Human never in mitosis gene A-related kinase 2

Target ID:

NEK2A

Aliases:

HsPK 21HsPK21NEK2ANLK1

Deposition Date:

Tuesday 25th April 2006

Families:

Protein Kinase

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2JAV

Authors:

A.C.W.PIKE,P.RELLOS,S.DAS,O.FEDOROV,E.PAPAGRIGORIOU,J.E.DEBRECZENI,A.P.TURNBULL,F.GORREC,J.BRAY,M.SUNDSTROM,C.H.ARROWSMITH,A.EDWARDS,J.WEIGELT,F.VON DELFT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2JAV

tabs

Structure Details

Nek2 was first identified as a cell cycle mutant (nimA) which arrested in late G2 phase in the multicellular filamentous fungus Aspergillus nidulans . The protein has later been cloned and identified as a Ser/Thr kinase called NIMA (Never in Mitosis). Surprisingly deletion of NIMA function leads to G2 arrest even in the presence of fully active Cdc2 kinase and mutational analysis subsequently also showed that activation of NIMA is Cdc2 dependent.

Nek2, a human NIMA homolog, is a 48-kDa protein with a catalytic kinase domain localized at the N-terminus of the molecule and a regulatory domain at the C-terminus. Two coiled coils regions are present within the regulatory domain. Nek2 regulation involves homodimerization through a leucine zipper coiled-coil motif which probably facilitates autophosphorylation in trans resulting in enzymatic activation. Autophosphorylation is however not the only mode of kinase activation: A study investigating meiotic progression in mouse spermatocytes showed that the MAPK/p90Rsk2 pathway is required to control Nek2 activation. Nek2 protein level is low in G1 and increases throughout S and G2. The protein is rapidly degraded at the pro-metaphase to metaphase transition of mitosis in an APC/C-Cdc20-dependent manner and with kinetics similar to Cyclin A.

Nek2 is a centrosomal protein that upon overexpression causes centrosome splitting, an event that is different from the physiological separation of centrosomes occurring at G2/M. During centrosome splitting mother and daughter centrioles are separated by > 2 mm, but this event is not followed by recruitment of motor proteins at centrosomes and assembly of a functional mitotic spindle. Nek2-driven centrosome splitting is likely accomplished through phosphorylation of C-Nap1, a protein interacting with Nek2 and associated with the proximal ends of mother and daughter centrioles.

Depletion of Nek2 by RNAi in mouse embryos revealed defects beginning at the second mitotic division. Many blastomeres arrested during M phase with abnormal spindle structures suggesting that Nek2 is essential for embryonic mitosis, especially for proper segregation of chromosomes.

Nek2 is overexpressed in a number of tumour cells including cell lines derived from Ewing's tumours (a paediatric osteosarcoma), non-Hodgkin lymphoma, breast, cervical and prostate carcinomas, lymphomas and invasive ductal carcinoma (IDC). In addition there is a correlation of increased Nek2 expression and poor prognosis of the patients. The role of Nek2 in tumour development makes this protein an interesting target for the development of inhibitors for the treatment of cancer.

We determined the structure of Nek2 in complex with the 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 ). This inhibitor 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 PDGFRβ (IC50 = 3 ηM), VEGFR2 (IC50 = 27 ηM), FGFR1 (IC50 = 170 ηM), and Kit family members (IC50 ~ 10-500 ηM) over EGFR (IC5020 μM).
The inhibitor has been reported to display anti-proliferative and pro-apoptotic properties in tumor cells and a related compound (SU11248, sunitinib malate) has been recently approved for the treatment of metastatic renal cancer.

Materials and Methods

NEK2: Human never in mitosis gene A-related kinase 2

PDB:2JAV

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP002488
Entry Clone Source:Steve Smerdon NIMR, London UK
SGC Clone Accession:
Tag:Tag sequence: Non -cleavable C-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:
Vector:pET22b

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 100 µg/ml ampicillin was used to inoculate 1 liter of LB media containing 100 µg/ml ampicillin. Cultures were grown at 37°C until the OD600 reached ~0.7. After that the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1mM IPTG. The cells were collected by centrifugation and the pellets were frozen.

Purification

Procedure
Column 1: Ni-affinity chromatography.
Procedure: The lysate was bound onto 5 ml of Ni-NTA Sepharose pre-equilibrated in lysis buffer. The resin was washed with 25 ml lysis buffer supplemented with 1 M NaCl, then with 25 ml of lysis buffer. Protein was eluted with lysis buffer supplemented with 150 mM imidazole. DTT was added to 5 mM, MnCl2 was added to an end concentration of 0.5 mM, Phosphates were removed adding 20 µl of Shrimp alkaline phosphatase and 100 µl of ?-phosphatase during an 12 h incubation at 4°C.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)
Procedure: The protein sample was concentrated by ultrafiltration (10K cutoff) and fractionated on a S75 superdex column equlibrated in 50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM NaPO4, 5 mM DTT, 5% glycerol. Eluted fractions were 95% pure as judged by SDS-PAGE, and confirmed by mass spectrometry as the unphosphorylated protein.

Protein concentration: Centricon with a 10kDa cut off in SEC-buffer

Extraction

Procedure
Cell pellets were resuspended in 25 ml of 50 mM HEPES pH 7.5, 5 mM Sodium phosphate, 300 mM NaCl, 20 mM Imidazole and 5% glycerol. The cells were lysed by sonication for a total of 3 min.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained at 4°C using vapor diffusion sitting drops at a protein concentration of 7.5 mg/ml containing 1 mM of 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 protein-ligand complex was crystallized by mixing protein and precipitant at ratios of 4:1, 3:1 and 2:1. The crystallization precipitant comprised 10% polyethylene glycol 6000, 200 mM MgCl2.
NMR Spectroscopy:
Data Collection:Crystals were vitrified in mother liquor supplemented with 25% ethylene glycol. Data were measured at SLS (Swiss Light Source).
Data Processing:

References

Ferrari, S. (2006). Protein kinases controlling the onset of mitosis. Cell Mol Life Sci.
Fry, A. M. (2002). The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 21 , 6184-6194.
Fry, A. M., Meraldi, P., and Nigg, E. A. (1998). A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators. Embo J 17 , 470-481.
Fry, A. M., Schultz, S. J., Bartek, J., and Nigg, E. A. (1995). Substrate specificity and cell cycle regulation of the Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA of Aspergillus nidulans. J Biol Chem 270 , 12899-12905.
Hayward, D. G., and Fry, A. M. (2005). Nek2 kinase in chromosome instability and cancer. Cancer Lett.
Quarmby, L. M., and Mahjoub, M. R. (2005). Caught Nek-ing: cilia and centrioles. J Cell Sci 118 , 5161-5169.