Human MAP Kinase 6

Target ID:

MAPK6A

Aliases:

DKFZp686F03189ERK3HsT17250p97MAPKPRKM6

Deposition Date:

Tuesday 29th August 2006

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2I6L

Authors:

P.FILIPPAKOPOULOS,A.P.TURNBULL,P.SAVITSKY,O.FEDOROV,G.BERRIDGE,S.DAS,J.ELKINS,M.SOUNDARARAJAN,X.W.YANG,D.DOYLE,E.PAPAGRIGORIOU,A.C.W.PIKE,G.BUNKONCZI,E.UGOCHUKWU,J.DEBRECZENI,F.VON DELFT,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,S.KNAPP,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2I6L

tabs

Structure Details

MAPK6 is an atypical member of the ERK family. Within this subfamily, it is mostly similar to ERK1 and ERK2;
however, MAPK6 has several unusual features, including S-E-G as the phosphorylation motif instead of the T-E-Y motif found in ERK1 and ERK2.
As a consequence, activation of MAPK6 does not require dual specificity phosphorylation activity since the activation segment tyrosine is mimicked by a glutamate residue.
In addition, S-P-R replaces the A-P-E motif, a highly conserved motif found C-terminal to the activation segment in most kinases;
however, the precise function of MAPK6 has not been established yet but it is thought to be involved in regulation of cell cycle and inhibition of DNA synthesis as well as S-phase entry.

MAPK6 shuttles between nucleus and cytoplasm and this nucleo-cytoplasmic shuttling is necessary for its inhibitory effect on DNA synthesis. The MAPK6 protein is highly unstable due to two regions (NDR1 and NDR2) located in the N-terminal lobe that have been described to mediate proteasomal degradation. The half life of the protein is dependent on the differentiation state of the cell. MAPK6 can be phosphorylated by MEK2 on serine and threonine residues.

MAPK6 expression in the lung is restricted to the distal lung epithelium during the pseudoglandular phase (E17) but shifts to the proximal airways, particularly Clara cells during the saccular stage (E21). Enhanced MAPK6 activity has been described in fibroblasts and upon treatment with serum or phorbol esters.

MAPK6 is over-expressed in several human cancers including oral squamous cell carcinoma , colon cancer, gastric cancer and high expression levels have been associated with tumor progression

Materials and Methods

MAPK6: Human MAP Kinase 6 (ERK3)

PDB:2I6L

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_002739
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfqSMNIHGFD LGSRYMDLKPLGCGGNGLVFSAVDNDCDK RVAIKKIVLTDPQSVKHALREIKIIRRLD HDNIVKVFEILGPSGSQLTDDVGSLTELN SVYIVQEYMETDLANVLEQGPLLEEHARL FMYQLLRGLKYIHSANVLHRDLKPANLFI NTEDLVLKIGDFGLARIMDPHYSHKGHLS EGLVTKWYRSPRLLLSPNNYTKAIDMWAA GCIFAEMLTGKTLFAGAHELEQMQLILES IPVVHEEDRQELLSVIPVYIRNDMTEPHK PLTQLLPGISREAVDFLEQILTFSPMDRL TAEEALSHPYMSIYSFPMDEPI
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Grow starter cultures from freshly transformed colonies in 10 ml LB, 50 mg/ml kanamycin. This started culture was diluted 1:1000 in fresh media and was grown at 37°C to a OD600 of 0.4 and than transferred to 18°C. Expression was induced at an OD600 of 0.6 - 0.7 using 1 mM IPTG (final concentration). Cells were harvested after 4h by centrifugation (15min, 6500rpm on a JLA 8.100 rotor), transferred to 50-ml tubes, and frozen at -20°C.

Purification

Procedure
Column 1 : DE52/Ni-NTA
Gravity feed chromatography: A DE52 column (10 g suspended in 100ml of 2.5M NaCl) was equilibrated with 100ml of Loading Buffer. A 5ml NiNTA column was equilibrated with 20ml of Loading Buffer. The lysed sample was applied to the DE-52 column and washed through with 50 ml loading buffer. The flow through was applied to the 5 ml Ni-NTA column which was washed with 2x10ml of wash buffer and eluted with elution buffer in 5 ml aliquots (Step elution using 50, 100, 150, 200 and 250 mM imidazole in the Elution Buffer)

Enzymatic treatment : Treated the IMAC elution(s) with TEV protease overnight.

Column 2 : SEC ( AKTA-prime)
Fractions containing MAPK6A collected from IMAC and treated with TEV protease overnight (identified by SDS PAGE ) were concentrated to about 1.5ml and directly applied to a S75 16/60 column equilibrated in 10 mM Hepes pH 7.5, 100 m NaCl. The flow rate was 1ml/min and the pure protein eluted at 55-70min.

Concentration : The combined samples from the SEC column (identified by SDS PAGE ) were concentrated using centricons with 10 kDa cut off.

Extraction

Procedure
The cell pellets (5 gr wet wt) were re-suspended in 50 ml extraction buffer containing a Protease Inhibitor Coctail tablet (Roche), and lysed in a high pressure cell disrupter. The supernatant was centrifuged for 45 minutes at 53k g in a JA 25.5 rotor
Concentration:
Ligand
MassSpec:LC- ESI -MStof confirmed the correct mass of 36237 expected for this construct after TEV treatment.
Crystallization:Crystals were grown at 4°C in 600nl sitting drops mixing 300 nl of MAPK6A (20 mg/ml in 10mM Hepes pH 7.5, 100mM NaCl ,10mM DTT) with 300 nl of a solution containing 1.8M MgSO4 and 0.1M MES pH 6.7. Cryo protection was achieved by adding to the crystallization mix Ethylene Glycole (20% final w/v).
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Bind, E., Kleyner, Y., Skowronska-Krawczyk, D., Bien, E., Dynlacht, B. D., and Sanchez, I. (2004). A novel mechanism for mitogen-activated protein kinase localization. Mol Biol Cell 15 , 4457-4466.