Structure and Regulation of the Human Nek2 Centrosomal Kinase^*

Protein kinase activity is crucial for the precise regulation of the eukaryotic cell cycle. Although cyclin-dependent kinases remain the master regulators of cell cycle progression, it is clear that a variety of other proteins kinases also play important roles. Nek2 is a member of the NIMA⁶-related kinase (or Nek) family of serine/threonine protein kinases, so named because all are related to the NIMA kinase of Aspergillus nidulans (for review, see Ref. 1). In Aspergillus, NIMA is essential for mitotic entry with temperature-sensitive nimA mutations leading to cells that are “never in mitosis” (2). Like Aspergillus, other fungi also express a single NIMA-related kinase, e.g. Kin3 in Saccharomyces cerevisiae and Fin1 in Schizosaccharomyces pombe. Although the yeast Neks do not appear to be essential for cell cycle progression, elegant studies on Fin1 have revealed key functions in chromosome segregation and mitotic exit (3, 4). In contrast, higher eukaryotes express multiple Neks with 11 genes (Nek1 to Nek11) encoded within the human genome (1). With the exception of human Nek6 and Nek7 and Nek10, all Neks have an N-terminal kinase domain followed by a C-terminal non-catalytic regulatory domain. However, the length and composition of motifs present within these C-terminal extensions vary considerably, reflecting diverse roles in cell regulation.

Of the human proteins, Nek2 is the most closely related to the fungal kinases, being 47% identical to NIMA within the amino acid sequence of their catalytic domains. Nek2 localizes to centrosomes. Here, it contributes to spindle pole formation during mitosis (5) through phosphorylation of proteins involved in centriolar cohesion, including C-Nap1 and rootletin, to allow spindle pole separation (6-8), and others such as Nlp, required for microtubule anchoring and spindle assembly (9). Interestingly, Nek2 homologues in Drosophila and Dictyostelium as well as both NIMA and Fin1 localize to the microtubule-organizing center, and functional studies support roles in centrosome organization (4, 10-12). Furthermore, it appears that other mammalian Nek family kinases may have microtubule-related functions in cell cycle progression or cilia formation (13, 14). In addition to acting at the centrosome, there is some evidence that Nek2 may contribute to other aspects of mitotic progression including chromatin condensation and spindle checkpoint signaling (15-17).

In line with functions in mitotic entry, Nek2 is a cell cycleregulated kinase with activity low in G₁, increased in S and G₂, and diminished after mitotic onset (18). At the G₁/S transition vertebrates express two major splice variants, Nek2A and Nek2B (19, 20). These variants differ in their extreme C termini, which has important implications for their regulation, as the C terminus of Nek2A, but not Nek2B, contains both a binding site for protein phosphatase 1 and motifs that target the protein for ubiquitin-mediated degradation after mitotic entry (21-23).

Like many protein kinases, Nek2 activity is also subject to control through its phosphorylation. Downstream of the kinase domain is an unusual leucine zipper motif that mediates dimerization as a prerequisite for efficient autophosphorylation and, thus, full Nek2 activity on exogenous substrates (24). Interaction of Nek2A with protein phosphatase 1 (PP1) can lead to dephosphorylation and inhibition of Nek2, suggesting that Nek2 may only become fully activated once PP1 is inactivated at the onset of mitosis (22, 25). Besides autophosphorylation, Nek2 may be regulated by upstream kinases. Indeed, the catalytic domain of Nek2 is phosphorylated in vitro by p90^Rsk2, an interesting observation as the activation of chromatin condensation by Nek2 in mouse spermatocytes is under the control of the mitogen-activated protein kinase/p90^Rsk2 pathway (26).

Despite the importance of Nek2 in cell cycle control, its regulation by phosphorylation remains poorly understood. Indeed, no structural information on any Nek kinase family has been reported despite the fact that together they constitute ∼2% of the entire human kinome. Here we report the structure of the kinase domain of human Nek2 in complex with a pyrrole-indolinone inhibitor SU11652 together with an analysis of the role of autophosphorylation in Nek2 regulation. Given that Nek2 is up-regulated in a number of human cancers and that down-regulation can inhibit cell proliferation (27), our observations have clear significance for the design of Nek2 inhibitors as potential anticancer agents and now provide a structural and mechanistic framework for understanding how this important cell cycle kinase is regulated, with implications for the control of Nek-family kinases as a whole.

