% SDS-PAGE on the same gel. The gel was analyzed by staining with Coomassie Blue for visualization of the bound GST fusion protein (a representative example is shown in the lowest panel) and by autoradiography for visualization of the bound radiolabeled protein (upper three panels).

Fig. 3. MAPK binding to MKK N termini

A, purified JNK1α1, JNK1β1, JNK2α2, or ERK2 or in vitro translated JNK3 was tested for binding to GST fusions to the N-terminal (N) non-catalytic domains of MEK1, MEK2, and MKK4. B, MAPK proteins (~1 pmol of JNK3 and ~10 pmol (0.5 μg) of the others) were tested for binding to 40 μg of GST (lane 2), GST-MEK1-(1–60) (M1 1–60; lane 3), or GST-MEK2-(1–64) (M2 1–64; lane 4) or 10 or 40 μg of GST-MKK4-(1–94) (MKK4 1–94; lanes 5 and 6). Co-sedimented MAPKs were separated by 10% SDS-PAGE and visualized by immunoblotting with anti-JNK or anti-ERK antibody or by autoradiography in the case of ³⁵S-labeled JNK3; 10% of the total amount of MAPK in the reactions is shown in lane 1. Coomassie Blue (CB) staining was used to visualize the bound GST fusion proteins (a representative example is shown in the lowest panel). C, the same experimental design as described for A was used, except that the GST fusions included GST-MKK4-(37–94) and GST-MKK4-(37–94)(EAE), and the MAPKs included in vitro translated, ³⁵S-labeled p38α and p38β. WT, wild-type.

Fig. 4. The MKK4 D-site is sufficient for binding to JNK

A, membrane-attached peptides containing the JNK-docking site of either wild-type (WT) MKK4 (shown as triangles) or a mutant (mut) version thereof were incubated with ³⁵S-labeled JNK3 to assess binding. B, shown are the sequences of the peptides used, corresponding to residues 37–51 of full-length MKK4. Residues mutated in the MKK4 mutant peptide are shown underlined. C, shown is an autoradiogram of representative peptide spots bound to ³⁵S-labeled JNK3. D, shown is a graphical representation of relative peptide binding, as quantified on a PhosphorImager. The average -fold above background binding of the peptides is shown. Three experiments like that shown in C were averaged; each contained triplicate spots. The error bars indicate S.D.

Fig. 5. Conserved residues in the JNK-docking site of MKK4 are required for high affinity JNK binding

A, MKK4 JNK-docking site mutants analyzed. Residues 38 –48 of MKK4 are shown. The positions of substitution mutations are shown underlined. The name of the mutant allele is on the left; a summary of the binding data (shown in B) is on the right. B, relative binding of radiolabeled JNK3 to GST-MKK4-(37–94) (wild-type (WT)) or mutant derivatives thereof. ³⁵S-Labeled JNK3 (~1 pmol) was incubated with 50 μg of GST or GST-MKK4 derivatives bound to glutathione-Sepharose beads, and the resulting bead-bound protein complexes were separated by SDS-PAGE and quantified on a PhosphorImager. The nonspecific background adsorption of each radiolabeled protein to GST alone was subtracted. Under these conditions, 16.0% of the input JNK3 bound to GST-MKK4-(37–94) (wild-type). Results were normalized by setting this value as 100%. Data shown are the average of at least four experiments for the single substitution mutants and at least two experiments for the multiple substitution mutants; error bars indicate S.E. C, representative data for experiments shown in B. Also shown are points containing 10 μg of GST-MKK4 derivatives. Input (10% of the amount used in the binding reactions) is shown in lane 1.

