Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites

Abstract

Protein kinase autophosphorylation of activation segment residues is a common regulatory mechanism in phosphorylation-dependent signalling cascades. However, the molecular mechanisms that guarantee specific and efficient phosphorylation of these sites have not been elucidated. Here, we report on three novel and diverse protein kinase structures that reveal an exchanged activation segment conformation. This dimeric arrangement results in an active kinase conformation in trans, with activation segment phosphorylation sites in close proximity to the active site of the interacting protomer. Analytical ultracentrifugation and chemical cross-linking confirmed the presence of dimers in solution. Consensus substrate sequences for each kinase showed that the identified activation segment autophosphorylation sites are non-consensus substrate sites. Based on the presented structural and functional data, a model for specific activation segment phosphorylation at non-consensus substrate sites is proposed that is likely to be common to other kinases from diverse subfamilies.

Figure 1

Domain organization, structural overview and inhibitors used in this study. (A) Domain organization of the four kinases. The kinase domain is highlighted in red and has been marked with KD, coiled-coil domains are shown in blue. The FHA domain in CHK2 is shown in orange. (B) Schematic representation of the structure of SLK. The secondary structure elements of the diphosphorylated form of SLK in complex with K00546 are shown. The N-terminal lobe (green), C-terminal lobe (red) and activation segment region (blue) are highlighted along with the observed phosphorylation sites. (C) Chemical structures of inhibitors used for successful co-crystallization. K00225 (Pyridone 6), (2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz(4,5-f]isoquinolin-7-one); K00546, 5-amino-3-(4-sulphamoyl-phenylamino)-(1,2,4]triazole-1-carbothioic acid (2,6-difluoro-phenyl)-amide; K00606, (4-(4-(5-cyclopropyl-1H-pyrazol-3-ylamino)-quinazolin-2-ylamino)-phenyl)-acetonitrile; K00593 (SU11724), 3-(1-(3,5-dimethyl-4-(4-methyl-piperazine-1-carbonyl)-2H-pyrrol-2-yl)-meth-(Z)-ylidene)-2-oxo-2,3-dihydro-1H-indole-5-sulphonic acid (3-chloro-phenyl)-methyl-amide.

Figure 2

Domain-exchanged kinase dimers. The activation segment regions from each monomer extend to form an extensive intermolecular interface. The arrangements of monomers within each dimer are distinct and the tip of each activation segment binds in a narrow cleft formed between helix αF and αG in the C-terminal lobe of the kinase. For each dimer, two views are shown separated by a 45° and a molecular surface (gold) is shown for one monomer, while the other monomer is shown in ribbon form (dark blue). Disordered regions are shown as dotted lines and bound inhibitors are depicted in space-filling form. Each dimer is shown in the same reference frame as that of SLK.

Figure 3

Conformation of activation segments. (A) Activation segment nomenclature. Schematic representation of the typical architecture of a kinase activation segment based on the active mono-phosphorylated STE kinase PAK4 (PDB: 2CDZ). The activation segment region runs from the magnesium-binding DFG motif (orange) to the αF helix (grey). The β9 strand (green), the activation segment (magenta), the P+1 loop (turquoise), APE (αEF) helix (blue) and αEF/αF loop (yellow) are highlighted. (B) Comparison of the activation segments of SLK (green), LOK (orange), DAPK3A (cyan) and CHK2 (magenta; PDB: 2CN5) after superimposition. The activation segments adopt a spectrum of orientations that are dictated by the relative orientation of each monomer with each respective dimer. The disordered region of LOK is indicated by a dotted line. For DAPK3, the activation segment from molecule B is shown as the corresponding region in molecule A is disordered. Inset: overall superposition of kinases indicating area of interest shown in main panel. (C) Structure-based sequence alignment of activation segment regions. The sequences of the activation segments of SLK, LOK, DAPK3, CHK2 and PAK4 are shown. Known phosphorylation sites are highlighted in orange. Secondary structure elements are shown for each kinase below the alignment and coloured using the same scheme as in panel B. The structural elements of PAK4 (PDB: 2CDZ), in which the activation segment adopts a classical intramolecular orientation, are shown for reference.

Figure 4

Kinase self-association in solution. (A) Size distribution of the sedimentation coefficient for 30 μM unphosphorylated LOK observed by an AUC velocity experiment. The first peak corresponds to the monomer and the second peak to the LOK dimer. A dimer concentration of approximately 17% was estimated. (B) Detection of dimers by cross-linking. The sizes for the dimers of CHK2 (lane 4), DAPK3 (lane 5) and LOK (lane 6) are indicated by an arrow. No band corresponding to dimers was observed for CSNK1G (lane 2) and STK38 (lane 3). Lane 1, molecular weight marker. (C) ESI-MS spectrum of wild-type SLK (black), wild-type SLK after autophosphorylation (blue), T183A (red) and T183A mutant after autophosphorylation (green) under identical conditions (48 h at 4°C). The expected position of the T183 autophosphorylation peak is indicated by an arrow. (D) Size distribution of the sedimentation coefficient for 30 μM SLK mutant Q185P (black line) and the corresponding wild-type protein (red line). Dimerization was only detected in the wild-type protein as indicated by a second peak at 3.4 S.

Figure 5

Comparison of active site and phosphorylated activation segment regions. (A) Superimposition of the active site regions for SLK (green), LOK (orange) and DAPK3 (cyan). Critical residues for kinase function are shown in ball and stick representation and the major secondary structure elements are labelled. (B, C) Active site stabilization via activation segment phosphorylation. (B) Details of the key interactions formed by the activating phosphate moiety in the STE kinase PAK4 (PDB id: 2CDZ) are depicted. The phosphate group on S474 is coloured red/orange and the activation segment is coloured according to the scheme in Figure 3A. (C) Details of key interactions formed by the two phosphate moieties in the dimer interface of the STE kinase SLK. The core of the kinase is shown along with the domain-exchanged activation segment from the adjacent molecule (residues indicated by ^*). The colour scheme is as in (B). Hydrogen bonds are indicated by dotted lines. The catalytic base (Asp155) is coloured with green carbons and labelled in red.

Figure 6

Substrate specificities of SLK, CHK2 and DAPK3. Biotinylated peptides bearing the indicated residue at the indicated position relative to a central S/T phosphor-acceptor site were subjected to phosphorylation using radiolabelled ATP. Aliquots of each reaction were subsequently spotted onto a streptavidin membrane, which was washed, dried and exposed to a phosphor screen.