EXPERIMENTAL PROCEDURES

Nek2 Kinase Domain Expression, Purification, and Crystallization—For expression of Nek2 constructs, DNA sequences were amplified by PCR and cloned between the NdeI and XhoI sites of pET22b, and the resulting plasmids were used to transform Escherichia coli BL21(DE3). Cultures were grown at 37 °C to an A₆₀₀ of ∼0.7, the temperature was adjusted to 18 °C, and expression was induced for 4 h using 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were collected by centrifugation, resuspended in buffer A (50 mm HEPES, pH 7.5, 5 mm sodium phosphate, 300 mm NaCl, 5% glycerol) supplemented with 20 mm imidazole, and lysed by sonication. Clarified lysate was loaded onto a nickel-nitrilotriacetic acid column equilibrated with lysis buffer and washed with the same buffer supplemented with 1 m NaCl. Bound Nek2 eluted in buffer A plus 150 mm imidazole. The protein sample was concentrated by ultrafiltration and fractionated on a S75 Superdex column equilibrated in 50 mm HEPES, pH 7.5, 300 mm NaCl, 10 mm sodium phosphate, 5 mm dithiothreitol, 5% glycerol. Eluted fractions were 95% pure as judged by SDS-PAGE. For crystallization, phosphates were removed by treatment with shrimp alkaline phosphatase and λ-phosphatase for 12 h at 4 °C.

Crystallization, Structure Determination, and Refinement—Crystals were obtained at 4 °C using vapor diffusion sitting drops at a protein concentration of 7.5 mg/ml containing 1 mm of a pyrrole-indolinone compound (5-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-(diethylamino) ethyl]-2,4 dimethyl-1H-pyrrole-3-carboxamide). The complex was crystallized by mixing with precipitant (10% polyethylene glycol 6000, 200 mm MgCl₂) at a ratio of 3:1. Monoclinic plates grew over 3-4 weeks to a maximum size of 75 × 75 × 10 μm³.

Crystals were vitrified in mother liquor supplemented with 25% ethylene glycol, and diffraction data were collected at 100 K on beamline X10A (λ = 0.95 Å) at the Swiss Light Source. Data were integrated, merged, and scaled using MOSFLM and SCALA. The structure was solved by molecular replacement with PHASER (28) using Aurora-A kinase (Protein Data Bank code 1OL7) as a search model. The C-terminal lobe was well resolved in the initial electron density maps, but density for the N-terminal lobe was poor due to conformational changes between the search and target structures. Attempts to place the isolated N-terminal lobe using PHASER failed. Instead, the domain was manually placed in the density, and its position was optimized using rigid-body refinement. Rounds of model building interspersed with restrained refinement incorporating TLS were carried out using REFMAC5 (29) and COOT (30). The coordinates are available in the Protein Data Bank (Protein Data Bank code 2JAV).

Autophosphorylation Site Mapping—For identification of autophosphorylation sites by mass spectrometry, an enriched subset of phosphorylated peptides was generated using gallium-chelated immobilized metal affinity chromatography spin columns (Pierce). The phosphorylated protein to be studied was first digested with sequencing grade modified trypsin (Promega) at 1:10 (trypsin:target w/w). The digestion was carried out in 10 mm ammonium bicarbonate overnight at room temperature. The tryptic digest was diluted 1:1 with 20% acetic acid and incubated on the gallium resin for 30 min at room temperature. The resin was washed with 10% acetic acid followed by 10% acetic acid plus 20% acetonitrile. The resin was then equilibrated with water followed by elution in 100 mm ammonium bicarbonate, pH 9.0, plus 10% acetonitrile. Identification of phosphorylated sites was achieved using a combination of electrospray ionization-mass spectroscopy (MS), matrix-assisted laser desorption ionization time-of-flight spectroscopy, and tandem MS-MS methods (supplemental “Experimental Procedures”.

