Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M609721200 on December 31, 2006

J. Biol. Chem., Vol. 282, Issue 9, 6833-6842, March 2, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/9/6833    most recent
M609721200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rellos, P.
Right arrow Articles by Smerdon, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rellos, P.
Right arrow Articles by Smerdon, S. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structure and Regulation of the Human Nek2 Centrosomal Kinase*Formula

Peter Rellos{ddagger}1, Frank J. Ivins§1, Joanne E. Baxter12, Ashley Pike{ddagger}, Timothy J. Nott§, Donna-Marie Parkinson, Sanjan Das{ddagger}, Steven Howell§, Oleg Fedorov{ddagger}, Qi Yu Shen, Andrew M. Fry3, Stefan Knapp{ddagger}4, and Stephen J. Smerdon§5

From the {ddagger}Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, United Kingdom, §Medical Research Council National Institute for Medical Research, Division of Molecular Structure, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom and Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom

Received for publication, October 16, 2006 , and in revised form, December 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The dimeric Ser/Thr kinase Nek2 regulates centrosome cohesion and separation through phosphorylation of structural components of the centrosome, and aberrant regulation of Nek2 activity can lead to aneuploid defects characteristic of cancer cells. Mutational analysis of autophosphorylation sites within the kinase domain identified by mass spectrometry shows a complex pattern of positive and negative regulatory effects on kinase activity that are correlated with effects on centrosomal splitting efficiency in vivo. The 2.2-Å resolution x-ray structure of the Nek2 kinase domain in complex with a pyrrole-indolinone inhibitor reveals an inhibitory helical motif within the activation loop. This helix presents a steric barrier to formation of the active enzyme and generates a surface that may be exploitable in the design of specific inhibitors that selectively target the inactive state. Comparison of this "auto-inhibitory" conformation with similar arrangements in cyclin-dependent kinase 2 and epidermal growth factor receptor kinase suggests a role for dimerization-dependent allosteric regulation that combines with autophosphorylation and protein phosphatase 1c phosphatase activity to generate the precise spatial and temporal control required for Nek2 function in centrosomal maturation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
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 p90Rsk2, an interesting observation as the activation of chromatin condensation by Nek2 in mouse spermatocytes is under the control of the mitogen-activated protein kinase/p90Rsk2 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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
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-beta-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 {lambda}-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 x 75 x 10 µm3.

Crystals were vitrified in mother liquor supplemented with 25% ethylene glycol, and diffraction data were collected at 100 K on beamline X10A ({lambda} = 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 TailorTM 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 beta-casein or the C-terminal domain of C-Nap1 and 1 µCi of [{gamma}-32P]ATP as substrates in 40 µl of kinase buffer (50 mM Hepes-KOH, pH 7.4, 5 mM MnCl2, 5mM beta-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-{gamma}-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
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
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.


Figure 1
View larger version (54K):
[in this window]
[in a new window]

  		FIGURE 1.
Structure and autophosphorylation of human Nek2. A, schematic showing the major structural and functional features Nek2A organization. Sites of autophosphorylation within both the catalytic and C-terminal regions are shown. PP1c, protein phosphatase 1c. B, a section of the 2Fo - Fc electron density map around the SU11652 inhibitor, contoured at 1{sigma}. C, structure of the Nek2 kinase domain-SU11652 complex. The N and C lobes are colored blue and red, respectively, the hinge region is highlighted in yellow, and the inhibitor is shown in green. Regions of disorder in the structure are included as dashed lines. D, phylogenetic tree showing the sequence relationships between the eleven human Nek-family members.

 
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 {lambda} 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 {alpha}C (Thr-45-Met-51), the loop connecting the two strands beta4 and beta5 (Arg-77-Thr-81), the region C-terminal to helix {alpha}E (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 {alpha}C helix in particular exhibits significant disorder, presumably due to mobility within the crystal, which is reflected by a high average temperature factor of 75 Å2. In comparison, the C-terminal domain (Glu-87-His-279) is well ordered with an average temperature factor of 33 Å2. Data and refinement statistics are shown in Table 1, and electron density for the inhibitor is shown in Fig. 1B.


