Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex

Abstract

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Keywords: ERK2; RSK1; protein kinase; signal transduction; structural biology.

Fig. 1.

Structure of the ERK2–RSK1 complex. (A) Crystal structure of the ERK2 (orange)–RSK1 (green) complex. The RSK1 linear motif region binds to the MAPK docking groove (interface 1, IF1). RSK1 forms a face-to-face antiparallel kinase dimer with the ERK2 kinase domain through IF2 where the catalytic aspartate (Asp149, shown in red) is located next to the RSK1 activation loop (shown in red, and Thr573 is shown with a black sphere). The nucleotide cofactor (AMPPNP) is colored blue. The unstructured part of the RSK1 loop is shown with a dashed line. Lower shows the complex from the back where the electron density for the linear motif (LM), αL, and the intervening linker region (colored in cyan) is shown with sigmaA-weighted omit map contoured at 1σ. (B) A close view of IF1 and IF2. At IF1, contacts are highlighted and labeled according to linear motif consensus (11). Black dashed lines indicate H-bond interactions. At IF2 contacts form between the APE motif of RSK1 and the P loop of ERK2 (Cα atoms of residues within van der Waals contacts are shown with spheres). The dephosphorylated activation loop of ERK2 blocks the RSK1 phosphorylation site (Thr573 in the TP motif) from accessing the ERK2 active site (Asp149, shown in stick representation in red) and substrate binding pocket. The ppERK2–RSK1 complex was generated by superposing the ppERK2 structure (PDB ID: 2ERK) with ERK2 from the crystallographic complex. The phosphorylated activation loop of ERK2 (with Tyr187 and Thr185) is shown in purple.

Fig. 2.

Experimental validation of ERK2–RSK1 contacts. (A) RSK1 mutants display reduced phosphorylation by preactivated ERK2. Phosphorylation rate of RSK1 was monitored by in vitro kinase assays (SI Appendix, Fig. S1). L714E has a mutation at IF1, whereas linker length mutants (Δ2–6) affect contacts at IF2 indirectly. The bar graph shows relative RSK1 phosphorylation, which was calculated based on initial phosphorylation rates that were normalized to wild type (WT), and error bars show the SDs from mean value; n = 3 experiments (SI Appendix, Fig. S1A). (B) RSK1 mutants display reduced activation capacity upon EGF stimulation in HEK293 cells. (Right) Representative set of three independent experiments where samples were taken at the indicated time points after EGF treatment of serum-starved HEK293 cells. Heterologous RSK1 and endogenous ERK activation, the latter as a control for EGF treatment, were monitored by Western blots with phospho-RSK1(Thr573) and ppERK2-specific antibodies, respectively. Transiently transfected RSK1 constructs had a FLAG tag, and anti-FLAG Western blotting was used to demonstrate equal load for different samples and uniform transfection efficiency for different experiments.

Fig. 3.

Experimental validation of surface contacts from the ppERK2–RSK1 MD model. (A) MD predicts that Ser452 and Glu623 play a role in the catalytic ppERK2–RSK1 complex. Phosphorylation rate of RSK1 was monitored by using in vitro kinase assays (SI Appendix, Fig. S3B). (B) The bar graph shows relative RSK1 phosphorylation, which was calculated based on initial phosphorylation rates that were normalized to wild type (WT), and error bars show the SDs from mean value; n = 3 experiments. (APE: RSK1 mutant with a modified APE motif; 452 or 623: Ser452 or Glu623 were changed to tryptophans; APE/623: two mutated regions are combined within one RSK1 construct.) (C) Results of RSK1 activation in EGF-stimulated cells. Blots show a representative set of three independent experiments where samples were taken at the indicated time points after EGF treatment of serum-starved HEK293 cells. Heterologous RSK1 and endogenous ERK activation or equal protein load were analyzed similarly as in Fig. 2 (and control blots for ppERK and RSK1 level are shown on SI Appendix, Fig. S3C). (For comparison, the blot for WT is the same as in Fig. 2.)

Fig. 4.

MD simulations on the ppERK2–RSK1 complex. Movements of the RSK1 activation loop are highlighted (in red). “Starting model” shows a close-up around the catalytic center; the “unrestrained” model shows the same region from the MD model that displayed the shortest distance between Thr573(RSK1) and Asp149(ERK2) (SI Appendix, Fig. S4B); and the “restrained model” was generated by modeling the RSK1 activation loop based on the DYRK1A–substrate peptide complex (14) (SI Appendix, Fig. S4B).

Fig. 5.

Role of the MAPKAPK APE motif and the MAPK P loop in substrate or activator kinase binding. (A) Inactive, unphosphorylated MAPKAPKs have a unique APE motif region different from related Ca²⁺/calmodulin-dependent protein kinases (CAMK) or from a canonical kinase. (B) Schematic model of a MAPK→MAPKAPK(CTD) signaling complex. The model depicts the dual role of the APE motif in activator kinase binding for unphosphorylated MAPKAPKs as well as in downstream substrate binding after MAPKAPK activation loop phosphorylation.

Fig. 6.

Model on the structural assembly of the signaling competent MAPK–MAPKAPK complex. The signaling competent ppERK2–RSK1 complex forms through hierarchical assembly of unique, group specific, and common kinase surface contacts. (This scheme is partly speculative but it is consistent with biochemical and structural data presented in this study.)