Site-directed Mutagenesis—Mutations were introduced by PCR-based mutagenesis of the pET22b-Nek2A, pGEM-Nek2A, or pRcCMV-myc-Nek2A plasmids using the Gene Tailor™ site-directed mutagenesis system (Invitrogen). All constructs were confirmed by DNA sequencing.

Kinase Assays—Myc-tagged Nek2 proteins were synthesized from the appropriate pGEM or pRcCMV constructs using coupled in vitro transcription-translation reactions according to manufacturer's instructions (Promega). Proteins were immunoprecipitated using anti-Myc antibodies (0.4 μg/ml; Cell Signaling) and a fraction of the immune complexes used for analysis by SDS-PAGE and Western blotting using an anti-Nek2 antibody (1 μg/ml; Zymed Laboratories Inc. (24)). The remaining immune complexes were used for kinase assays. Specifically, 5-10 μl of washed immune complex beads were incubated with 5 μg of β-casein or the C-terminal domain of C-Nap1 and 1 μCi of [γ-³²P]ATP as substrates in 40 μl of kinase buffer (50 mm Hepes-KOH, pH 7.4, 5 mm MnCl_2, 5mm β-glycerophosphate, 5 mm NaF, 4 μm ATP, 1 mm dithiothreitol) at 30 °C for 30 min. Reactions were stopped with 50 μl of protein sample buffer and analyzed by SDS-PAGE and autoradiography. Western blots were quantified using NIH image software, and substrate phosphorylation was quantified by scintillation counting of proteins excised from dried gels.

Cell Culture and Transfection—U2OS osteosarcoma and HeLa cervical carcinoma cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heatinactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO₂ atmosphere. Transient transfections were performed with either Lipofectamine 2000 (Invitrogen) or FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions.

Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as previously described (31). Primary antibodies used were anti-γ-tubulin (0.15 μg/ml; Sigma) and anti-Myc (0.4 μg/ml) antibodies. Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse IgGs (1 μg/ml; Invitrogen). Fluorescence images were captured on a TE300 inverted microscope (Nikon) using an ORCA ER CCD camera (Hamamatsu) using Openlab 3.5.1 software (Improvision, Coventry, UK) and processed using Adobe Photoshop (San Jose, CA).

RESULTS AND DISCUSSION

Bacterial Expression of Nek2 Constructs and Phosphorylation Site Mapping—Initial attempts to overexpress full-length human Nek2A or a 271-residue fragment encompassing just the predicted kinase domain in E. coli were unsuccessful due to apparent toxicity of the recombinant enzymes. However, we were able to circumvent these problems by constructing mutant kinase domains with substitutions at the highly conserved catalytic Asp-141 (D141A) or Lys-37 (K37R) that could be expressed and purified in high yield. In contrast to the D141A protein, which showed no autophosphorylation at all, electrospray ionization-mass spectroscopy analysis of the K37R domain showed autophosphorylation at an average of four sites (supplemental Fig. S1A). Of these, one site, Thr-175 within the “activation” or T-loop of Nek2, was unambiguously identified by tandem mass spectroscopy analysis. Expression of fulllength K37R Nek2A yields an unstable product, but a fulllength construct containing a T175A substitution showed both low toxicity and increased stability. Purified protein showed phosphorylation on an average of 13 sites. Two sites (Thr-170 or Ser-171, Thr-179) map to the Nek2 T-loop region, and a third, Ser-241, is located within the kinase C-lobe (see below). A further six phosphorylation sites were assigned within the C-terminal regulatory region (Fig. 1A and supplemental Fig. S1B). Four occur around the KEN-box, suggesting a potential autoregulatory role at the level of Nek2 stability. In addition, Ser-356 and Ser-428 lie within predicted regions of coiledcoil and may, therefore, be involved in modulating Nek2A dimerization and localization. These ideas are currently under investigation.