View this table:
[in this window]
[in a new window]

  		TABLE 1
Crystallographic data and refinement statistics

 
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 [PDB] ) 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 C{alpha} positions. The two structures differ mainly in the length and location of secondary structural elements, particularly in the C-lobes. For example, {alpha}G is extended by one helical turn in Nek2 (residues Gly-101-Glu-105), and the helix inserted between sheet beta3 and {alpha}C 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 {alpha}-helix that we refer to as {alpha}T. 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 {alpha}E. Intriguingly, this additional element of secondary structure is wedged between {alpha}C, 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 {alpha}T. 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 {alpha}T to equivalent motifs found in these kinases strongly suggest that {alpha}T 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 {alpha}C pushes it toward the ATP/substrate binding cleft with concomitant disruption of the inhibitory helix. This allows Glu-51CDK2 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 {alpha}T unfolds into a more extended conformation in the active form.


Figure 2
View larger version (57K):
[in this window]
[in a new window]

  		FIGURE 2.
Structural comparisons. A, least-squares superposition of Nek2 with the inactive Aurora-A-ATP complex (Protein Data Bank code 1OL6) reveals differences distributed throughout the two structures, most obviously the presence of {alpha}T in Nek2 (circled) that is absent in Aurora-A. B, the Nek2 {alpha}T helix packs against the N lobe and catalytic helix {alpha}C and prevents access of Glu-55 to the active site. Glu-55 makes salt-bridge interactions with Arg-164 that is predicted to contact Thr(P)-175 in the active enzyme conformation. C, an inhibitory {alpha}T-like helix is observed in CDK2 (top), EGFR kinase (bottom), and Src/Hck family kinases (not shown). In each case hydrophobic residues at the equivalent positions to Nek2 Leu-162, Ile-165, and Leu-166 positions (see the inset) stabilize the inhibitory conformation through nonpolar interactions with residues from the kinase N lobe, including the P-loop, and {alpha}C itself. AMPPNP, adenosine 5'-(beta,{gamma}-imino)triphosphate.

 
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).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 3.
Structural basis of inhibition. The SU11652 inhibitor interacts with the ATP binding cleft through a network of hydrogen-bonding interactions with main-chain atoms from the kinase hinge region and van der Waals interactions with residues from the N and C lobe together with Leu-162 from the {alpha}T helix. The structure of the inhibitor is shown in the inset. The (diethylamino)ethyl moiety (highlighted) is disordered in the crystal structure.

 
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 beta, 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 Km of ~90 µM and kcat 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 Km 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 {alpha}T 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 {alpha}T 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 {alpha}T 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 {gamma}-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 beta-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.


Figure 4
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 4.
Regulation of Nek2A kinase activity by phosphorylation. A, U2OS cells were transfected with Myc-tagged Nek2 constructs (as indicated) for 24 h before being fixed and stained using anti-Myc (red) antibodies to detect transfected cells and anti-{gamma}-tubulin (green) antibodies to detect centrosomes. Approximately 100 transfected cells were scored for split centrosomes (>2 µm apart) in three independent experiments; untransfected cells (-ve) were used as a negative control. S.E. are indicated. Similar results were obtained using HeLa cells (data not shown). Representative merged images, including DNA stained with Hoechst 33258 (blue), are shown in the lower panel with insets showing magnified views of the centrosome staining. Arrowheads indicate centrosomes; scale bar, 5 µm. WT, wild type; KR, K37R. B, Myc-tagged Nek2A proteins were generated by coupled in vitro transcription-translation reactions and used in kinase assays against beta-casein as substrate as described under "Experimental Procedures." The activity of different Nek2A constructs is expressed as a percentage of wild-type activity normalized to the amount of precipitated protein. Each construct was assayed at least three times, and S.E. are indicated. Similar results were obtained using the C-terminal domain of C-Nap1 as a substrate (data not shown).

