Human serine/threonine kinase 10

Target ID:

STK10A

Aliases:

LOKPRO2729

Deposition Date:

Monday 7th July 2014

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

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Authors:

Elkins, J.M., Salah, E., Szklarz, M., vonDelft, F., Canning, P., Raynor, J., Bountra, C., Edwards, A.M., Knapp, S.

Site:

Oxford

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tabs

Structure Details

STK10, also known as lymphocyte-oriented kinase (LOK), is a Ser/Thr protein kinase expressed predominantly in lymphocytes (Kuramochi et al., 1997; Qian et al., 1998). LOK deficiency promoted LFA-1 mediated adhesions of mitogen-stimulated T cells (Endo et al., 2000). LOK may be involved in regulation of PLK1 (Johnson et al., 2008; Walter et al., 2003), and may be the dominant kinase phosphorylating ERM proteins (ezrin, radixin or moesin) in lymphocytes (Belkina et al., 2009; Viswanatha et al., 2012). The off-target effects of the kinase inhibitor Erlotinib (an EGFR inhibitor) on LOK is responsible for the enhanced lymphocytic response that leads to skin disorders with Erlotinib treatment (Yamamoto et al., 2011).
As part of efforts to develop chemical tools to interrogate LOK function, we have determined the co-crystal structure of LOK with inhibitor SB-633825. SB-633825 was identified as a reasonably selective LOK inhibitor through screening of compounds in the GSK Published Kinase Inhibitor Set (PKIS). Two crystal structures were determined. In structure 4USD the kinase LOK is in an inactive conformation, characterised by a flipped DFG motif (shown in red in the figure), while in structure 4USE the kinase has its DFG motif in an active conformation.

Materials and Methods

STK10

PDB:4USD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:SGC Oxford
SGC Clone Accession:
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:SMRKSREYEHVRRDLDPNEVWEIVGELGDGAFGKVYKAKNKETGALAAAKVIETKSEEELEDYIVEIEILATCDHPYIVKLLGAYYHDGKLWIMIEFCPGGAVDAIMLELDRGLTEPQIQVVCRQMLEALNFLHSKRIIHRDLKAGNVLMTLEGDIRLADFGVSAKNLKTLQKRDSFIGTPYWMAPEVVMCETMKDTPYDYKADIWSLGITLIEMAQIEPPHHELNPMRVLLKIAKSDPPTLLTPSKWSVEFRDFLKIALDKNPETRPSAAQLLEHPFVSSITSNKALRELVAEAKAEVMEE The N-terminal residues, SM, derive from the vector.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Cell Lysis: The resuspended cell pellet was thawed and lysed by sonication. PEI (polyethyleneimine) was added to a final concentration of 0.15 %. The cell debris and precipitated DNA were spun down.Lysis Buffer: 50 mM Hepes pH 7.4, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP. Purification: Column 1: 5 ml of Ni-Sepharose in a 2.5 cm diameter gravity flow column. Column 1 Buffers: Binding Buffer: 50 mM Hepes pH 7.4, 200 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP. Wash Buffer 1: As Binding Buffer except 1 M NaCl and 40 mM imidazole.Wash Buffer 2: As Binding Buffer except 60 mM imidazole. Elution Buffer: As Binding Buffer except 250 mM imidazole. Column 1 Procedure: The clarified supernatant was passed through the column. The column was washed with 100 mL of Binding Buffer and 50 mL each of Wash Buffer 1 and 2. 25 ml of Elute Buffer was passed through to elute the protein. TEV protease was added and the sample left overnight. Column 2: S200 16/60 Gel Filtration (GE Healthcare) Column 2 Buffers: GF Buffer: 50 mM Hepes pH 7.5, 300 mM NaCl, 0.5 mM TCEP Column 2 Procedure: The eluted protein was concentrated to 5 ml volume and injected onto the column. Column 3: Ni-Sepharose. The pooled fractions from gel filtration were passed through the resin.

Extraction

Buffers
Procedure
Expression strain: BL-21(DE3)-R3-lambda-ppase (coexpresses lambda phosphatase). Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure. Expression: Colonies were used to inoculate 50 mL of LB media containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol, which was placed in a 37°C shaker overnight. The next day 4x 10 mL of this starter culture was used to inoculate 4x 1L of LB media containing 37.5 µg/mL kanamycin in 2L baffled shaker flasks. When the OD600 was approximately 0.45, the temperature was reduced to 20°C and when the OD600 was approximately 0.6 the cells were induced by the addition of 0.5 mM IPTG. The expression was continued overnight. Cell harvest: Cells were spun at 5000rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C.
Concentration:The protein was concentrated to 13.6 mg/ml (measured by 280 nm absorbance).
Ligand
MassSpec:
Crystallization:Compound SB-633825 was added to 1 mM. Crystals grew from a 1:2 ratio of protein and precipitant solution (0.20M NH4Cl; 0.1M TRIS pH 8.0; 20.0% PEG 6K; 10.0% Ethylene Glycol), using the vapour diffusion method.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol, and then flash frozen in liquid nitrogen.
Data Processing:

References

Belkina, N. V, Liu, Y., Hao, J.-J., Karasuyama, H., and Shaw, S. (2009). LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation. PNAS 106, 4707-4712.

Endo, J., Toyama-Sorimachi, N., Taya, C., Kuramochi-Miyagawa, S., Nagata, K., Kuida, K., Takashi, T., Yonekawa, H., Yoshizawa, Y., Miyasaka, N., et al. (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.

Johnson, T.M., Antrobus, R., and Johnson, L.N. (2008). Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP). Biochemistry 47, 3688-3696.

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.

Qian, Y.-W., Erikson, E., and Maller, J.L. (1998). Purification and Cloning of a Protein Kinase That Phosphorylates and Activates the Polo-Like Kinase Plx1. Science (80-. ). 282, 1701-1704.

Viswanatha, R., Ohouo, P.Y., Smolka, M.B., and Bretscher, A. (2012). Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells. J. Cell Biol. 199, 969-984.

Walter, S. a, Cutler, R.E., 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.

Yamamoto, N., Honma, M., and Suzuki, H. (2011). Off-target serine/threonine kinase 10 inhibition by erlotinib enhances lymphocytic activity leading to severe skin disorders. Mol. Pharm. 80, 466-475.