Structural Analysis of the Nek2 Kinase Domain—To maximize yields of recombinant protein and minimize structural perturbations around the ATP-binding site, crystallization efforts were focused on the T175A mutant of the Nek2 kinase domain. The purified recombinant protein is heterogeneously phosphorylated, and crystallization required prior dephosphorylation by alkaline and λ phosphatase. Crystallization of complexes with non-hydrolysable ATP analogues was unsuccessful. However, diffracting crystals were obtained of a complex with a single inhibitor, SU11652, identified using temperature shift assays (supplemental “Experimental Procedures”). The structure was solved by molecular replacement using the Aurora-A kinase structure as a search model and refined against diffraction data extending to 2.1 Å of resolution. The final model comprises the entire kinase domain (residues 3-271), the C-terminal hexahistidine tag, 107 water molecules, and the pyrrole-indolinone ligand. Residues within the loop N-terminal to αC (Thr-45-Met-51), the loop connecting the two strands β4 and β5 (Arg-77-Thr-81), the region C-terminal to helix αE (Asp-132-Arg-140), and the activation segment residues between Asn-167 and Val-177 are not visible in the electron density and are presumably disordered. The N-terminal lobe (Ser-3-Met-86) and the αC helix in particular exhibits significant disorder, presumably due to mobility within the crystal, which is reflected by a high average temperature factor of 75 Ų. In comparison, the C-terminal domain (Glu-87-His-279) is well ordered with an average temperature factor of 33 Ų. Data and refinement statistics are shown in Table 1, and electron density for the inhibitor is shown in Fig. 1B.

The Nek2 catalytic domain exhibits a typical bilobal protein kinase fold (Fig. 1C). As a family, the Nek kinases form a subclade with pairwise sequence identities of 35-42% within the catalytic domains (Fig. 1D and supplemental Fig. S2). Their closest homologues in the human kinome are microtubule-associated regulatory kinases (MARK) along with the Polo-like kinases and Aurora-family kinases that are centrosome-associated and known to play important roles in mitotic regulation. Of kinases with known structure, Nek2 is most similar at the sequence level to Aurora-A (31% identity) and MARK2 (31% identity), and a structural overlap of the Nek2 complex with that of the inactive but ATP-bound Aurora-A (Protein Data Bank code 1OL6) is shown in Fig. 2A. As expected for kinases that require phosphorylation of specific residues in the activation segment, these regions are not completely ordered in both cases. The overall structures are similar and superimpose with a root mean square deviation of 1.8 Å for 202 matched Cα positions. The two structures differ mainly in the length and location of secondary structural elements, particularly in the C-lobes. For example, αG is extended by one helical turn in Nek2 (residues Gly-101-Glu-105), and the helix inserted between sheet β3 and αC in Aurora-A is not well defined in Nek2, which shows only half a helical turn before the chain becomes disordered.

Perhaps the most notable difference occurs around the highly conserved DFG motif that lies at the N-terminal end of the T-loop. Functionally, this motif serves to both anchor the N-terminal end of the T-loop and supply the aspartate as a ligand for a bound divalent metal ion. In the ATP-bound Aurora-A structure, the DFG motif adopts an extended conformation as is generally observed in other inactive kinases. However, in Nek2 the DFG motif and the following five residues constitute an extended motif (DFGLARIL) that is largely conserved in Nek-family members and which folds into a short region of α-helix that we refer to as αT. As a consequence of this arrangement the short sheet structure after the DFG motif is missing in Nek2, resulting in disorder of the region at the end of helix αE. Intriguingly, this additional element of secondary structure is wedged between αC, which contains the catalytically important Glu-55, and the ATP-binding site (Fig. 2B). In this conformation Glu-55 projects away from the active site and is held by a salt-bridging interaction with Arg-164 from αT. Clearly, this conformation is incompatible with catalytic activity, and the helix must, therefore, move or be disrupted in the activated form. Although unusual, a helical structure after the DFG motif is not unique to Nek-family kinases and is structurally related to a similar helical “insert” observed previously in structures of cyclin-free CDK2 bound to ATP (32) (Fig. 2C, upper panel) and in inactive forms of both the EGFR kinase (33) (Fig. 2C, lower panel) and Src/Hck (Ref. 34 and references therein). The remarkable similarity in the structure, location, and sequence of Nek2 αT to equivalent motifs found in these kinases strongly suggest that αT is a feature of the Nek2 inactive conformation, which is selected for, rather than induced by inhibitor binding. In the case of CDK2 activation, binding of an activating cyclin to the characteristic PSTAIRE portion of αC pushes it toward the ATP/substrate binding cleft with concomitant disruption of the inhibitory helix. This allows Glu-51_CDK2 access to the active site and Arg-150 (equivalent to Arg-164 in Nek2) to contact Thr(P)-160 within the T-loop. The latter interaction is common in kinases activated by T-loop phosphorylation to the extent that the presence of an arginine at a position equivalent to 164 in Nek2 is considered predictive of this mode of activation (35). The fact that Nek2 appears to be activated by T-loop phosphorylation at Thr-175 (see below) strongly suggests that αT unfolds into a more extended conformation in the active form.

