On the molecular mechanisms of mitotic kinase activation

Abstract

During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein-protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.

Keywords: mitosis; protein kinases; protein structures; regulation.

Figure 1.

An illustration of the protein kinases that are discussed in this review and the mitotic events they coordinate. Many of the kinases play roles in multiple stages of mitosis. Progress through the cell cycle is restricted by checkpoints that serve to maintain the fidelity of genetic transmission (red boxes).

Figure 2.

Conserved structural features of the active conformation of a protein kinase. The four panels use the following colour scheme: kinase main chain and Lys/Glu pair (beige), HRD motif (blue), DFG motif side chains (red), activation segment (cyan), phosphorylated residue on activation loop (yellow), hydrophobic spine (green), ATP (magenta), substrate protein (grey). (a) Architecture of the archetypal protein kinase A (PKA) catalytic domain in cartoon representation, with key residues shown as sticks and the ADP ligand shown as spheres (PDB code 1JBP). The side chains of the four residues that comprise the hydrophobic spine are surrounded by a wire mesh. This and all subsequent structure figures were produced using PyMOL. (b) Magnified view of the active site and residues involved in regulation. Key interactions between residues are indicated with dashed black lines; these do not necessarily imply hydrogen bonds. (c) Schematic that summarizes the major features of the kinase active conformation. Versions of this diagram will be used throughout the text to illustrate differences between inactive and active states of protein kinases. Three key interactions are labelled in black. (d) Schematic diagram to illustrate the canonical interactions between the phosphorylated residue of the activation loop and basic residues in the αC-helix, β9 and the HRD motif. These interactions are not all conserved in mitotic kinases. For example, Aurora-A and Plk1 lack the basic residue on β9, and instead have a basic residue on the activation loop (shown with fainter colouring). The different parts of the activation segment are labelled, such as the P+1 loop that recognizes the P+1 residue of the substrate protein.

Figure 3.

A schematic to summarize Cdk2 activation. (a) A simplified scheme showing two-step activation of Cdk2 by Cyclin A binding and CAK phosphorylation. The first step of Cdk2 activation is through binding of a cyclin partner protein, which induces a number of conformational changes, resulting in a partially active kinase that bears many of the hallmarks of an active kinase structure. Cyclin binding also exposes Thr160 within the activation loop to solvent, allowing access to an activating kinase. The second step of Cdk2 activation is phosphorylation on Thr160, by a variety of kinases collectively called Cdk-activating kinase (CAK). (b) A schematic of inactive Cdk2 alone (PDB code 1HCK), following the same conventions as figure 2c. The structure of Cdk2 appears as if incompletely folded, with many structural elements displaced from their expected positions. (c) The structure of Cyclin A-bound Cdk2 (PDB code 1FIN) reveals a partly active conformation, with the Lys–Glu salt bridge in place. Cyclin A is coloured green, with key structural elements that bind Cdk2 labelled. (d) The phosphorylated Cdk2/Cyclin A structure (PDB code 1JST) resembles that of active PKA, with all the expected interactions in place.

Figure 4.

Autoinhibited structures in which the activation segment forms an ordered structure that disrupts the Lys–Glu pair. In each panel, the name of the kinase and the ligand bound to the active site are shown. The activation loop is coloured green–blue, and the Ser/Thr residue that is phosphorylated upon kinase activation is coloured yellow in structures in which it was modelled. At the far right of each panel, the sequence of the DFG motif plus five residues is shown vertically, with hydrophobic residues that block the formation of the Lys–Glu salt bridge circled. (a) (i) The autoinhibited conformation of Cdk2 (PDB code 1HCK). (ii) The active conformation of Cdk2-Cyclin A (PDB code 1JST), with the cyclin shown as a green surface. (b) Two potential autoinhibited conformations of Nek2: (i) the activation segment is partially disordered (PDB code 2JAV); (ii) the activation segment was fully modelled (PDB code 2W5B). (c) Two potential autoinhibited conformations of Aurora-A: (i) the activation segment is partially disordered (PDB code 1MUO); (ii) the activation segment was fully modelled (PDB code 2WTV).

Figure 5.

The β4-strand component of the hydrophobic spine is displaced in Nek7 and Bub1. (i) The structures in cartoon representation, with key residues shown as sticks; (ii) the same information in a schematic representation, based on figure 2c. (a) The structure of Nek7 (PDB code 2WQM) reveals an autoinhibited ‘Tyr-down’ conformation, in which the side chain of Tyr97, the β4-strand component of the hydrophobic spine, points into the active site, which displaces the C-helix from its active position. In order for Nek7 to be catalytically active, Tyr97 must change conformation to adopt the position shown for the equivalent side chain in Nek2 (Tyr70, yellow, PDB code 2W5B). (b) The structure of Bub1 (PDB code 3E7E) reveals a hydrophobic spine comprising only three side chains (green mesh). Phe852 (which is equivalent to Tyr97 on Nek7) adopts the down position, but this does not disrupt the Lys–Glu pair, and a continuous hydrophobic spine is formed (green mesh). This is because the side chain of Phe852 fills the void that is left by the absence of a side chain on the αC component of the spine (Gly834). The extended N-terminus of the Bub1 kinase domain (beige), which is crucial for activity, interacts with the activation segment. Thr960 and Thr968 are candidates to form interactions with Asp917 in the active conformation.