 
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)-{Phi}, where {Phi} 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.


Figure 5
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 5.
Autophosphorylation in the T-loop. A, conservation within the T-loop of vertebrate Nek2 kinases (Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Gg, Gallus gallus; Xl, Xenopus laevis), the 11 human Nek kinases, and T-loop sequences of MARK4, Chk2, and protein kinase A (PKA). T-loop phosphorylation at Nek2 position 175 is a conserved feature in many serine/threonine protein kinases (green circle), whereas phosphorylation at the equivalent position to Thr-179 (red circle) has only been previously reported in MARK4 as an inhibitory modification (Thr-202) by an upstream kinase (52) and in Chk2 (Thr-387) (53) where it has previously been taken as an activating modification. The remaining sites in Nek2 (Thr-170 and/or Ser-171) are highlighted with a blue circle. B, the structure of a transition state analogue of phosphoryl transfer in protein kinase A shows that Nek2 Thr-179 is likely to play a similar role to protein kinase A Thr-201 in stabilizing the configuration of catalytic residues. Phosphorylation is incompatible with efficient catalysis due to its proximity to the pentavalent intermediate in the transition-state. Nek2 residue numbers are in parentheses.

 
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 {alpha}H 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, {alpha}H 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 {alpha}T 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 {alpha}T helical segment forms a barrier to access of a conserved catalytic glutamate (Nek2 Glu-55) on {alpha}C to the catalytic site. Furthermore, the interactions that stabilize the position of the inhibitory helix with respect to the N-lobe and {alpha}C 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 {alpha}T unfolding.

What, then, is responsible for disruption of {alpha}T 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 {alpha}C/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 Km (190 µM) and kcat (8.4 min-1) on the GTF peptide substrate and a resulting 4-fold decrease in kcat/Km. 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, {alpha}H within the C-lobe of one monomer packs in a cyclin-like fashion against {alpha}C of the other, thus disrupting the intervening {alpha}T-like helix. Such a model is attractive since Nek2 Ser-241 that is targeted as a site of inhibitory autophosphorylation is also located on {alpha}H. 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 {alpha}T-like structures have been observed together with the overall similarity in {alpha}T 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.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2JAV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by grants (to A. M. F.) from The Wellcome Trust, the Association for International Cancer Research, and Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental material, including supplemental Figs. S1-S3. Back

1 These authors contributed equally to this work. Back

2 Supported by a Ph.D. studentship from Millennium Pharmaceuticals Inc. (Boston). Back

3 To whom correspondence may be addressed. Tel.: 44-116-229-7069; E-mail: amf5{at}leicester.ac.uk.

4 To whom correspondence may be addressed. Tel.: 44-1865-227978; E-mail: Stefan.Knapp{at}sgc.ox.ac.uk.

5 To whom correspondence may be addressed. Tel.: 44-20-8816-2533; E-mail: stephen.smerdon{at}nimr.mrc.ac.uk.

6 The abbreviations used are: NIMA, never in mitosis A; MARK, microtubule-associated regulatory kinase; CDK2, cyclin-dependent kinase; EGFR, epidermal growth factor receptor. Back