Pyrrole-indolinone Inhibitor Binding to an Inactive-like Conformation of Nek2—Nek2 expression has been reported to be significantly elevated in a number of tumor cells including Ewing tumor (pediatric osteosarcoma) (36) and primary breast cancer tissues (37). Furthermore, high expression levels of Nek2 have been strongly associated with disease progression in non-Hodgkin lymphomas (38). Moreover, deregulation of Nek2 has been linked to centrosome abnormalities and aneuploidy. These hallmarks of tumor progression are generally associated with poor prognostic outcome since the lack of control over chromosome segregation leads to chromosomal instability, a general mechanism by which tumors select for cells with more malignant phenotypes. The strong link of Nek2 to cellular events critical for tumor progression and a recent report demonstrating that depletion of Nek2 leads to an apparent arrest in HeLa cell proliferation and an increase in apoptosis suggest that Nek2 may be a viable target for the development of novel anticancer drugs (39, 40).

We determined the structure of a non-activating Nek2 mutant in complex with the pyrrole-indolinone compound SU11652. Binding of this compound was discovered during screens of a panel of generic kinase inhibitors for their ability to structurally stabilize the Nek2 T175A kinase domain (data not shown). The structure of the Nek2 complex shows the inhibitor bound at the back of the ATP binding cleft, forming two hydrogen bonds with main chain atoms from Glu-87 and Cys-89 within the hinge region between the N and C lobes and making hydrophobic interactions with the active site residues Met-86, Val-68, Phe-148, Cys-89, and Leu-162 (Fig. 3). In this position it forms two hydrogen bonds with main-chain atoms from Glu-87 and Cys-89 within the hinge. These interactions are common in kinase-inhibitor complexes and mimic the contacts formed by main-chain atoms from hinge residues with the N1 and N6 atoms of the adenine base in ATP-bound complexes. An intramolecular hydrogen bond is also formed between the indole oxygen and the pyrrole ring that stabilizes the overall planarity of the extended ring system.

SU11652 has been described as a cell-permeable compound that acts as a potent and ATP-competitive tyrosine kinase receptor and angiogenic inhibitor that exhibits activity for platelet-derived growth factor receptor β, vascular endothelial growth factor receptor 2, fibroblast growth factor receptor, EGFR, and Kit family members with a broad range of IC₅₀ values ranging from 3 nm to 20 μm (41-43). Later reports showed that SU11652 not only inhibits tyrosine kinases for which it was originally designed but also a number of serine/threonine protein kinases possibly linked to tumorigenesis (44). SU11652 has been reported to display antiproliferative and pro-apoptotic properties in tumor cells, and a closely related compound (SU11248, Sunitinib malate) has been recently approved for the treatment of metastatic renal cancer. We determined IC₅₀ values for both compounds using a substrate peptide GTFRSSIRRLSTRRRY (GTF peptide; the phosphorylated residue is underlined) derived from analysis of substrate sites of NIMA kinase (45). This substrate is efficiently phosphorylated by the recombinant full-length Nek2 T175A mutant kinase, with a K_m of ∼90 μm and k_cat of ∼17 min^-1 (supplemental Fig. S3). This is comparable with the value of 20 μm determined for wild-type Aspergillus NIMA, indicating that the substrate specificity of these kinases is similar and that Nek2 specificity is not significantly perturbed by the T175A mutation. Indeed, the sequence specificity of the full-length Nek2 T175A mutant determined using degenerate peptide libraries (in preparation) shows strong selection for Phe/Leu/Met in the Ser(P)-3 position much like that observed for NIMA (45). We determined an apparent K_m for ATP to be 115 μm (supplemental Fig. S3) and, using the same peptide substrate, measured IC₅₀ values for SU11652 and SU11248 of ∼8 and ∼12 μm, respectively (data not shown), indicating that the pyrrole-indolinones are potentially useful lead inhibitors for further optimization and that some of the efficacy of Sunitinib malate in the treatment of tumors might be associated with Nek2 inhibition.

