Human Mitogen Activated Protein 3 Kinase

Target ID:

MAP3K5A

Aliases:

ASK1MAPKKK5MEKK5

Deposition Date:

Friday 28th April 2006

Families:

Protein Kinase

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2CLQ

Authors:

G.BUNKOCZI,E.SALAH,O.FEDOROV,A.PIKE,O.GILEADI,F.VON DELFT,C.ARROWSMITH,A.EDWARDS,M.SUNDSTROM,J.WEIGELT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2CLQ

tabs

Structure Details

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAP3K5) which plays an essential role in stress and immune response.
ASK1 is activated by various stimuli including liposaccharides (LPS), oxidative stress (ROS), ER stress, influx of calcium ions and various cytokines such as tumor necrosis factor and Fas ligands.
Signalling through the TRAF6â€“ASK1â€“p38 pathway has been shown to be essential for inflammatory response upon LPS stimulation.
This role has been convincingly demonstrated by ASK1 -/- mice which show no macroscopic or microscopic phenotype but are resistant to LPS induced septic shock.

Human ASK1 is a protein of 1374 amino acids with a central serine/threonine kinase domain.
Over-expression of wild-type or constitutively active ASK1 results in activation of the kinases MKK3/MKK6 and MKK4/MKK7 and in induction of apoptosis.
However, the downstream events of ASK1 are variable depending on the cell types and cellular context and may lead also to cell differentiation, neurite outgrowth and induction of cytokines. In its inactive form ASK1 forms a homo-oligomer through its C-terminal coiled-coil domain. An ubiquitously expressed reduction/oxidation (redox) protein thioredoxin binds to the N-terminal of ASK1 and inhibits its activity. Upon oxidative stress stimulation formation of an intramolecular disulfide bridge leads to dissociation of that complex and ASK1 subsequent activation. Apart from it role in apoptosis ASK1 is an essential regulator of TLR-4 (Toll like receptor) mediated innate immunity.

Furthermore, ASK1 plays also an important role in polyglutamine (polyQ) diseases known to be the cause of at least nine inherited neurodegenerative disorders, including Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and six spinocerebellar ataxias as well as SCA3/Machado-Joseph disease [MJD]). PolyQ fragments accumulate as aggregates in the cytoplasm and/or nucleus and induce stress leading to neuronal cell death. ASK1 is also a potential therapeutic target for malignant fibrous histiocytomas, an particularly malignant form of undifferentiated liposarcomas.

Materials and Methods

MAP3K5

PDB:2CLQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|5174547
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:Final protein sequence: mhhhhhhssgvdlgtenlyfqsmRSTEEGDCESDLLEYDYEYDENGDRVVLGKGTYGIVYAGRDLSNQVRIAIKEIPERDSRYSQPLHEEIALHKHLKHKNIVQYLGSFSENGFIKIFMEQVPGGSLSALLRSKWGPLKDNEQTIGFYTKQILEGLKYLHDNQIVHRDIKGDNVLINTYSGVLKISDFGTSKRLAGINPCTETFTGTLQYMAPEIIDKGPRGYGKAADIWSLGCTIIEMATGKPPFYELGEPQAAMFKVGMFKVHPEIPESMSAEAKAFILKCFEPDPDKRACANDLLVDEFLKVSSKKKKTQPKL
Vector:pLIC-SGC1

Growth

Medium:
Antibiotics:
Procedure:1 ml from a 10 ml overnight culture containing 100 µg/ml kanamycin was used to inoculate 1 liter of TB media containing 100 µg/ml kanamycin. Cultures were grown at 37°C until the OD600 reached ~2.0. After that the temperature was adjusted to 25°C. Expression was induced for 4 hours using 1mM IPTG. 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.
The column was equilibrated with binding buffer. The lysate was applied to the column which was subsequently washed with wash buffer 1. MAP3K5 was eluted with elution buffer. The eluted protein was collected and analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM. 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 ml 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. MAP3K5 eluted at a retention time corresponding to the monomeric protein. 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 using 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 13 mg/ml containing and 1 mM of staurosporine in SEC buffer. 100nl of the concentrated protein were mixed with 100nl of a well solution containing 80% MPD, 100 mM Hepes pH 7.8
NMR Spectroscopy:
Data Collection:X-ray source: Swiss Light Source beamline BL10, single wavelength.
Data Processing:

References

Harada, C., Nakamura, K., Namekata, K., Okumura, A., Mitamura, Y., Iizuka, Y., Kashiwagi, K., Yoshida, K., Ohno, S., Matsuzawa, A. , et al. (2006). Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo. Am J Pathol 168 , 261-269.

Hayakawa, T., Matsuzawa, A., Noguchi, T., Takeda, K., and Ichijo, H. (2006). The ASK1-MAP kinase pathways in immune and stress responses. Microbes Infect.

Kutuzov, M. A., Andreeva, A. V., and Voyno-Yasenetskaya, T. A. (2005). Regulation of apoptosis signal-regulating kinase 1 (ASK1) by polyamine levels via protein phosphatase 5. J Biol Chem 280 , 25388-25395.

Masutani, H., Ueda, S., and Yodoi, J. (2005). The thioredoxin system in retroviral infection and apoptosis. Cell Death Differ 12 Suppl 1 , 991-998.

Matsuzawa, A., Saegusa, K., Noguchi, T., Sadamitsu, C., Nishitoh, H., Nagai, S., Koyasu, S., Matsumoto, K., Takeda, K., and Ichijo, H. (2005). ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol 6 , 587-592.

Sekine, Y., Takeda, K., and Ichijo, H. (2006). The ASK1-MAP Kinase Signaling in ER Stress and Neurodege-nerative Diseases. Curr Mol Med 6 , 87-97.

Sumbayev, V. V., and Yasinska, I. M. (2005). Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species. Arch Biochem Biophys 436 , 406-412.

Takeda, K., Matsuzawa, A., Nishitoh, H., and Ichijo, H. (2003). Roles of MAPKKK ASK1 in stress-induced cell death. Cell Struct Funct 28 , 23-29.