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, 288 (12), 8596-609

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Affiliations

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Gábor Glatz et al. J Biol Chem.

Abstract

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.

Figures

FIGURE 1.
FIGURE 1.
A PB1 domain in a MAPK kinase is unique to the ERK5 signaling module. A, the cladogram for MAPKs and their specific MKK activators. Cladograms were calculated based on the sequences of kinase domains for MAPKs or MKKs found in KinBase (57). CDK, cyclin-dependent kinase. (Atypical MAPKs are ERK3/4/7 or NLK (58), STE11 family is comprised of MAPK kinase kinases.). B, schematic organization of the MEKK2/3-MKK5-ERK5 MAPK module. PB1 domains from MEKK3 and MKK5 are shown with colored squares, and a box next to the ERK5 kinase domain indicates the long non-catalytic C-terminal tail (407–816) of ERK5. C, all MKKs contain a linear D-motif: their sequences are shown for the seven human MKKs. In the MKK D-motif consensus Ψ, Φ, and X denotes basic, hydrophobic, and any amino acids, respectively. In addition, MKK5 also contains an evolutionary conserved PB1 globular domain. The panel on the right shows the MAPK docking surface colored according to its electrostatic potential (positive in blue and negative in red) from the ERK2 crystal structure complexed with a peptide containing the MKK2 linear D-motif (shown in black with the side chains of the consensus motif forming amino acids indicated) (26).
FIGURE 2.
FIGURE 2.
Characterization of the MKK5-ERK5 interaction. A, three distinct MKK5 regions contribute to its interaction with ERK5. MBP pulldown results with the ERK5 kinase domain (residues 1–431) as prey and different deletion constructs (A, A–G panels) of MKK5 as baits. Interaction of bait and prey was detected on an SDS-PAGE gel stained with Coomassie protein dye. Arrows indicate the position of ERK5 in the pulldown experiments. MBP control was used as negative control to assess unspecific binding of the prey. Bands appearing in addition to baits or preys are degradation products of MBP-fusion proteins. The panel shows the results of a representative MBP pulldown assay from two independent experiments. B, schematic diagram of direct (top panels) and competitive (bottom panels) FP-based titration experiments for determining protein-peptide or protein-protein binding affinities (Kd). Panels on the right show binding isotherms for direct (on the top) and for competitive (on the bottom) ERK5·PB1-D binding experiments. Errors indicate uncertainty in the fit, and error bars on the binding isotherms show S.D. based on three independent measurements. C, competitive binding experiments to monitor binding of MKK5_KD, MKK5_ΔPB1, and MKK5_FL to ERK5. The panel shows the competitive binding curve using unlabeled PB1-D as the competitor and labeled ERK5·PB1-D complex as the reporter (PB1-D labeled with 5-iodoacetamidofluorescein, IAF). The y axis shows the degree of complex formation that was determined based on arbitrary FPmin and FPmax units from numerical fits to competitive binding equations as shown on B. Data were fit to a competition binding equation. Errors indicate uncertainty in the fit, and error bars on the binding isotherms show S.D. based on three independent measurements. D, interactions contributing to the formation of the MKK5·ERK5 binary complex: (i) the MKK5 PB1 domain binds to the ERK5 kinase domain, (ii) MKK5 D-motif presumably binds in the ERK5 docking groove, and (iii) the MKK5 kinase domain binds to the ERK5 kinase domain on an area distinct from the PB1 and D-motif-interacting surfaces. This latter interaction might speculatively form between the MKK5 active site and the ERK5 activation loop (shown with an asterisk or with a black line, respectively). conc., concentration.
FIGURE 3.
FIGURE 3.
Crystal structure of the ERK5·PB1-D complex. A, transparent surface representation of the ERK5·PB1-D complex. The ERK5 kinase domain is shown in gray, and its C-terminal region (from 385 to 399) is shown in red. The MKK5 PB1-D construct is shown in orange. B, zoomed-in view of the protein-protein interface in the MAPK docking groove (IF1). C, zoomed-in view of the protein-protein interface on the PB1 domain (IF2). Only amino acid side chains appearing to play an important role at the interfaces are indicated. Specific hydrogen bonds are shown with dashed lines, and the non-hydrolyzable ATP analog, AMP-PNP, is shown in pink. D, representative examples of electron density maps shown around the MKK5 D-motif at IF1 and the ERK5 C-terminal extension at IF2. 2FoFc maps were calculated with the ERK5·PB1-D final model and contoured at 1σ. E, results of binding affinity measurements on ERK5·PB1-D complex formation. The table displays the binding affinities between wild-type (see also Fig. 2B) or modified versions of MKK5 PB1-D and ERK5 kinase domain constructs. ERK5ΔC385–399 lacks the C-terminal ERK5 kinase domain region shown in red on A and C. PB1-Dmut is an MKK5 PB1-D construct in which Arg-115, Leu-120, and Ile-122 are mutated to alanines. (− indicates no detectable binding; Kd > 100 μm). Errors indicate uncertainty in the fit, and error bars on the binding isotherms show S.D. based on three independent measurements. mut, mutant.
FIGURE 4.
FIGURE 4.