A number of highly selective inhibitors have been developed targeting the inactive conformation of kinases (for review, see Ref. 46). These so-called “type II” inhibitors exploit an allosteric site present only in the inactive “DFG-out” conformation, and their clinical success has initiated efforts to develop new families of kinase inhibitors that combine classical type I scaffolds with chemical moieties that are known to stabilize DFG-out conformations. This particular conformation of the activation loop is not accessible for every kinase and opens a binding pocket with much larger sequence variability, making the development of specific inhibitors more likely. The structure of Nek2 in complex with SU11652 offers two possibilities for the development of inhibitors that stabilize the inactive conformation. First, the type I pyrrole-indolinone scaffold in combination with more bulky type II inhibitor moieties in place of the exocyclic chlorine could be used to enlarge the allosteric binding pocket that is rather small in the Nek2·SU11652 complex. Second, the surface presented by the αT helix could also be targeted to create more stabilizing interactions. Indeed, the structure shows that the chlorine of the SU11652 indolinone ring makes favorable interactions with Leu-162 of the Nek2 αT helix (Fig. 3). SU11248 contains a fluorine atom in place of chlorine that might be expected to weaken this interaction due to a much smaller van der Waals radius and increased polarity consistent with the small difference in their IC₅₀ values. Because αT prevents access of Glu-55 to the active site, inhibitors that interact with and stabilize the helical conformation might be expected to provide increased potency and selectivity.

Nek2 Activation by Autophosphorylation—As described earlier, mass spectrometric analysis identified four sites of autophosphorylation within the catalytic domain. To establish their functional significance, we carried out localization and activity assays in U2OS cells expressing Myc-tagged full-length, wildtype, and K37R mutant Nek2A and mutants containing substitutions of Thr-170, Ser-171, Thr-175, Thr-179, or Ser-241 with alanine or glutamate/aspartate as phosphomimics. To assess the activity of Nek2 proteins in cells, we scored transfected cells for premature centrosome splitting (CS) (Fig. 4A). When stained with antibodies against γ-tubulin, centrosomes usually appear as two adjacent dots close to the nucleus in interphase cells. However, it has been previously observed that expression of wild-type Nek2A for 24 h in either HeLa or U2OS cells induces CS by >2 μmin ∼50-60% of the cells compared with untransfected cells in which ∼10% show split centrosomes over the same time period (5, 47). Because catalytically compromised mutants of Nek2 fail to induce CS, this effect appears to be dependent on phosphorylation of centrosomal components (6-8). By way of comparison, we also wished to assess the relative catalytic activities of the mutant full-length Nek2 proteins in vitro. To do this we immunoprecipitated each Nek2 variant from in vitro translation extracts and assayed for kinase activity using β-casein (Fig. 4B). Importantly, all Nek2 mutants examined show appropriate centrosomal staining in these experiments, demonstrating that any effects of mutation within the kinase domain do not seriously impinge on Nek2 localization.

As expected, the K37R mutation results in an enzyme with reduced activity in vitro and which shows CS activity comparable with untransfected cell controls. In contrast, T175E resulted in an activated kinase in vitro that also showed an elevation in splitting in vivo. The overall increase in CS is small, reflecting the fact that splitting efficiency of 50-60% appears to be close to the maximum observable, and mechanisms are likely to exist that prevent centrosome separation at higher frequencies. Regardless, the importance of Thr-175 suggested by its conservation in many other kinases is underlined by the T175A null mutant that shows reduced kinase activity and a significant reduction in efficiency of CS.

