Human SLK (Ste20-like kinase) mono-phosphorylated

Target ID:

SLKA

Aliases:

bA16H23.1KIAA0204MGC133067se20-9STK2

Deposition Date:

Friday 2nd February 2007

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2JFM

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2JFM

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. Here we determined the structure of unphosphorylated SLK in complex with the inhibitor 5-amino -3-((4-(aminosulfonyl) phenyl)amino)-N- (2,6-difluorophenyl) 1,2,4-triazole -1-carbothioamide as well as the structure of mono-phosphorylated SLK in complex with a pyrazol quinazoline inhibitor. Both structures revealed a flipped out activation segment conformation binding to the substrate binding site of a symmetry related molecule. In addition we determined the structure of essentially mono-phosphorylated apo-SLK and di-phosphorylated SLK with 5-amino -3-((4-(aminosulfonyl) phenyl)amino)-N- (2,6-difluorophenyl) 1,2,4-triazole -1-carbothioamide. The activation segment of all structures domain exchanges with a symmetry related molecule to form an active kinase in trans.

Materials and Methods

Entry Clone Source: FivePrime

Entry Clone Accession: NM_014720 Variant

SGC Construct ID: SLKA-c004

GenBank GI number: gi|41281453

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [GenBank]

Tags and additions: Tag sequence:
mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence:
mhhhhhhssgvdlgtenlyfqsmKQYEHV
TRDLNPEDFWEIIGELGDGAFGKVYKAQN
KETSVLAAAKVIDTKSEEELEDYMVEIDI
LASCDHPNIVKLLDAFYYENNLWILIEFC
AGGAVDAVMLELERPLTESQIQVVCKQTL
DALNYLHDNKIIHRDLKAGNILFTLDGDI
KLADFGVSAKNTRTIQRRDSFIGTPYWMA
PEVVMCETSKDRPYDYKADVWSLGITLIE
MAEIEPPHHELNPMRVLLKIAKSEPPTLA
QPSRWSSNFKDFLKKCLEKNVDARWTTSQ
LLQHPFVTVDSNKPIRELIAEAKAEVTEE
VEDGKE

The highlighted residue (red) corresponds to mutation (K/T) with respect to the reference sequence

Host: BL21 (DE3)

Growth medium, induction protocol: 1ml from a 10 ml overnight culture (BL21 Rosetta strain) 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.

Extraction buffer, extraction method: Cell pellets were lysed by sonication. The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 300mM NaCl,, 20 mM Imidazole. Wash buffer 1: 50 mM HEPES pH 7.5, 1M NaCl, 20mM Imidazole. Wash buffer 2 : as for lysis buffer . Elution buffer: 50mM HEPES pH 7.5, 300mM NaCl, 150 mM Imidazole.

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. 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)

SEC-Buffers: 50 mM Hepes, pH 7.5, 300 mM NaCl, 5 mM DTT.

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

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 of the 4-(4- (5-cyclopropyl- 1h-pyrazol-3- ylamino)- quinazolin-2- ylamino)- benzonitrile complex were obtained using essentially mono-phosphorylated protein a protein concentration of 10 mg/ml containing 1 mM of the inhibitor by mixing 100 nl of protein solution with 50 nl of a buffer containing 18% PEG3350, 0.15M KSCN, 0.1M BisTris propane pH 6.5 and 10 % ethylene glycol. In order to obtain crystals of phosphorylated apo-SLK and the di-phosphorylated complex with 4-(4- (5-cyclopropyl- 1h-pyrazol-3- ylamino)- quinazolin-2- ylamino)- benzonitrile the recombinant protein was incubated with 5 mM ATP/Mg overnight. Apo-SLK crystallized using 0.20M KSCN; 0.1M BisTris propane pH 7.5; 20.0% PEG 3350; 10.0% ethylene glycol. Crystals appeared after a couple of days at 4°C.

Data Collection: Crystals were cryo-protected using the well solution supplemented with an additional 15% ethylene glycol and flash frozen in liquid nitrogen. All diffraction data were collected at the SLS beam line X10 (λ=0.979 Å).

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.