   ACKNOWLEDGMENTS
 
We thank members of the Structural Genomics Consortium for assistance with plasmid preparation and diffraction data collection. The Structural Genomics Consortium is a registered charity (no. 1097737) funded by the Wellcome Trust, GlaxoSmith-Kline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, VINNOVA, The Knut and Alice Wallenberg Foundation, and the Swedish Foundation for Strategic Research and Karolinska Institutet. S. J. S. is grateful to the Medical Research Council, UK for continuing support, Dr. Steve Martin for advice on kinase assays, and laboratory members for discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. O'Connell, M. J., Krien, M. J., and Hunter, T. (2003) Trends Cell Biol. 13, 221-228[CrossRef][Medline] [Order article via Infotrieve]
  2. Morris, N. R., Osmani, S. A., Engle, D. B., and Doonan, J. H. (1989) BioEssays 10, 196-201[CrossRef][Medline] [Order article via Infotrieve]
  3. Grallert, A., and Hagan, I. M. (2002) EMBO J. 21, 3096-3107[CrossRef][Medline] [Order article via Infotrieve]
  4. Grallert, A., Krapp, A., Bagley, S., Simanis, V., and Hagan, I. M. (2004) Genes Dev. 18, 1007-1021[Abstract/Free Full Text]
  5. Fry, A. M., Meraldi, P., and Nigg, E. A. (1998) EMBO J. 17, 470-481[CrossRef][Medline] [Order article via Infotrieve]
  6. Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y. D., Tanaka, K., and Nigg, E. A. (1998) J. Cell Biol. 141, 1563-1574[Abstract/Free Full Text]
  7. Mayor, T., Hacker, U., Stierhof, Y. D., and Nigg, E. A. (2002) J. Cell Sci. 115, 3275-3284[Abstract/Free Full Text]
  8. Bahe, S., Stierhof, Y. D., Wilkinson, C. J., Leiss, F., and Nigg, E. A. (2005) J. Cell Biol. 171, 27-33[Abstract/Free Full Text]
  9. Rapley, J., Baxter, J. E., Blot, J., Wattam, S. L., Casenghi, M., Meraldi, P., Nigg, E. A., and Fry, A. M. (2005) Mol. Cell. Biol. 25, 1309-1324[Abstract/Free Full Text]
  10. Prigent, C., Glover, D. M., and Giet, R. (2005) Exp. Cell Res. 303, 1-13[Medline] [Order article via Infotrieve]
  11. Wu, L., Osmani, S. A., and Mirabito, P. M. (1998) J. Cell Biol. 141, 1575-1587[Abstract/Free Full Text]
  12. Graf, R. (2002) J. Cell Sci. 115, 1919-1929[Abstract/Free Full Text]
  13. Roig, J., Groen, A., Caldwell, J., and Avruch, J. (2005) Mol. Biol. Cell 16, 4827-4840[Abstract/Free Full Text]
  14. Quarmby, L. M., and Mahjoub, M. R. (2005) J. Cell Sci. 118, 5161-5169[Abstract/Free Full Text]
  15. Chen, Y., Riley, D. J., Zheng, L., Chen, P. L., and Lee, W. H. (2002) J. Biol. Chem. 277, 49408-49416[Abstract/Free Full Text]
  16. Lou, Y., Yao, J., Zereshki, A., Dou, Z., Ahmed, K., Wang, H., Hu, J., Wang, Y., and Yao, X. (2004) J. Biol. Chem. 279, 20049-20057[Abstract/Free Full Text]
  17. Di Agostino, S., Fedele, M., Chieffi, P., Fusco, A., Rossi, P., Geremia, R., and Sette, C. (2004) Mol. Biol. Cell 15, 1224-1232[Abstract/Free Full Text]
  18. Fry, A. M., Schultz, S. J., Bartek, J., and Nigg, E. A. (1995) J. Biol. Chem. 270, 12899-12905[Abstract/Free Full Text]
  19. Hames, R. S., and Fry, A. M. (2002) Biochem. J. 361, 77-85[CrossRef][Medline] [Order article via Infotrieve]
  20. Uto, K., Nakajo, N., and Sagata, N. (1999) Dev. Biol. 208, 456-464[CrossRef][Medline] [Order article via Infotrieve]