Figure 6.

Sequence alignment of mitotic kinase activation segments, compared with protein kinase A (PKA). The alignment was produced using ClustalW2. Absolutely conserved residues are marked with an asterisk, conservatively substituted residues with a colon and semi-conservative substitutions with a dot. Potential sites of activating phosphorylation are highlighted in yellow. Sites that have been confirmed to have an activating role in a crystal structure are marked in bold type. The ‘GT’ motif in the P+1 loop is underlined.

Figure 7.

The mechanism of Plk1 and Aurora-A activation is mainly through stabilization of the activation segment conformation. (a) Summary of the one-step activation mechanism of Plk1, based on the crystal structures in unphosphorylated state (PLK1-unphos, blue, PDB code 3D5U) and phosphorylated state (PLK1-phos, orange, PDB code 3D5W). Upon phosphorylation, the activation loop changes conformation from an inactive position inconsistent with substrate protein binding (dashed line) to an active position (solid line). (b) Summary of the two-step activation mechanism of Aurora-A, based on crystal structures of the unphosphorylated state (AURA-unphos, blue, PDB codes 1MUO, 1MQ4), phosphorylated state (AURA-phos, orange, PDB code 1OL7) and phosphorylated state in complex with TPX2 (AURA-phos/TPX2, red, PDB code 1OL5). Note that there is currently no crystal structure of unphosphorylated Aurora-A bound to TPX2 (yellow). The activation segment of unphosphorylated Aurora-A is partially disordered; the activation segment of phosphorylated Aurora-A is ordered, but in an inactive conformation incompatible with substrate protein binding; and the addition of TPX2 locks the activation segment into a conformation compatible with protein substrate binding. (c) Superposed crystal structures of PLK1-phos (orange) and PLK1-unphos (blue), shown in the vicinity of the activation segment. Phosphorylated Thr210 is shown in yellow, and key interactions are shown as dashed lines. Conformational differences are mostly restricted to the activation segment. In addition, the side chain of Lys97 also moves closer to the phosphorylated form of Thr210, although the functional significance of this is unclear. (d) Schematic diagrams summarizing the key features of (i) PLK1-unphos and (ii) PLK1-phos. Note that the unphosphorylated Plk1 structure has many of the features of an active kinase, with the exception of the activation loop conformation. (e) Superposed crystal structures of phosphorylated Aurora-A alone (orange) and bound to TPX2 aa1–43 (red). Phosphorylated Thr288 is shown in yellow, and the position of pThr288 in the presence of TPX2 is labelled (+TPX2). Key interactions are shown as dashed lines. The side chain of Arg286 is only shown in the TPX2-bound form of Aurora-A because, if it were to be shown in the other structure, its position would obscure the interactions of pThr288. (f) Schematic summarizing the key features of (i) AURA-phos and (ii) AURA-phos/TPX2. Note that the AURA-phos structure bears a close resemblance to PLK1-unphos, and AURA-phos/TPX2 is similar to PLK1-phos.

Figure 8.

Structures of Mps1/TTK and Haspin. (a) Structure of Mps1/TTK captured in an inactive conformation (PDB code 2ZMC). (i) The structure viewed in the vicinity of the activation segment (light blue). (ii) The key features are summarized in the schematic illustration. (b) Structure of Haspin in an active conformation, in the absence of phosphorylation or any other external factor (PDB code 2VUW). (i) The structure viewed in the vicinity of the activation segment (light blue), which adopts a highly unusual structure. (ii) Key residues and interactions are summarized.

Figure 9.

Comparison of activation mechanisms of Aurora-A and Aurora-B. (a) Crystal structure of phosphorylated Aurora-A (red, activation segment in green–blue) bound to TPX2 (green), based on PDB code 1OL5. (b) Structure of phosphorylated Aurora-B (grey, activation segment in green–blue) bound to INCENP (green), based on PDB code 2BFX. The three α-helices of the INCENP fragment are labelled α1, α2 and α3. (c) Structure of INCENP/Aurora-B complex in the vicinity of the activation segment. It is not known whether INCENP affects the conformation of the activation segment, which it does not directly contact. It is thought that Phe837 of INCENP causes a rotation of the αC helix that prevents Lys–Glu salt bridge formation. Note that Xenopus laevis Aurora-B has residue Met156, equivalent to human Leu140; all the other residues are labelled with human protein numbering. (d) Schematic illustration of the INCENP/Aurora-B complex.