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CSNK1G2: Human Casein Kinase 1 Gamma 2 in complex with 5-Iodotubercidin

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PDB Code 2C47 Target Class Protein Kinase

Target CSNK1G2A
Alias CK1g2
Disease Area/Function signalling, metabolism
Date Deposited 2005-10-16
Authors G.BUNKOCZI, P.RELLOS, S.DAS, E.UGOCHUKWU, O.FEDOROV, F.SOBOTT, J.ESWARAN, A.AMOS, L.BALL, F.VON DELFT, A.BULLOCK, J.DEBRECZENI, A.TURNBULL, M.SUNDSTROM, J.WEIGELT, C.ARROWSMITH, A.EDWARDS, S.KNAPP

Struc Details Tabs

Structure Details
CKI represents a unique group of serine/ threonine protein kinases that is ubiquitously expressed in eukaryotic organisms. Seven mammalian CKI isoforms (á, â, ã1, ã2, ã3, ä and å) and various splice variants have been identified to date. CKI 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.

The characterization of the substrate specificity of CKI isoforms initially led to the identification of the canonical consensus sequence S/T(P)-X1-2-S/T, indicating that modification of serine or threonine residues by CKI requires the preceding phosphorylation of amino acid residues N-terminal of the target site. This requirement of a priming phosphorylation by another kinase restricted CKI to a function in the hierarchical phosphorylation of substrates; however, further studies revealed that a cluster of acidic amino acids N-terminal of the target serine/threonine and an acidic amino acid in position n-3 could substitute for the phosphoamino acid efficiently.
This non-canonical motif consisting of the sequence SLS has been shown to be recognized by CKI in combination with a cluster of acidic amino acid residues C-terminal of the phosphoacceptor site. The CKI substrates NF-AT and â-catenin exhibit such motifs, but phosphorylation of this non-canonical motif is 15–25 fold less efficient compared to the motif primed by a phosphoamino acid.

Recently, it was shown that rat CKI-ã2 interacts with the adaptor protein NCK via a proline- rich sequence in rat CKI-ã2 called the PXXP motifs (VHPKVPSQPPHR), which are not present in either CKI-ã1 or CKI-ã3. This interaction contributes to the substrate specificity of CKI-ã2 bringing the kinase in close proximity with its downstream targets. For example it was shown that upon PDGF stimulation, CKI-ã2 phosphorylates the PDGF receptor which also associates with LCK, leading to receptor inactivation.

CKI-ã2 may affect the development of brain and has been associated with vesicular trafficking and neurotransmitter release from small synaptic vesicles. In addition, 10 single nucleotide polymorphisms (SNPs) have been identified and were linked to occurrence of febrile seizures during early childhood.

See also

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.

Bioukar EB, Marricco NC, Zuo D, Larose L. (1999). Serine phosphorylation of the ligand-activated beta-platelet-derived growth factor receptor by casein kinase I-gamma2 inhibits the receptor's autophosphorylating activity. J Biol. Chem. 274, 21457-21463.

Lussier G, Larose L. (1997). A casein kinase I activity is constitutively associated with Nck. J Biol. Chem. 272, 2688-2694.

Materials & Methods
StructureCSNK1G2
PDB Code2C47
Entry clone accession gi|21314778
Entry clone source synthetic DNA
Tag mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Construct sequencemhhhhhhssgvdlgtenlyfqsmGPNFRVGKKIGCGNFGELRLGKNLYTNEYVAIKLEPIKSRAPQLHLEYRFYKQLSAT
EGVPQVYYFGPCGKYNAMVLELLGPSLEDLFDLCDRTFTLKTVLMIAIQLITRMEYVHTKSLIYRDVKPENFLVGRPGTK
RQHAIHIIDFGLAKEYIDPETKKHIPYREHKSLTGTARYMSINTHLGKEQSRRDDLEALGHMFMYFLRGSLPWQGLKADT
LKERYQKIGDTKRATPIEVLCENFPEEMATYLRYVRRLDFFEKPDYDYLRKLFTDLFDRS GFVFDYEYDWAGKPLPTPIG
TVHTDLPSQPQLRD
Vector pLIC- SGC1
Expression host BL21 (DE3)
Growth method 1mL from a 10 mL overnight culture containing 100 µg/mL ampiciline was used to inoculate 1 liter of LB media containing 100 µg/mL ampiciline. Cultures were grown at 37°C until the OD600 reached ~0.3. After that the temperature was adjusted to 18°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; 300 mM NaCl; 20 mM imidazole.
Extraction procedure Cell pellets were lysed using a high pressure cell disrupter. The lysate was centrifuged at 19,000 rpm for 60 minutes and the supernatant collected for purification.
Purification procedure Column 1: Ni-affinity chromatography

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 300mM NaCl,, 20 mM Imidazole. Wash buffer 1: 50 mM HEPES pH 7.5, 1M NaCl, 20mM Imidazole. Wash buffer 2: as for lysis buffer. Elution buffer: 50mM HEPES pH 7.5, 300mM NaCl, 150 mM Imidazole. 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. CSNK1G2 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 his6-tag was cleaved by incubating the protein overnight with TEV protease.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

SEC-Buffers: 50 mM Hepes, pH 7.5, 300 mM NaCl, 5 mM DTT. 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. CSNK1G2 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

Crystallization Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/mL containing 1 mM 5-Iodotubercidin by mixing 100nl of the concentrated protein with 100nl of a well solution containing 19% PEG10K, 0.2M magnesium sulfate, 0.1M cacodylate pH 7.3. Crystals appeared after 3 days at 4°C.
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