Like T175E, phosphomimic mutations of either Thr-170 or Ser-171 produced elevated kinase activity and maximal levels of CS, indicating that these sites may have functional significance. However, in both cases no significant effect of alanine mutation at these positions was observed in either assay. These residues are largely conserved in metazoan Nek2 kinases but are rather variable in other human Nek-family members (Fig. 5A). Interestingly, Nek6 activation has been reported to depend primarily on phosphorylation at Thr-206, the structural equivalent of Nek2 Thr-175, but full activity appears to require phosphorylation of Ser-202 (Nek2 Ser-171) by Nek9 in a “Nek-kinase cascade” (48). Thus, it appears that autophosphorylation of Thr-170 and/or Ser-171 in Nek2 may fine-tune overall activity of Nek2 in vivo. We also note that the sequence context of Ser-171 is also highly conserved in Nek2 orthologues (His-Asp-Thr-Ser-Phe; Ser-171) and matches closely the specificity motif for Plk1 ((K/R)(E/D)X(pS/pT)-Φ, where Φ is hydrophobic, and p is phosphorylated (49)). Given that Plk1 phosphorylation of Nek2 has been proposed to regulate the efficiency of Nek2-mediated CS (50), our data suggest that Ser-171 may be a target for phosphorylation of Nek2 by Plk1 at the centrosome.

A Role for Inhibitory Autophosphorylation in Nek2 Regulation—In contrast to the activating Thr-170/Ser-171 and Thr-175 phosphorylation described above, our data also indicate that autophosphorylation at Thr-179, also within the T-loop, and Ser-241 in the C-lobe may act to negatively regulate Nek2. In marked contrast to the Thr-170/Ser-171 and Thr-175 mutants, both T179A and T179E substitutions reduce kinase and CS activity (Fig. 4). Threonine residues at this position play a conserved role in serine/threonine protein kinases, serving as an anchor point for the C-terminal end of the T-loop in the activated, substratebound kinase. Ternary complex structures of protein kinase A (51) show that this residue stabilizes the conformation of catalytically important residues during phosphoryl transfer, explaining why phosphorylation of Thr-179 is inhibitory (Fig. 5B). Although autophosphorylation at this position may appear surprising, we note that phosphorylation of the structurally equivalent Thr-206 in the related MARK4 by an upstream kinase appears to be important for downregulation of MARK4 activity in vivo (52). In addition, the DNA damage kinase Chk2 undergoes T-loop autophosphorylation on Thr-383, the conserved activating site and on Thr-387 (53) that is equivalent to Nek2 Thr-179. This suggests that, like Nek2 and MARK4, Chk2 is also controlled positively and negatively by T-loop phosphorylation and supports the notion that this regulatory mechanism may be more common than has previously been appreciated.

The second inhibitory site is Ser-241, located at the N-terminal end of αH within the C-terminal domain. Serine or threonine is present at the equivalent structural location in many other kinases, but there is also a good deal of variability, and we are unaware of any previous study that has demonstrated regulatory phosphorylation of this site. S241A and S241E/S241D mutations markedly reduce kinase activity and CS (Fig. 4), but the extent of these effects is surprising for the following reasons. First, the x-ray structure shows that although loss of the hydrogen-bonding potential or introduction of a phosphoryl group might be expected to result in local perturbations, αH is, nonetheless, rather remote from the active site. Second, Ser-241 substitutions do not seem to compromise Nek2 stability since these mutants localize to the centrosome correctly and are as abundant as the wild-type kinase in extracts used for kinase assays. It is possible that the observed effects of Ser-241 phosphorylation on catalytic activity are manifested through conformation changes that are propagated through the C-lobe to the active site some 30 Å distant. However, Ser-241 phosphorylation may play a more direct role in Nek2 regulation as described below.

Kinase-Kinase Interactions in the Nek2 Dimer—Perhaps the most striking feature of the Nek2 kinase domain structure is the presence of the αT helix at the N-terminal end of the T-loop, a conformation that has only been previously been observed in inactive forms of CDK2, EGFR kinase, and Src/Hck family kinases. In all cases, the αT helical segment forms a barrier to access of a conserved catalytic glutamate (Nek2 Glu-55) on αC to the catalytic site. Furthermore, the interactions that stabilize the position of the inhibitory helix with respect to the N-lobe and αC are also very similar in each of these kinases (Fig. 2). The presence of the helical insert in cyclin-free CDK2 is insensitive to Thr-160 phosphorylation (32), suggesting that T-loop phosphorylation is unlikely to be responsible for Nek2 αT unfolding.

