Human SNF1 sucrose nonfermenting like kinase (yeast)

Target ID:

SLKA

Aliases:

bA16H23.1KIAA0204MGC133067se20-9STK2

Deposition Date:

Monday 7th July 2014

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

4USF

Authors:

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

Site:

Oxford

4USF

tabs

Structure Details

SLK (STK20-like kinase) is a Ser/Thr kinase capable of auto-activation by phosphorylation (Pike et al., 2008; Yamada et al., 2000). It is widely expressed and extended overexpression promoted apoptosis (Sabourin and Rudnicki, 1999). SLK may have roles in ErbB2-driven breast cancer cell motility (Roovers et al., 2009) and activation of ERM (ezrin, radixin or moesin) proteins (Machicoane et al., 2014).
As part of efforts to develop chemical tools to interrogate SLK function, we have determined the co-crystal structure of LOK with inhibitor SB-440719. This inhibitor was identified as a reasonably selective LOK/SLK inhibitor after optimisation of a screening hit obtained from the GSK Published Kinase Inhibitor Set (PKIS). The structure shows SLK in an inactive conformation, with an unusual P-loop conformation of the kinase, which curves around the inhibitor, and extensive involvement of the activation loop in binding the inhibitor.

Materials and Methods

SLK

PDB:4USF

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:SMKQYEHVTRDLNPEDFWEIIGELGDGAFGKVYKAQNKETSVLAAAKVIDTKSEEELEDYMVEIDILASCDHPNIVKLLDAFYYENNLWILIEFCAGGAVDAVMLELERPLTESQIQVVCKQTLDALNYLHDNKIIHRDLKAGNILFTLDGDIKLADFGVSAKNTRTIQRRDSFIGTPYWMAPEVVMCETSKDRPYDYKADVWSLGITLIEMAEIEPPHHELNPMRVLLKIAKSEPPTLAQPSRWSSNFKDFLKKCLEKNVDARWTTSQLLQHPFVTVDSNKPIRELIAEAKAEVTEEVEDGKE 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, 500 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, 500 mM NaCl, 20 mM Imidazole, 0.5 mM TCEP. Wash Buffer 1: As Binding Buffer except 40 mM imidazole and 1M NaCl.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 50 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 was dialysed into GF Buffer overnight. GF Buffer: 50 mM Hepes pH 7.5, 300 mM NaCl, 0.5 mM TCEP Column 2: Ni-Sepharose. The digested sample was passed through the resin, which was eluted with GF Buffer containing 10mM, 20mM, 30mM and then 40mM imidazole. Column 3: S200 16/60 Gel Filtration (GE Healthcare) Column 3 Buffers: 50 mM HEPES, 300 mM NaCl, 5 mM DTT Column 3 Procedure: The eluted protein was concentrated to 5 ml volume and injected onto the column.

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 mg/ml (measured by 280 nm absorbance).
Ligand
MassSpec:
Crystallization:Compound SB-440719 was added to 1 mM. Crystals grew from a 1:2 ratio of protein and precipitant solution (20% PEG6000, 10% ethylene glycol, 0.1M MES pH 6.0, 0.1M magnesium chloride), using the vapour diffusion method. Crystals were cryo-protected by equilibration into precipitant solution containing 25% ethylene glycol, and then flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Machicoane, M., de Frutos, C. a, Fink, J., Rocancourt, M., Lombardi, Y., Garel, S., Piel, M., and Echard, A. (2014). SLK-dependent activation of ERMs controls LGN-NuMA localization and spindle orientation. J. Cell Biol. 205, 791-799.

Pike, A.C.W., Rellos, P., Niesen, F.H., Turnbull, A., Oliver, A.W., Parker, S. a, Turk, B.E., Pearl, L.H., and Knapp, S. (2008). Activation segment dimerization: a mechanism for kinase autophosphorylation of non-consensus sites. EMBO J. 27, 704-714.

Roovers, K., Wagner, S., Storbeck, C.J., O'Reilly, P., Lo, V., Northey, J.J., Chmielecki, J., Muller, W.J., Siegel, P.M., and Sabourin, L. a (2009). The Ste20-like kinase SLK is required for ErbB2-driven breast cancer cell motility. Oncogene 28, 2839-2848.

Sabourin, L.A., and Rudnicki, M.A. (1999). Induction of apoptosis by SLK, a Ste20-related kinase. Oncogene 18, 7566-7575.

Yamada, E., Tsujikawa, K., Itoh, S., Kameda, Y., Kohama, Y., and Yamamoto, H. (2000). Molecular cloning and characterization of a novel human STE20-like kinase, hSLK. Biochim. Biophys. Acta 1495, 250-262.