Human Death Associated Kinase 3

Target ID:

DAPK3A

Aliases:

DAPK3FLJ36473ZIPZIPK

Deposition Date:

Tuesday 31st October 2006

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2J90

Authors:

A.P.TURNBULL,G.BERRIDGE,O.FEDOROV,A.C.W.PIKE,P.SAVITSKY,J.ESWARAN,E.PAPAGRIGORIOU,E.UGOCHUKWA,F.VON DELFT,O.GILEADI,C.H.ARROWSMITH,A.EDWARDS,J.WEIGELT,M.SUNDSTROM,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2J90

tabs

Structure Details

The death-associated protein (DAP) kinase family includes three protein kinases, DAP kinase, DAP kinase-related protein 1, and DAPK3 (ZIPK), also called dual leucine zipper kinase . Members of this family mediate cell death initiated by various external and internal signals, including tumor necrosis factor alpha (TNF-α), Fas, transforming growth factor β and detachment from the extracellular matrix. One common feature of DAPK induced death is membrane blebbing, which has been attributed to increased phosphorylation of the regulatory light chain of myosin II (MLC). Sequence alignment of the catalytic domains showed that DAPKs share a unique stretch of basic amino acids at the N-terminus unique to these kinases referred to as the "fingerprint" of the DAPK family.

DAPK3 kinase localizes to both the nucleus and the cytoplasm and fractionates as monomeric and trimeric forms. Significantly, modification of the DAP kinase phosphorylation sites influences both the localization and oligomerization status of ZIP kinase. DAPKs are Ca2+/calmodulin (CaM)-regulated kinases associated with actin microfilaments. However, DAPK3 lacks the CaM regulatory domain, suggesting different modes of activation. In addition to the N-terminal catalytic domain, DAPK3 contains a leucine zipper structure at its C terminus which is believed to mediate homodimerization.

DAPK3 may be present in the cytoplasm as well as in the nucleus. Similar to other DAPKs, cytoplasmic DAPK3 has death-inducing properties, including phosphorylation of MLC and induction of membrane blebbing. L ocalization of DAPK3 is regulated during the cell cycle and DAPK3 has been reported to be associated with centrosomes, centromeres and contractile ring during prophase and early metaphase. In the nucleus DAPK3 has been shown to bind to the ATF4 transcription factor and it phosphorylates centromere-specific histones during mitosis.

Interestingly DAPK3 has also been identified as a regulator of Ca2+ sensitization in smooth muscle cells by controlling the phosphorylation of myosin phosphatase (SMPP-1M) and the inhibitor protein CPI17. Phosphorylation of SMPP-1M and CPI17 by DAPK3 inhibits phosphatase activity towards myosin and causes profound Ca2+ sensitization and contraction in smooth muscle. DAPK3 also directly phosphorylates both muscle and non-muscle myosin. The highly selective actions of DAPK3 in the control of myosin phosphorylation makes this enzyme an interesting candidate for the development of novel therapeutics to treat smooth muscle related disorders such as hypertension or asthma.

We determined the structure (2J90) of DAPK3 in complex with the inhibitor Pyridone 6 (2-(1,1-Dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one). A potent cell-permeable, and ATP-competitive inhibitor that has been described to inhibit Janus protein tyrosine kinases (JAKs) and the related receptor tyrosine kinase Tyk2.

In addition, we determined the structure of unphosphorylated DAPK3 in complex with a beta-carboline (3BHY) and an imidazo pyridazine (3BQR) inhibitor, respectively.

Materials and Methods

DAPK3: Human Death Associated Kinase 3 (zipper interacting protein kinase - ZIPK)

PDB:2J90

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4557511
Entry Clone Source:FivePrime
SGC Clone Accession:
Tag:N-terminal his6 tag with TEV cleavage site.
Host:BL21 (DE3) R3 (Phage resistant strain)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq^smVEDHY EMGEELGSGQFAIVRKCRQKGTGKEYAAK FIKKRRLSSSRRGVSREEIEREVNILREI RHPNIITLHDIFENKTDVVLILELVSGGE LFDFLAEKESLTEDEATQFLKQILDGVHY LHSKRIAHFDLKPENIMLLDKNVPNPRIK LIDFGIAHKIEAGNEFKNIFGTPEFVAPE IVNYEPLGLEADMWSIGVITYILLSGASP FLGETKQETLTNISAVNYDFDEEYFSNTS ELAKDFIRRLLVKDPKRRMTIAQSLEHSW IKAIRRRNVRGEDSG
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 20 ml overnight was used to inoculate 1 l of TB medium containing 50µg/ml Kanamycine . E .coli cells were grown in 2.5-L baffled flasks at 37°C until OD reached 2.0. The cells were cooled to 25°C and expression of DAPK3 was induced adding 0.5 mM IPTG at an OD600 of 2.2

Purification

Procedure
Column 1 : : Ni-affinity, HisTrap, 1 ml (GE/Amersham Biosciences )
Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gelfiltration, Hiload 16/60 Superdex 200 prep grade, 120 ml (GE/ Amersham Bioscience)
Procedure: The eluted fractions from the Ni-affinity Histrap column were loaded on the gel filtration column at 0.80 ml/min. Eluted proteins were collected in 2 ml fractions.

Concentration : 5 mM DTT was added to the diluted sample before concentrating the protein in Amicon (30 K) to 10.3 mg/ml. The protein concentration was determined spectrophotometrically using the calculated molar extinction coefficient.

Extraction

Procedure
Frozen cell pellets were thawed at 37°C and re-suspended in a total volume of 100 ml lysis buffer. The cells were disrupted by a high pressure cell disrupter (20 kpsi). Nucleic acids and cell debris were removed by adding 0.15% PEI , followed by centrifugation for 30 minutes at 40 000xg. The supernatant was further clarified by filtration (0.2 µm).
Concentration:
Ligand
MassSpec:The mass of the recombinant protein was in agreement with the mass calculated from the expected sequence.
Crystallization:Crystals were grown by vapor diffusion at 4°C from a sitting drop consisting of 150 nl protein (10.3 mg/ml)containing 1 mM Pyridone 6 (2-(1,1-Dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one) and 50 nl well solution. The drop was equilibrated against well solution containing 30 % PEG1000, 0.1M SPG buffer, pH8.0. The crystal was transferred to a cryo-protectant composed of 20% ethylene glycol before flash-cooling in liquid nitrogen .
NMR Spectroscopy:
Data Collection:Resolution: 2.0 Å diffraction data were collected at the Swiss light source (SLS) beam line BM10.
Data Processing:

References

An, J., Xu, Q. Z., Sui, J. L., Bai, B., and Zhou, P. K. (2005). Silencing of DNA-PKcs alters the transcriptional profile of certain signal transduction genes related to proliferation and differentiation in HeLa cells. Int J Mol Med 16 , 455-462.

Haystead, T. A. (2005). ZIP kinase, a key regulator of myosin protein phosphatase 1. Cell Signal 17 , 1313-1322.

Page, G., Kogel, D., Rangnekar, V., and Scheidtmann, K. H. (1999). Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis. Oncogene 18 , 7265-7273.

Shani, G., Marash, L., Gozuacik, D., Bialik, S., Teitelbaum, L., Shohat, G., and Kimchi, A. (2004). Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions. Mol Cell Biol 24 , 8611-8626.