What, then, is responsible for disruption of αT in the activated form of Nek2? As mentioned, conformational changes in CDK2 required to unwind the “blocking” helix are effected allosterically through cyclin binding to the αC/PSTAIRE helix. In the case of Nek2, no activating subunit is known nor is one necessary since even the purified full-length T175A mutant kinase is highly active (supplemental Fig. S3). However, although the isolated T175A Nek2 kinase domain autophosphorylates efficiently, it nevertheless shows significantly lower values for both K_m (190 μm) and k_cat (8.4 min^-1) on the GTF peptide substrate and a resulting 4-fold decrease in k_cat/K_m. Because our mass spectrometric analysis shows that the isolated and dimerized recombinant T175A kinase domains show rather similar levels of autophosphorylation, it appears that dimerization may play a direct role in relieving the autoinhibited conformation observed in the x-ray structure. Interestingly, recent studies of EGFR kinase activation (33) show how ligand-induced receptor dimerization allows activation through an “asymmetric” interaction between EGFR kinase domains. Here, αH within the C-lobe of one monomer packs in a cyclin-like fashion against αC of the other, thus disrupting the intervening αT-like helix. Such a model is attractive since Nek2 Ser-241 that is targeted as a site of inhibitory autophosphorylation is also located on αH. This would potentially explain the initially surprising observation that alanine or aspartate substitutions of Ser-241 are so detrimental to Nek2 activity in vitro and in vivo despite its distance from the active site and the fact that a variety of substitutions are found at the equivalent position in other kinases.

Clearly, further work will be necessary to test the veracity of these ideas. Nonetheless, the fact that allosteric effects have been shown to be necessary for formation of active kinase conformations in CDK2, EGFR, and Src/Hck, the only other systems where αT-like structures have been observed together with the overall similarity in αT sequence, structure, and interactions between Nek2 and these kinases suggest that kinase-kinase interactions in dimeric Nek2 may play an important regulatory role.

Concluding Remarks—Precise spatial and temporal control of Nek2 activity is necessarily complex, involving autophosphorylation of the kinase domain and its dephosphorylation by protein phosphatase 1c isoforms that form tight complexes with the Nek2A C-terminal region. In addition, protein phosphatase 1 (PP1) itself is negatively regulated by Nek2 phosphorylation, an interplay of opposing activities that creates a “bi-stable switch” that maintains the Nek2A dimer in an inactive state until PP1c is released. In light of the data presented here, we now suggest that extant models of Nek2 control are likely to be inadequate and that regulation acts at many different levels. First, our observation of multiple autophosphorylation sites within the C-terminal region may indicate that Nek2 dimerization and protein phosphatase 1 binding along with stability/turnover and centrosomal localization may all be subject to phospho-dependent control, and these ideas are currently being tested. Second, our data now suggest a model in which activating autophosphorylation and/or phosphorylation by upstream kinases (Thr(P)-170/Ser-171 and Thr(P)-175) is counterbalanced not only by protein phosphatase 1c activity but also inhibitory autophosphorylation events that may directly alter stereochemistry at the site of phosphoryl transfer or disrupt potential kinase-kinase interactions within the Nek2 dimer required for allosteric activation. With complexity comes opportunity, and all of the regulatory mechanisms described here may be amenable to therapeutic intervention. The importance of Nek2 in the centrosome cycle, its overexpression in a number of tumor cell types, and the prevalence of chromosomal instability and aneuploid defects in cancer cells suggest that it may constitute a valid anti-cancer target. That clinically relevant anti-cancer agents such as Sunitinib and its derivatives are active against Nek2 together with the unexpected features of the inactive Nek2 conformation gives some confidence that effective and specific inhibitors of the Nekfamily kinases are likely to be achievable in the future.