Human casein kinase gamma 1

Target ID:

CSNK1G1A

Deposition Date:

Monday 15th May 2006

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2CMW

Authors:

G.BUNKOCZI,P.RELLOS,S.DAS,P.SAVITSKY,F.NIESEN,F.SOBOTT,O.FEDOROV,A.C.W.PIKE,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2CMW

tabs

Structure Details

CK1 represents a unique group of serine/threonine protein kinases ubiquitously expressed in eukaryotic organisms. Seven mammalian CK1 isoforms (α, β, γ1, γ2, γ3, δ and ε) and various splice variants have been identified to date. CK1 family members contain a highly conserved N-terminal catalytic domain coupled to a variable C-terminal region that ranges in size from 40 to 180 amino acids. Casein kinases have been described to act as monomeric, constitutively active enzymes. In the CK1 family the Asp-Pro-Glu motif of domain VIII, which is common to most Ser/Thr kinases is replaced by Ser-Ile-Asn. CK1-γ1 is the most abundant serine/threonine kinase in eukaryotic cell extracts. Several splice variants exist from which two splice variants have been described in more detail: CK1-γ1S encoding 393 amino acids and CK1-γ1L encoding 422 amino acids. CK1-γ1L has a characteristic sequence of 50 amino acids at the C-terminal end and this motif was shown to be shared by the CK1-γ2 and γ3 from rat and human, suggesting that it is a signature sequence of the gamma-isoforms.

CK1-γ1 is widely expressed with high RNA levels detected in liver, skeletal muscle, heart and kidney. The short splice variant is mainly detected in testis.

Mutations and deregulation of CK1 expression and activity has been linked to various diseases including neurodegenerative disorders such as Alzheimerâ€™s and Parkinsonâ€™s disease, sleeping disorders and proliferative diseases such as cancer.

We determined the structure of CK1-γ1 in complex with an inhibitor of the purine class, a potent, cell-permeable, and selective inhibitor of the cell cycle-regulating kinase, Cdc28p (IC50 = 7 μM), and the related Pho85p kinase (IC50 = 2 μM).

Materials and Methods

CSNK1G1A: Human casein kinase gamma 1

PDB:2CMW

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:
Tag:
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmRVGKKIGCGNFGELRLGKNLYTNEYVAIKLEPIKSRAPQLHLEYRFYKQLGSAGEGLPQVYYFGPCGKYNAMVLELLGPSLEDLFDLCDRTFTLKTVLMIAIQLLSRMEYVHSKNLIYRDVKPENFLIGRQGNKKEHVIHIIDFGLAKEYIDPETKKHIPYREHKSLTGTARYMSINTHLGKEQSRRDDLEALGHMFMYFLRGSLPWQGLKADTLKERYQKIGDTKRNTPIEALCENFPEEMATYLRYVRRLDFFEKPDYEYLRTLFTDLFEKKGYTFDYAYDWVGRPIPTPVGSVHVDSGASAITRE
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1 ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 liter of LB media containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD 600 reached ~2.0. After that the temperature was adjusted to 20°C. Expression was induced for 4 hours using 1mM IPTG. The cells were collected by centrifugation and the pellet was frozen for until processed.

Purification

Procedure
Column 1: Ni-affinity chromatography.
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. CSNK1G1 was eluted with 25 mls of 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)
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. CSNK1G1 eluted at 65 minutes corresponding to a retention time of a monomeric protein of that size. Eluted fractions were 95% pure as judged by SDS-PAGE.

Protein concentration: Centricon with a 10kDa cut off in SEC-buffer

Extraction

Procedure
25 ml of 50 mM HEPES pH 7.5, 300 mM NaCl, 5 mM NaPO4, 20 mM Imidazole. The cells were lysed by sonication for 3 min (40% max, 20 sec on 1 m in off).
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml containing 1 mM 2-(2-Hydroxyethylamino)-6-(3-chloroanilino)-9-isopropylpurine by mixing 100nl of the concentrated protein with 100nl of a well solution containing 12% PEG10K, 0.05M ammonium acetate, 0.1M bis tris pH 5.5. Crystals appeared after 3 days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution and 20% PEG400 and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 at a single wavelength (0.9765 Å).
Data Processing:

References

Knippschild U, Gocht A, Wolff S, Huber N, Lohler J, Stoter M. (2005) The casein kinase 1 family: participation in multiple cellular processes in eukaryotes. Cell Signal 17(6), 675-689.
Knippschild U, Wolff S, Giamas G, Brockschmidt C, Wittau M, Wurl PU, Eismann T, Stoter M. The role of the casein kinase 1 (CK1) family in different signaling pathways linked to cancer development. Onkologie, 10, 508-514.
Gray NS, Wodicka L, Thunnissen AM, Norman TC, Kwon S, Espinoza FH, Morgan DO, Barnes G, LeClerc S, Meijer L, Kim SH, Lockhart DJ, Schultz PG. (1998). Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 281:533-538.