Specificity of the ERK5 MAPK docking groove compared with ERK2. A, ERK5 and ERK2 binding affinities of chemically synthesized peptides compared with the binding affinities of the MKK5 PB1-D construct. Asterisks indicate peptides for which ERK2 binding affinities are listed from Ref. . A minus sign indicates no detectable binding; Kd > 100 μm. Amino acids corresponding to the ϕxϕ motif are shown in boldface type, and basic amino acids binding to the negatively charged common docking (CD) groove are underlined and shown in boldface type. Note that linear motifs may bind into the MAPK docking groove in two different N-to-C-terminal orientations. In addition to the classical D-motif, reverse D-motifs also exist (e.g. pepMNK1 or pepRSK1) (23). Shown are the results of PB1-D (B) and MEF2A (C) docking peptide binding experiments with ERK5 and ERK2, ERK5 competitive titration experiments with various chemically synthesized peptides (D), ERK2 binding experiment with pepMKK5 (E). Errors indicate uncertainty in the fit, and error bars on the binding isotherms show S.D. based on three independent measurements. F, MKK1, MKK2, and MKK5 phosphorylate only their cognate MAPK in vitro. Recombinant ERK5 and ERK2 (5 μm) were incubated as substrates with constitutively activated forms of MKK enzymes (MKK1_EE, MKK2_EE, MKK5_DD; 0.5 μm) in the presence of radioactive [γ-32P]ATP. G, phosphorylation of different MAPK substrates by ERK5 and ERK2. Recombinantly expressed wild-type MAPKs (1 μm) were preincubated with ATP and constitutively active MKK5 or MKK1 enzymes (1–1 μm) creating activated MAPKs (*), in turn phosphorylation of MEF2A, MNK1, and RSK1 phosphorylation reporters was monitored by adding 5-fold excess of substrates (5 μm) in the presence of radioactive [γ-32P]ATP. F and G show typical phosphorimaging results of in vitro kinase assays from two independent experiments.
FIGURE 5.
FIGURE 5.
Different sequence and surface features set ERK5 apart from ERK2. A, sequence alignment of the ERK5 kinase domain and full-length human ERK2. Sequence regions that are important and responsible for different ERK5 versus ERK2 protein-protein binding surface topography are highlighted in red. The threonine and tyrosine phosphorylation target sites of the upstream kinase, which are located in the MAPK activation loop, are shown in boldface type. B, comparison of the ϕxϕ MAPK docking groove region from apo ERK5 and the ERK5·MKK5(PB1-D) complex. Note that the conformation of the Q-loop (shown between two small arrows) differs between the apo (red) and the complexed (gray) crystal structures. (This panel shows the conformation of the ERK5 Q loop from one of the non-crystallographic symmetry-related ERK5 molecules, whereas this region is disordered in the other molecule.) C, comparison of the ϕxϕ MAPK docking groove region from the ERK5·MKK5(PB1-D) and the ERK2-pepMKK2 complex crystal structures (26). Note the difference in the width of the ϕxϕ binding groove. The width is defined by the distance between two H-bond forming amino acids from the MAPK specificity loop on top and from the so-called Q loop from below. These two H-bonds are observed in all known MAPK-docking peptide complex structures, and they are shown with dashed lines. Sequence logos on B and C show the evolutionarily conserved but distinct ERK2 and ERK5 Q-loop sequences (from various annotated vertebrate, primitive metazoan, and opisthokont MAPKs.) D, superposition of the ERK5·MKK5(PB1-D) and the ERK2-pepMKK2 complexes. The figure shows the Cα trace of the two MAPKs when they bind to MKK5 or MKK2 constructs. Note the different width of the MAPK docking groove, which is set by the amino acid supporting the Q loop (Arg-258 in ERK5 and Asn-224 in ERK2). E, 2FoFc electron density map contoured at 1σ and shown around the Q loop from apo ERK5. F, 2FoFc maps contoured at 1σ and shown around the Q loop (left) or around Arg-258 (right) from the ERK5·PB1-D complex. Dashed lines show H-bonds.
FIGURE 6.
FIGURE 6.
The MKK5 PB1 domain serves as an adaptor between MEKK3 and ERK5. A, results of in vitro reconstituted kinase assays with MEKK3-MKK5-ERK5 kinase module components (0.1 μm MEKK3, 0.2 μm MKK5, and 2 μm ERK5). ERK5 phosphorylation was detected by anti-phospho-ERK5 Western blots. Panels show the phosphorimaging results of kinase activity experiments on a general kinase substrate. MEKK3_FL, full-length; MEKK3_KD, kinase domain; MyBP, myelin basic protein. Panels show a representative set of results from two independent experiments. B, ERK5 kinase domain, PB1-D from MKK5, and the PB1 domain from MEKK3 form a ternary complex. Results of a GST pulldown experiment, with GST-MEKK3(PB1) as the bait and the ERK5 MAPK domain and a MBP fusion of MKK5(PB1-D) as prey, are shown in the SDS-PAGE gel stained with Coomassie protein dye (top). Lanes 1 and 2 corresponding to prey show the position of ERK5 and MBP-PB1-D proteins. Control lanes (lanes 3 and 4) indicate the lack of unspecific binding to GST protein-loaded glutathione beads. The panel shows the results of a representative GST pulldown assay from two independent experiments. The panel below shows the results of a fluorescence polarization-based binding assay where complex formation between ERK5 and a labeled MKK5 PB1-D construct was attempted to be competed by the addition of increasing amounts of GST-MEKK3(PB1). Error bars on the binding isotherms show S.D. based on three independent measurements. Results of this experiment are also consistent with the existence of a ternary ERK5·PB1-D·MEKK3(PB1) complex. C, role of PB1 domains in ERK5 activation: (i) synergism between the linear MKK5 D-motif and the MKK5 PB1 domain contributes to high affinity MAPK specific binding (ii) adapter/scaffold function of the MKK5 PB1 domain ensures efficient phosphorylation of ERK5 initiated by MEKK3. An asterisk indicates kinase active sites. P in red highlights ERK5 activation through activation loop phosphorylation by MKK5. D, model of the ERK5·PB1-D·MEKK3(PB1) ternary complex (shown in surface representation). The binary complex crystal structure of the MKK5 and MKK3 PB1 domains (Protein Data Bank code 2O2V) was superimposed on the ERK5·PB1-D complex via the MKK5 PB1 domain.

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