  21. Hames, R. S., Wattam, S. L., Yamano, H., Bacchieri, R., and Fry, A. M. (2001) EMBO J. 20, 7117-7127[CrossRef][Medline] [Order article via Infotrieve]
  22. Helps, N. R., Luo, X., Barker, H. M., and Cohen, P. T. (2000) Biochem. J. 349, 509-518[CrossRef][Medline] [Order article via Infotrieve]
  23. Hayes, M. J. Y. K., Wattam, S. L., Lindon, C., Mao, G., Yamano, H., and Fry, A. M. (2006) Nat. Cell Biol. 8, 607-614[CrossRef][Medline] [Order article via Infotrieve]
  24. Fry, A. M., Arnaud, L., and Nigg, E. A. (1999) J. Biol. Chem. 274, 16304-16310[Abstract/Free Full Text]
  25. Eto, M., Elliott, E., Prickett, T. D., and Brautigan, D. L. (2002) J. Biol. Chem. 277, 44013-44020[Abstract/Free Full Text]
  26. Di Agostino, S., Rossi, P., Geremia, R., and Sette, C. (2002) Development 129, 1715-1727[Abstract/Free Full Text]
  27. Hayward, D. G., and Fry, A. M. (2006) Cancer Lett. 237, 155-166[CrossRef][Medline] [Order article via Infotrieve]
  28. Storoni, L. C., McCoy, A. J., and Read, R. J. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 432-438[CrossRef][Medline] [Order article via Infotrieve]
  29. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  30. Emsley, P., and Cowtan, K. (2004) Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132[CrossRef][Medline] [Order article via Infotrieve]
  31. Faragher, A. J., and Fry, A. M. (2003) Mol. Biol. Cell 14, 2876-2889[Abstract/Free Full Text]
  32. Brown, N. R., Noble, M. E., Lawrie, A. M., Morris, M. C., Tunnah, P., Divita, G., Johnson, L. N., and Endicott, J. A. (1999) J. Biol. Chem. 274, 8746-8756[Abstract/Free Full Text]
  33. Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J. (2006) Cell 125, 1137-1149[CrossRef][Medline] [Order article via Infotrieve]
  34. Sicheri, F., and Kuriyan, J. (1997) Curr. Opin. Struct. Biol. 7, 777-785[CrossRef][Medline] [Order article via Infotrieve]
  35. Johnson, L. N., Noble, M. E., and Owen, D. J. (1996) Cell 85, 149-158[CrossRef][Medline] [Order article via Infotrieve]
  36. Wai, D. H., Schaefer, K. L., Schramm, A., Korsching, E., Van Valen, F., Ozaki, T., Boecker, W., Schweigerer, L., Dockhorn-Dworniczak, B., and Poremba, C. (2002) Int. J. Oncol. 20, 441-451[Medline] [Order article via Infotrieve]
  37. Hayward, D. G., Clarke, R. B., Faragher, A. J., Pillai, M. R., Hagan, I. M., and Fry, A. M. (2004) Cancer Res. 64, 7370-7376[Abstract/Free Full Text]
  38. de Vos, S., Hofmann, W. K., Grogan, T. M., Krug, U., Schrage, M., Miller, T. P., Braun, J. G., Wachsman, W., Koeffler, H. P., and Said, J. W. (2003) Lab. Investig. 83, 271-285[Medline] [Order article via Infotrieve]
  39. Fletcher, L., Cerniglia, G. J., Yen, T. J., and Muschel, R. J. (2005) Biochim. Biophys. Acta 1744, 89-92[Medline] [Order article via Infotrieve]
  40. Fletcher, L., Cerniglia, G. J., Nigg, E. A., Yend, T. J., and Muschel, R. J. (2004) Radiat. Res. 162, 128-135[CrossRef][Medline] [Order article via Infotrieve]
  41. Heryanto, B., Lipson, K. E., and Rogers, P. A. (2003) Reproduction 125, 337-346[Abstract]
  42. Sun, L., Liang, C., Shirazian, S., Zhou, Y., Miller, T., Cui, J., Fukuda, J. Y., Chu, J. Y., Nematalla, A., Wang, X., Chen, H., Sistla, A., Luu, T. C., Tang, F., Wei, J., and Tang, C. (2003) J. Med. Chem. 46, 1116-1119[CrossRef][Medline] [Order article via Infotrieve]
