Human mitogen-activated protein kinase kinase kinase 10 (SH3 domain)

Target ID:

MAP3K10A

Aliases:

MAP3K10MLK2MST

Deposition Date:

Thursday 27th September 2007

Families:

Protein KinaseLinkers

Structure type:

apo

Download Coordinates:

2RF0

Authors:

Ugochukwu, E., Eswaran, J., Elkins, J., Keates, T., Pike, A. C. W., Berridge, G., Savitsky, P., Sundstrom, M., Arrowsmith, C. H., Weigelt, J., Edwards, A., von Delft, F., Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

2RF0

tabs

Structure Details

The mixed lineage kinase MAP3K10 belongs to the TKL ( Tyrosine Kinase Like) family of serine threonine kinases. The full-length protein consists of an N-terminal SH3 domain, a kinase catalytic domain, a double leucine zipper motif and a Rac/Cdc42 GTPase-binding (CRIB) motif.

MAP3K10 has been linked to a number of diverse cellular functions including a proposed role in neuronal apoptosis, in enhancement of trans-repression of alien and nuclear receptors, a function in vesicle formation and in endosome recycling by binding to clathrin. In addition, MAP3K10 has been suggested to be involved in neuronal toxicity induced by polyglutamine-expanded huntingtin as observed in Huntington disease and other neurodegenerative diseases.

Here we determined the structure of the SH3 domain of MAP3K10. This protein interaction domain has been shown to play a role in the auto-regulation of MAP3K10. SH3-defective mutants of the closely related protein MLK3 exhibited increased catalytic activity compared with wild type MLK3 suggesting that the SH3 domain negatively regulates MLK3 activity by interacting with the Cdc42/Rac binding motif in the C-terminus of mixed linage kinases.

Materials and Methods

MAP3K10

PDB:2RF0

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_002446 Variant
Entry Clone Source:Origene
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable ( * ) N-terminal his6 tag.
Host:BL21 (DE3) phage resistant Rosetta strain

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smGTTPAGPVWTAVFDYEAAGDEELTLRRGDRVQVLSQDCAVSGDEGWWTGQLPSGRVGVFPSNYVAP
Vector:pNIC28-Bsa4

Growth

Medium:1ml from a 10 ml overnight culture 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; 500 mM NaCl; 5% glycerol, 20 mM imidazole.
Antibiotics:
Procedure:

Purification

Procedure
Column 1: Ni-affinity chromatography.Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 5% Glycero, 20mM Imidazolel. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 30 mM Imidazole , 5% glycerol. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl , 50 to 250 mM imidazole, 5% Glycerol .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. The protein was eluted by gravity flow by applying 5 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150mM, 250 mM); fractions were collected until essentially all protein was eluted. The eluted protein was analyzed by SDS - PAGE. DTT was added to the protein sample to a final concentration of 10mM.Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)SEC-Buffers: 50 mM Hepes, pH 7.5, 150 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.

Extraction

Procedure
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.
Concentration:Centricon with a 10kDa cut off in SEC-buffer.
Ligand
MassSpec:The mass of the protein determined by ESI -MS-tof corresponded to the theoretical mass of Dalton 9724.5 calculated for the protein before TEV cleavage.
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 20 mg/ml by mixing 150nl of the concentrated protein with 50nl of a well solution containing: 0.20M Na(malonate); 0.1M BTProp pH 8.5; 20.0% PEG 3350 and 10.0% EtGly in the presence of 1 mM of the peptide: \"DRERPHVRRRRG\".
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution supplemented with an additional 20% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line SAX10 to 2.0 Å resolution.
Data Processing:

References

Liu, Y.F., Dorow, D., and Marshall, J. (2000). Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin. The Journal of biological chemistry 275 , 19035-19040.

Maroney, A.C., Finn, J.P., Connors, T.J., Durkin, J.T., Angeles, T., Gessner, G., Xu, Z., Meyer, S.L., Savage, M.J., Greene, L.A. , et al. (2001). Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family. The Journal of biological chemistry 276 , 25302-25308.

Poitras, L., Jean, S., Islam, N., and Moss, T. (2003). PAK interacts with NCK and MLK2 to regulate the activation of jun N-terminal kinase. FEBS letters 543 , 129-135.

Xu, Z., Maroney, A.C., Dobrzanski, P., Kukekov, N.V., and Greene, L.A. (2001). The MLK family mediates c-Jun N-terminal kinase activation in neuronal apoptosis. Molecular and cellular biology 21 , 4713-4724.

Zhang, H., and Gallo, K.A. (2001). Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain. The Journal of biological chemistry 276 , 45598-45603.