Fig. 6. Inhibition of MKK-mediated MAPK phosphorylation by D-site peptides

A, D-site peptides (triangle) were used to inhibit MKK4 or MEK2 phosphorylation of JNK1 or ERK2, respectively. B, the sequences of the soluble synthetic peptides used in this study are shown; substitution mutations are underlined. The name of the peptide is on the left; the residues of the corresponding full-length protein are on the right. C, shown is the inhibition of MKK4-dependent phosphorylation of JNK1 by D-site peptides. Purified unactivated JNK1 (2 μM) was incubated with purified active MKK4 (~11 nM) and [γ-³²P]ATP for 20 min at 30 °C in the absence (black bar) or presence of 200μM JIP-1, MKK4, and MKK4(EAG) peptides (gray bars) or MEK1, MEK2, and MEK2(EEAA) peptides (white bars). Results are plotted as percent phosphorylation relative to that observed in the absence of any added peptide. JNK1 phosphorylation was analyzed by SDS-PAGE and quantified on a PhosphorImager. Data are the average of two to four experiments, with triplicate data points in each experiment. D, shown is the inhibition of MEK2-dependent phosphorylation of ERK2 by D-site peptides. Purified, catalytically inactive ERK2 (1 μM) was incubated with purified active MEK2 (~30 nM). Peptides were added to a concentration of 100 μM; other details are as described for C.

Fig. 7. Inhibition of MKK4 binding to GST-JNK3 by MKK4 and JIP-1 D-site peptides

The experimental design was as described in the legend to Fig. 2, except that D-site peptides were added to most reactions to see if they would inhibit the binding of full-length MKK4 to GST-JNK3. A, ³⁵S-radiolabeled full-length MKK4 protein (~1 pmol) was prepared by in vitro translation, partially purified by ammonium sulfate precipitation, and then incubated with 30 μg of purified GST or GST-JNK3 prebound to glutathione-Sepharose beads in the absence or presence of the specified concentrations of the indicated peptides (peptides were added to the beads prior to the addition of the radiolabeled protein). Bead-bound protein complexes were isolated by sedimentation and resolved on 10% SDS-polyacrylamide gels. A portion of the in vitro translated MKK4 protein corresponding to 10% of the amount added to the binding reactions is shown in the left lane. Gels were analyzed by staining with Coomassie Blue for visualization of the bound GST fusion protein to verify equal amounts in each reaction (data not shown) and by autoradiography for visualization of the bound radiolabeled proteins. B, relative binding of radiolabeled MKK4 to GST-JNK3 was determined using a PhosphorImager in the absence of peptide (black bar) or in the presence of 25 or 100 μM peptide (gray bars). After subtracting the nonspecific background adsorption to GST alone, 8.7% of the input MKK4 bound to wild-type GST-JNK3 in the absence of peptide. Results were normalized by setting this value as 100%. Data are the average of three or four experiments; error bars indicate S.E.

Fig. 8. Inhibition of MAPK-dependent phosphorylation of transcription factors by D-site peptides

A, D-site peptides (triangle) were used to inhibit JNK2 phosphorylation of c-Jun or ATF2 or ERK2 phosphorylation of Elk-1. B and C, purified GST-c-Jun (1 μM) was incubated with purified active JNK2 (~5 nM) and [γ-³²P]ATP for 20 min in the absence or presence of the specified concentrations of the indicated peptides (see Fig. 6B). B, results are plotted as percent phosphorylation relative to that observed in the absence of any added peptide. c-Jun phosphorylation was analyzed by SDS-PAGE and quantified on a Phosphor-Imager. Data are the average of two to five experiments, with triplicate data points in each experiment. C, shown is an autoradiogram of a representative experiment. D and E, purified GST-ATF2 (1 μM) was incubated with purified active JNK2 (~5 nM); other details are as described for B and C. F and G, shown is the inhibition of ERK2-dependent phosphorylation of Elk-1 by D-site peptides. Purified GST-Elk-1 (1 μM) was incubated with purified active ERK2 (~1 nM) and [γ-³²P]ATP for 20 min in the absence or presence of the specified concentrations of the indicated peptides. F, results are plotted as percent phosphorylation relative to that observed in the absence of any added peptide. Elk-1 phosphorylation was analyzed by SDS-PAGE and quantified on a PhosphorImager. The fastest migrating band (see G), which appeared to be unaffected by peptide inhibition, was omitted from the quantification. Data are the average of two to four experiments, with duplicate or triplicate data points in each experiment. G, shown is an autoradiogram of a representative experiment.