Human SLK (Ste20-like kinase)

Target ID:

SLKA

Aliases:

bA16H23.1KIAA0204MGC133067se20-9STK2

Deposition Date:

Friday 8th September 2006

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2J51

Authors:

A.C.W.PIKE,P.RELLOS,O.FEDOROV,T.KEATES,E.SALAH,P.SAVITSKY,E.PAPAGRIGORIOU,A.P.TURNBULL,J.E.DEBRECZENI,F.VON DELFT,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2J51

tabs

Structure Details

The STE20 kinase SLK is a ser/thr kinase and a member of group II of germinal center kinases (GCKs), which includes MST1-3, SOK1 and LOK. SLK has been reported to be involved in actin stress fiber dissolution and mediation of programmed cell death in a kinase-independent manner. Depletion of SLK by small interfering RNAs has shown that SLK is required for progression through G2 phase and a functional role for SLK during muscle development as well as in adult skeletal muscle has been suggested.

SLK is highly expressed in the muscle mass and neuronal lineages of developing embryos. The protein can be detected at E10.5 and E12.5 in the forebrain, midbrain and hindbrain of the developing CNS. At later developmental stages, it is expressed in the hypothalamus region, all layers of the neural tube, dorsal root ganglion and in the proliferating ependymal layers. Following middle cerebral artery occlusion, SLK expressing neuronal cells are lost and SLK is localized to phagocytic macrophages.

In adult muscle tissue, SLK is found at myofibrillar striations of specific subsets of myofibers. In addition it is highly expressed in the nerve at neuromuscular junctions as well as in tubular epithelial cells, in fetal and adult kidneys and in developing and mature podocytes. SLK activity is increased during the G2 phase, after incubation with serum or after exposure to chemical anoxia whereas SLK expression is decreased in quiescent and differentiating cells. SLK resides in the cytoplasm and co-localises with the mitotic spindle during mitosis.

SLK is cleaved by caspase 3 in vitro and in vivo during c-Myc-, tumor necrosis factor alpha, and UV-induced apoptosis. Cleavage of SLK results in two domains with distinct activities: an activated N-terminal kinase domain that promotes apoptosis and cytoskeletal rearrangements and a C-terminal domain that disassembles actin stress fibers. SLK has been reported to interact with the neuronal transmembrane protein calsyntenin and has been reported to phosphorylate P olo-like kinase 1.

SLK has been identified as marker for Cutaneous T-cell lymphomas (CTCL), which are a group of skin neoplasms that originate from T lymphocytes predominantly and include mycosis fungoides (MF) and the Sezary Syndrome (SS). In addition, SLK is induced during recovery from acute renal failure and has been suggested to be involved in the pathogenesis of podocyte-specific glomerulopathies like membranous nephropathy.

Materials and Methods

SLK

PDB:2J51

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|41281453
Entry Clone Source:synthetic DNA
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfqsmKQYEHV KRDLNPEDFWEIIGELGDGAFGKVYKAQN KETSVLAAAKVIDTKSEEELEDYMVEIDI LASCDHPNIVKLLDAFYYENNLWILIEFC AGGAVDAVMLELERPLTESQIQVVCKQTL DALNYLHDNKIIHRDLKAGNILFTLDGDI KLADFGVSAKNTRTIQRRDSFIGTPYWMA PEVVMCETSKDRPYDYKADVWSLGITLIE MAEIEPPHHELNPMRVLLKIAKSEPPTLA QPSRWSSNFKDFLKKCLEKNVDARWTTSQ LLQHPFVTVDSNKPIRELIAEAKAEVTEE VEDGKE
Vector:pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycine was used to inoculate 1 liter of LB media containing 50 µg/ml kanamycine. Cultures were grown at 37°C until the OD 600 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.
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. SLK 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)
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.

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:Centricon with a 10kDa cut off in SEC-buffer
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml containing 1 mM Cdk1/2 Inhibitor III (CalbioChem) by mixing 100nl of the concentrated protein with 50nl of a well solution containing 16% PEG3350, 0.15M KSCN, 0.1M BisTris propane pH 6.5 and 10 % ethylene glycol. Crystals appeared after a couple of days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with an additional 15% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 ( l =0.979 Å) to 2.1Å
Data Processing:

References

Chaar, Z. Y., O'Reilly, P., Gelman, I. , and Sabourin, L. A. (2006). v-src-dependent downregulation of the ste20-like kinase SLK by casein kinase II. J Biol Chem.
Cybulsky, A. V., Takano, T., Papillon, J., Khadir, A., Bijian, K., Chien, C. C., Alpers, C. E., and Rabb, H. (2004). Renal expression and activity of the germinal center kinase SK2. Am J Physiol Renal Physiol 286 , F16-25.
Ellinger-Ziegelbauer, H., Karasuyama, H., Yamada, E., Tsujikawa, K., Todokoro, K., and Nishida, E. (2000). Ste20-like kinase (SLK), a regulatory kinase for polo-like kinase (Plk) during the G2/M transition in somatic cells. Genes Cells 5 , 491-498.
Hao, W., Takano, T., Guillemette, J., Papillon, J., Ren, G., and Cybulsky, A. V. (2006). Induction of apoptosis by the Ste20-like kinase SLK, a germinal center kinase that activates apoptosis signal-regulating kinase and p38. J Biol Chem 281 , 3075-3084.
O'Reilly, P. G., Wagner, S., Franks, D. J., Cailliau, K., Browaeys, E., Dissous, C., and Sabourin, L. A. (2005). The Ste20-like kinase SLK is required for cell cycle progression through G2. J Biol Chem 280 , 42383-42390.
Sabourin, L. A., and Rudnicki, M. A. (1999). Induction of apoptosis by SLK, a Ste20-related kinase. Oncogene 18 , 7566-7575.
Storbeck, C. J., Daniel, K., Zhang, Y. H., Lunde, J., Scime, A., Asakura, A., Jasmin, B., Korneluk, R. G., and Sabourin, L. A. (2004). Ste20-like kinase SLK displays myofiber type specificity and is involved in C2C12 myoblast differentiation. Muscle Nerve 29 , 553-564.
Wagner, S., Flood, T. A., O'Reilly, P., Hume, K., and Sabourin, L. A. (2002). Association of the Ste20-like kinase (SLK) with the microtubule. Role in Rac1-mediated regulation of actin dynamics during cell adhesion and spreading. J Biol Chem 277, 37685-37692.