Human Serine Threonine kinase 10 (STK10, LOK)

Target ID:

STK10A

Aliases:

LOKPRO2729

Deposition Date:

Tuesday 17th October 2006

Families:

Protein Kinase

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2J7T

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2J7T

tabs

Structure Details

STK10 is a member of the STE20 family of serine/threonine kinases and the human homolog of the murine kinase LOK, a serine/threonine kinase highly expressed in lymphocytes. STK10 is closely related to the polo-like kinase kinase SLK and has been shown to associate with Plk1 and phosphorylate it in vitro.

STK10 knockout mice do not exhibit any obvious histopathologic abnormalities and show normal distribution of B cells, T cells and their precursors in the bone marrow, thymus, spleen and lymph nodes as well as normal lymphocytes development.

STK10 has also been shown to play a role in modifying TcR/CD28 signals for activation of cytokine transcription and it is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion. STK10 has been found to be over-expressed in a number of tumor cells and a heterozygous somatic missense mutation (K277E) within the kinase domain has been found in human testicular germ-cell tumors.

STK10 has been co-crystallized with the cell-permeable pyrrole indolinone SU11274 ( (3Z)-N- (3-Chlorophenyl)- 3-((3,5-dimethyl- 4-((4-methylpiperazin -1-yl)carbonyl)-1H- pyrrol-2-yl)methylene) -N-methyl-2-oxo -2,3-dihydro-1H- indole-5-sulfonamide) which has been described as a potent inhibitor of Met kinase activity (IC50= 20 nM). SU11274 has been shown to inhibit HGF/SF-stimulated cellular signaling and Met-mediated tumorigenesis in various cancer cell lines.

Materials and Methods

STK10: Human Serine Threonine kinase 10 (STK10, LOK)

PDB:2J7T

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|5174701
Entry Clone Source:synthetic DNA
SGC Clone Accession:
Tag: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:The following mutations have been detected in that clone with respect to the reference sequence: V62A, E136V, G317E. The mutations have been confirmed by electron density and ESI-MS.
Sequence:smRKSREYEHVRRDLDPNEVWEIVGELGDGAFGKVYKAKNKETGALAAAKVIETKSEEELEDYIVEIEILATCDHPYIVKLLGAYYHDGKLWIMIEFCPGGAVDAIMLELDRGLTEPQIQVVCRQMLEALNFLHSKRIIHRDLKAGNVLMTLEGDIRLADFGVSAKNLKTLQKRDSFIGTPYWMAPEVVMCETMKDTPYDYKADIWSLGITLIEMAQIEPPHHELNPMRVLLKIAKSDPPTLLTPSKWSVEFRDFLKIALDKNPETRPSAAQLLEHPFVSSITSNKALRELVAEAKAEVMEE
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture (BL21 DE3) containing 50 µg/ml kanamycine and 35 µg/ml chloramphenicole was used to inoculate 1 liter of LB media containing the same concentration of antibiotics. Cultures were grown at 37°C until the OD600 reached ~0.3. After that the temperature was adjusted to 20°C. Expression was induced for 4 hours using 1mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 300 mM NaCl; 20 mM imidazole.

Purification

Procedure
Column 1: Ni-affinity chromatography.
Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 50 ml binding buffer. The lysate was applied to the column which was subsequently washed with 50 ml wash buffer 1 and 2. STK10 was eluted with 25 mls of elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 10mM. The N-terminal his 6 -tag was cleaved by incubating the protein overnight with TEV protease.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)
Procedure: The fractions eluted of the Ni-affinity chromatography were concentrated to about 4 mls using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 0.8 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

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

Extraction

Procedure
Cell pellets were lysed by sonication. The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 12.83 mg/ml containing 1 mM SU11274 ( (3Z)-N-(3- Chlorophenyl)-3-((3,5-dimethyl -4-((4-methylpiperazin -1-yl)carbonyl) -1H-pyrrol-2-yl)methylene) -N-methyl-2-oxo -2,3-dihydro-1H-indole -5-sulfonamide) by mixing 100nl of the concentrated protein with 50nl of a well solution containing 45% PEG300, 0.24M CaAc, 0.1M Na cacodylate pH 6.5) Crystals appeared after a couple of days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were directly flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 (λ=0.999 Å) to 2.0Å
Data Processing:

References

Berthou, S., Aebersold, D. M., Schmidt, L. S., Stroka, D., Heigl, C., Streit, B., Stalder, D., Gruber, G., Liang, C., Howlett, A. R. , et al. (2004). The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants. Oncogene 23 , 5387-5393.
Endo, J., Toyama-Sorimachi, N., Taya, C., Kuramochi-Miyagawa, S., Nagata, K., Kuida, K., Takashi, T., Yonekawa, H., Yoshizawa, Y., Miyasaka, N., and Karasuyama, H. (2000). Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes. FEBS Lett 468 , 234-238.
Kuramochi, S., Matsuda, Y., Okamoto, M., Kitamura, F., Yonekawa, H., and Karasuyama, H. (1999). Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues. Immunogenetics 49 , 369-375.
Kuramochi, S., Moriguchi, T., Kuida, K., Endo, J., Semba, K., Nishida, E., and Karasuyama, H. (1997). LOK is a novel mouse STE20-like protein kinase that is expressed predominantly in lymphocytes. J Biol Chem 272 , 22679-22684.
Ma, P. C., Jagadeeswaran, R., Jagadeesh, S., Tretiakova, M. S., Nallasura, V., Fox, E. A., Hansen, M., Schaefer, E., Naoki, K., Lader, A. , et al. (2005). Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res 65 , 1479-1488.
Schreiner, B., Baur, D. M., Fingerle, A. A., Zechner, U., Greten, F. R., Adler, G., Sipos, B., Kloppel, G., Hameister, H., and Schmid, R. M. (2003). Pattern of secondary genomic changes in pancreatic tumors of Tgf alpha/Trp53+/- transgenic mice. Genes Chromosomes Cancer 38 , 240-248.
Walter, S. A., Cutler, R. E., Jr., Martinez , R., Gishizky, M., and Hill, R. J. (2003). Stk10, a new member of the polo-like kinase kinase family highly expressed in hematopoietic tissue. J Biol Chem 278 , 18221-18228.