Human Ca2+/calmodulin-dependent protein kinase I-delta

Target ID:

CAMK1DA

Aliases:

CaM-K1CaMKIDCKLiK

Deposition Date:

Wednesday 20th December 2006

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2JC6

Authors:

J.E.DEBRECZENI,P.RELLOS,O.FEDOROV,F.H.NIESEN,C.BHATIA,L.SHRESTHA,E.SALAH,C.SMEE,S.COLEBROOK,G.BERRIDGE,O.GILEADI,G.BUNKOCZI,E.UGOCHUKWU,A.C.W.PIKE,F.VON DELFT,S.KNAPP,M.SUNDSTROM,J.WEIGELT,C.H.ARROWSMITH,A.EDWARDS

Site:

Oxford

ICM Datapack:

ICM Datapack

2JC6

tabs

Structure Details

The Ser/Thr kinases Ca2+/calmodulin-dependent protein kinase I (CaMKI) constitutes a family of closely related isoforms (alpha, beta, delta and gamma). CAMK1D, also known as CKLIK is expressed in polymorphonuclear leukocytes (PMNs) and may be part of the chemokine signal transduction pathway that regulates granulocyte function. CAMK1D is enzymatically active and its activity is enhanced by Ca2+ and calmodulin. Expression of this kinase is increases during differentiation of CD34 positive cord blood stem cells toward a neutrophilic lineage and is maximal during terminal differentiation. In addition, CAMK1D activity is induced by IL8 in myeloid cell lines stably expressing this kinase. Transfection of COS cells with constitutively active CAMK1D showed that activation of this kinase results in downstream activation of ERK1 mitogen-activated protein kinase activity.

Here we present the structure of CaMK1D in complex with the ATP competitive inhibitor(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine. This inhibitor has been initially described as a potent GSK3 inhibitor with a Ki of 24 nM.

Materials and Methods

CAMK1D - Human Ca2+/calmodulin-dependent protein kinase I-delta

PDB:2JC6

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|9966875
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:Rosetta (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq^sMARENG ESSSSWKKQAEDIKKIFEFKETLGTGAFS EVVLAEEKATGKLFAVKCIPKKALKGKES SIENEIAVLRKIKHENIVALEDIYESPNH LYLVMQLVSGGELFDRIVEKGFYTEKDAS TLIRQVLDAVYYLHRMGIVHRDLKPENLL YYSQDEESKIMISDFGLSKMEGKGDVMST ACGTPGYVAPEVLAQKPYSKAVDCWSIGV IAYILLCGYPPFYDENDSKLFEQILKAEY EFDSPYWDDISDSAKDFIRNLMEKDPNKR YTCEQAARHPWIAGDTALNKNIHESVSAQ IRKNFAKSKWRQAFNATAVVRHMRKLHLG SSLDSSNA ^:cleavage site for TEV protease
Vector:pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Growth

Medium:
Antibiotics:
Procedure:3ml from a 50 ml overnight culture was used to inoculate each of two flasks containing 1 litre of LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. Cultures were grown at 37°C to an OD600 of ~0.4 and then cooled to 18°C. Expression was induced for 4 hours using 0.5mM IPTG at an OD 600 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; 5 mM imidazole.

Purification

Procedure

Extraction

Procedure
Cell pellets in binding buffer plus 1 mM PMSF, 0.5 mM TCEP were lysed using sonication. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification. Prior to purification the lysate was passed through a DE52 column (10g/L resin) to remove DNA.
Concentration:
Ligand
MassSpec:
Crystallization:The inhibitor (5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine was added to the protein in 3-fold excess and the complex concentrated to 10 mg/ml. Crystals were obtained in sitting drops using the vapor diffusion method by mixing 75nl of the concentrated protein with 75nl of a well solution containing 0.1M citrate pH 5.6; 20% isopropanol; 20% PEG 4K. Crystals appeared after several days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution and 15% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 at a single wavelength (0.99 nm).
Data Processing:

References

Kruidering, M., Schouten, T., Evan, G. I., and Vreugdenhil, E. (2001). Caspase-mediated cleavage of the Ca2+/calmodulin-dependent protein kinase-like kinase facilitates neuronal apoptosis. J Biol Chem 276, 38417-38425.
Pierce, A. C., ter Haar, E., Binch, H. M., Kay, D. P., Patel, S. R., and Li, P. (2005). CH...O and CH...N hydrogen bonds in ligand design: a novel quinazolin-4-ylthiazol-2-ylamine protein kinase inhibitor. J Med Chem 48, 1278-1281.
Verploegen, S., Lammers, J. W., Koenderman, L., and Coffer, P. J. (2000). Identification and characterization of CKLiK, a novel granulocyte Ca(++)/calmodulin-dependent kinase. Blood 96, 3215-3223.
Verploegen, S., Ulfman, L., van Deutekom, H. W., van Aalst, C., Honing, H., Lammers, J. W., Koenderman, L., and Coffer, P. J. (2005). Characterization of the role of CaMKI-like kinase (CKLiK) in human granulocyte function. Blood 106, 1076-1083.
Yamada, T., Suzuki, M., Satoh, H., Kihara-Negishi, F., Nakano, H., and Oikawa, T. (2004). Effects of PU.1-induced mouse calcium-calmodulin-dependent kinase I-like kinase (CKLiK) on apoptosis of murine erythroleukemia cells. Exp Cell Res 294, 39-50.