Structure of the human protein kinase MPSK1 reveals an atypical activation loop architecture

Abstract

The activation segment of protein kinases is structurally highly conserved and central to regulation of kinase activation. Here we report an atypical activation segment architecture in human MPSK1 comprising a beta sheet and a large alpha-helical insertion. Sequence comparisons suggested that similar activation segments exist in all members of the MPSK1 family and in MAST kinases. The consequence of this nonclassical activation segment on substrate recognition was studied using peptide library screens that revealed a preferred substrate sequence of X-X-P/V/I-phi-H/Y-T*-N/G-X-X-X (phi is an aliphatic residue). In addition, we identified the GTPase DRG1 as an MPSK1 interaction partner and specific substrate. The interaction domain in DRG1 was mapped to the N terminus, leading to recruitment and phosphorylation at Thr100 within the GTPase domain. The presented data reveal an atypical kinase structural motif and suggest a role of MPSK1 regulating DRG1, a GTPase involved in regulation of cellular growth.

Figure 1

The Domain Architecture of MPSK1 (A) Schematic drawing of full-length MPSK1. Regions predicted to be membrane anchoring (N-myristoyl glycine at position 2 and S-palmitoyl cysteines at positions 6 and 8) are shown as red boxes. The location of the activation segment helix is shown in blue, and identified autophosphorylation sites are indicated. (B) Secondary structure elements labeled for MPSK1 amino acid sequence. Sheets are indicated in green, helices in red, and 3₁₀ helices in magenta. The N-terminal tag sequence is indicated by small cursive letters and the C terminus not visible in the electron density is shown as small blue cursive letters. The autophosphorylation sites are highlighted in red. (C) Ribbon diagram showing an MPSK1 monomer. The two staurosporine molecules binding to the MPSK1 kinase domain are indicated using ball-and-stick representation. The activation segment is highlighted in blue. (D) Interactions of staurosporine binding to the ATP binding site. Hydrogen bonds are shown as dotted lines. The hydrogen bond between the two conserved residues Lys49 and Glu65 typically found in active kinases is also shown. (E) Detailed view of the interactions formed by the two staurosporine molecules binding between symmetry-related protein molecules.

Figure 2

MPSK1 Activation Loop Architecture (A) Structural overlay of the activation loop of active Aurora A (PDB ID code: 1OL7) (shown in magenta) with MPSK1 (shown in red). The main structural elements are labeled. (B) Hydrogen bonds and hydrophobic interactions stabilizing the activation segment of MPSK1 are shown as dotted lines and the residues involved in stabilization are shown in ball-and-stick representation. The interacting β sheet (β11), the P + 1 loop, and the ASCH, as well as the helix αEF, are labeled. (C) Interface of the ASCH interacting with the lower kinase lobe. Hydrophobic residues are indicated as solid white surfaces. (D) Prediction of similar activation loop helices present in the kinome. Secondary structure elements predicted to be smaller than three residues have been deleted. The experimentally determined secondary structure (MPSK1exp) and the predicted one of MPSK1 are also shown. The activation segment helix and helix αEF were predicted accurately, whereas the β sheet secondary structure was not recognized by the prediction program. One representative member of the MAST kinase family predicted to contain an activation loop helix is also shown. Hydrophobic residues, buried in the interface between the ASCH and the lower kinase lobe, are indicated (^∗) in the sequence alignment.

Figure 3

Phosphorylation Motifs for MPSK1 Biotinylated peptides bearing the shown residue at the indicated position relative to a central serine/threonine phosphoacceptor site were subjected to phosphorylation by MPSK1 using radiolabeled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried, and exposed to a phosphor screen. The row marked “0” indicates either no peptide (−) or peptides with fixed Ser or Thr at the phosphoacceptor position.

Figure 4

DRG1 Is an Interaction Partner for MPSK1 (A) GST pull-down experiment using recombinant His-MPSK1 and DRG1 expressed in bacteria. GST-DRG1 was saturated with the nonhydrolyzable GTP/GDP analogs, β-S-GDP or γ-S-GTP and loaded onto a glutathione-Sepharose column. After the column was stringently washed, GST-DGR1 was eluted with glutathione. Copurifying proteins were separated by SDS-PAGE and analyzed by western blotting using a specific MPSK1 antiserum. GST and a bacterial extract expressing only the His₆ fusion partner were used as negative controls. (B) Interaction of MPSK1 and DRG1 in vivo: NIH 3T3 cells were transiently transfected with plasmids overexpressing MPSK1 and DRG1 as well as with an empty vector serving as negative control. MPSK1 was immunoprecipitated under native conditions. After extensive washing, the immunoprecipitated (bound) and the nonimmunoprecipitated fractions (unbound) were separated by SDS-PAGE and analyzed by western blot using a specific anti-DRG1 antiserum.

Figure 5

Domain Architecture of DRG1 and Phosphorylation by MPSK1 (A) DRG1 domains highlighting the glycine-rich region (black box) the GTPase domain (MMR-HSR) as well as the ThrRS, GTPase, and SpoT (TGS) domain. Boundaries of the C-terminal truncated constructs used for pull-down assays are indicated. (B) Pull-down experiment using His-MPSK1 and full-length DRG1 and DRG1-N65. Purified His-MPSK1 along with GST-DRG1 or DRG1-N65 were loaded onto the Ni-NTA column separately. After the column was stringently washed, His-MPSK1 was specifically eluted with 200 mM imidazole and the copurifying proteins were separated by SDS-PAGE. (C) ESI-MS spectrum of DRG1 treated with MPSK1, ATP, and Mg²⁺ for 1 hr at 30°C. The mass corresponding to monophosphorylated DRG1 is indicated. (D) Sequence alignment of DRG1 orthologs and human DRG2 showing the identified phosphorylation site (Thr100) (highlighted in bold). DRG2 contains a cysteine residue at that position which is highly conserved in DRG2 orthologs.

Figure 6

Autophosphorylation of MPSK1 and Mapping of Phosphorylation Sites (A) ESI-MS spectrum recorded for unphosphorylated MPSK1 (black line), after autophosphorylation for 1 hr (red line), 3 hr (blue), and 18 hr (green). (B–D) Location of MPSK1 autophosphorylation sites in MPSK1. Phosphorylated residues are highlighted in red and neighboring residues are shown in ball-and-stick representation. A spectrum of the two identified peptides is shown in (C) and (D), respectively. Identified peptides in MPSK1 are indicated in bold letters in the sequence shown below the panels. Phosphorylated residues are highlighted in red.