  43. Liao, A. T., Chien, M. B., Shenoy, N., Mendel, D. B., McMahon, G., Cherrington, J. M., and London, C. A. (2002) Blood 100, 585-593[Abstract/Free Full Text]
  44. Fabian, M. A., Biggs, W. H., 3rd, Treiber, D. K., Atteridge, C. E., Azimioara, M. D., Benedetti, M. G., Carter, T. A., Ciceri, P., Edeen, P. T., Floyd, M., Ford, J. M., Galvin, M., Gerlach, J. L., Grotzfeld, R. M., Herrgard, S., Insko, D. E., Insko, M. A., Lai, A. G., Lelias, J. M., Mehta, S. A., Milanov, Z. V., Velasco, A. M., Wodicka, L. M., Patel, H. K., Zarrinkar, P. P., and Lockhart, D. J. (2005) Nat. Biotechnol. 23, 329-336[CrossRef][Medline] [Order article via Infotrieve]
  45. Lu, K. P., Kemp, B. E., and Means, A. R. (1994) J. Biol. Chem. 269, 6603-6607[Abstract/Free Full Text]
  46. Liu, Y., and Gray, N. S. (2006) Nat. Chem. Biol. 2, 358-364[CrossRef][Medline] [Order article via Infotrieve]
  47. Meraldi, P., and Nigg, E. A. (2001) J. Cell Sci. 114, 3749-3757
  48. Belham, C., Roig, J., Caldwell, J. A., Aoyama, Y., Kemp, B. E., Comb, M., and Avruch, J. (2003) J. Biol. Chem. 278, 34897-34909[Abstract/Free Full Text]
  49. Lowery, D. M., Lim, D., and Yaffe, M. B. (2005) Oncogene 24, 248-259[CrossRef][Medline] [Order article via Infotrieve]
  50. Zhang, W., Fletcher, L., and Muschel, R. J. (2005) J. Biol. Chem. 280, 42994-42999[Abstract/Free Full Text]
  51. Madhusudan Akamine, P., Xuong, N. H., and Taylor, S. S. (2002) Nat. Struct. Biol. 9, 273-277[CrossRef][Medline] [Order article via Infotrieve]
  52. Timm, T., Li, X. Y., Biernat, J., Jiao, J., Mandelkow, E., Vandekerckhove, J., and Mandelkow, E. M. (2003) EMBO J. 22, 5090-5101[CrossRef][Medline] [Order article via Infotrieve]
  53. Schwarz, J. K., Lovly, C. M., and Piwnica-Worms, H. (2003) Mol. Cancer Res. 1, 598-609[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
M. Yu. Zakharova, N. A. Kuznetsov, S. A. Dubiley, A. V. Kozyr, O. S. Fedorova, D. M. Chudakov, D. G. Knorre, I. G. Shemyakin, A. G. Gabibov, and A. V. Kolesnikov
Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches
J. Biol. Chem., July 3, 2009; 284(27): 17902 - 17913.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Li, I. A. Taylor, J. Lloyd, J. A. Clapperton, S. Howell, D. MacMillan, and S. J. Smerdon
Chk2 Oligomerization Studied by Phosphopeptide Ligation: IMPLICATIONS FOR REGULATION AND PHOSPHODEPENDENT INTERACTIONS
J. Biol. Chem., December 19, 2008; 283(51): 36019 - 36030.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
O. Fedorov, B. Marsden, V. Pogacic, P. Rellos, S. Muller, A. N. Bullock, J. Schwaller, M. Sundstrom, and S. Knapp
A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases
PNAS, December 18, 2007; 104(51): 20523 - 20528.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. Wu, J. E. Baxter, S. L. Wattam, D. G. Hayward, M. Fardilha, A. Knebel, E. M. Ford, E. F. da Cruz e Silva, and A. M. Fry
Alternative Splicing Controls Nuclear Translocation of the Cell Cycle-regulated Nek2 Kinase
J. Biol. Chem., September 7, 2007; 282(36): 26431 - 26440.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/9/6833    most recent
M609721200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rellos, P.
Right arrow Articles by Smerdon, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rellos, P.
Right arrow Articles by Smerdon, S. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement