Human CDC2-Like Kinase 3

Target ID:

CLK3A

Aliases:

FLJ22858PHCLK3PHCLK3/152

Deposition Date:

Friday 28th October 2005

Families:

Protein Kinase

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2EU9

Authors:

E.PAPAGRIGORIOU,P.RELLOS,S.DAS,E.UGOCHUKWU,A.TURNBULL,F.VONDELFT,G.BUNKOCZI,F.SOBOTT,A.BULLOCK,O.FEDOROV,C.GILEADI,P.SAVITSKY,A.EDWARDS,C.AERROWSMITH,J.WEIGEL,M.SUNDSTROM,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2EU9

tabs

Structure Details

The Clk family consists in human of four kinases that display so-called dual specificity, i.e., they are capable of phosphorylating both Ser/Thr as well as Tyr residues. CLK homologues have been isolated from a number of organisms including Arabidopsis, Drosophila, mouse and human. The kinase domain of CLK family members is located at the C terminus and contains the family signature motif â€˜EHLAMMERILG', giving rise to the name of these kinases: â€˜LAMMER' kinases.

It has been shown that CLK family members play a pivotal role controlling splicing by regulating members of the SR family of splicing factors. Both CLK3 and CLK1 have been shown to tightly associate with splicing factors and to phosphorylate them at multiple sites. It is therefore not surprising that inactivating mutation in CLK results in drastic phenotypes. In Drosophila mutation in the Lammer kinase DOA (darkener of apricot) alter sexual differentiation and activity of DOA is required for development of embryonic tissues. In addition, non-functional DOA leads to defects in body segmentation, eye formation, and neuronal development.

High levels of CLK3 expression are found in mature spermatozoa where CLK3 is localized in the spermatozoa acrosome and tail. Interestingly CLK3 is released from sperm during the acrosome reaction suggesting that CLK3 may play role in the fertilization process.

Furthermore it has been shown that expression of all CLK family members is up-regulated during hexamethylene bisacetamide induced murine erythroleukemia cell differentiation and a potential therapeutic use of CLK3 has been suggested for the treatment of frontotemporal dementia and parkinsonism linked to chromosome 17.

See other kinases.

Materials and Methods

CLK3

PDB:2EU9

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_003983
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) . TEV-cleavable (*) N-terminal hexaHis tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqSMQSSKRSSRSVEDDKEGHLVCRIGDWLQERYEIVGNLGEGTFGKVVECLDHARGKSQVALKIIRNVGKYREAARLEINVLKKIKEKDKENKFLCVLMSDWFNFHGHMCIAFELLGKNTFEFLKENNFQPYPLPHVRHMAYQLCHALRFLHENQLTHTDLKPENILFVNSEFETLYNEHKSCEEKSVKNTSIRVADFGSATFDHEHHTTIVATRHYRPPEVILELGWAQPCDVWSIGCILFEYYRGFTLFQTHENREHLVMMEKILGPIPSHMIHRTRKQKYFYKGGLVWDENSSDGRYVKENCKPLKSYMLQDSLEHVQLFDLMRRMLEFDPAQRITLAEALLHPFFAGLTPEERSFHT
Vector:pLIC- SGC 1

Growth

Medium:
Antibiotics:
Procedure:1 ml from a 10 ml overnight culture in LB, 100 µg/ml ampicillin was used to inoculate 1 litre of LB medium containing 100 µg/ml ampicillin. Cultures were grown at 37°C until they reached an OD600 of 0.3 and then cooled to 18°C. Expression was induced for 4 hours using 1 mM IPTG at an OD600 of 0.6. The cells were collected by centrifugation, transferred to 50 ml tubes, resuspended in 30 ml binding buffer, and frozen. Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu.

Purification

Procedure
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample.

Buffers: Binding buffer: 50 mM HEPES, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu.

Procedure: Supernatant was applied at gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.

Column 2: Ni-affinity. Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Buffers : Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% glycerol, 50 mM L-Arg and L-Glu. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol, 50 mM L-Arg and L-Glu.

Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 3 x 10 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted. After elution DTT was added to a final concentration of 10 mM.

Enzymatic treatment : (Dephosphorylation and His tag cleavage) Samples containing CLK3 were pooled and 20 µg GST-lambda phosphatase and 20 µg TEV protease added for overnight incubation at 4°C: protein solution contained 10 mM DTT and 0.05 mM MnCl 2

Column 3: Size Exclusion Chromatography

Buffers: Fractions containing CLK3 collected from IMAC were concentrated and directly applied to a S75 16/60 HiLoad gel filtration column equilibrated in 50 mM Hepes pH 7.5, 500 mM NaCl, 50 mM L-glutamic acid, 50 mM L-arginine

Procedure : AKTA-prime

Column 4: Anion Exchange Chromatography

Buffers: Fractions containing CLK3 collected from SEC were diluted to a final concentration of 50 mM HEPES pH 7.5, 50 mM NaCl and applied to a MonoQ 5/50 GL equilibrated in 50 mM Hepes pH 7.5, 50 mM NaCl. The potein was eluted using an NaCl gradient

Procedure : AKTA-express

Extraction

Procedure
The frozen cells were thawed on ice and binding buffer (plus 1 mM PMSF) added to a final volume of 50 ml. Cells were lysed using a high pressure cell disruptor. The lysate was centrifuged at 18,500 RPM for 50 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown at 4°C in 150nl sitting drops mixing 75 nl of CLK3 11mg/ml in 50mM Hepes pH 7.5, 200mM NaCl,10mM DTT, with 75 nl of a solution containing 27%PEG 3350, 30 mM Ammonium acetate, 100 mM Bis Tris pH 5.5
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Gracia-Sacristan, A., Fernandez-Nestosa, M.J., Hernandez, P., Schartzman, J.B., Krimer, DB (2005). Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias towards the skipped splice variant characterizes postcommitment stages. Cell Research 15 :495-503.

Menegay, H., Moeslein, F., Landreth, G. (1999). The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa. Experimental Cell Research 253 :463-473.

Howell BW, Afar DE, Lew J, Douville EM, Icely PL, Gray DA, Bell JC. (1991). STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases. Mol Cell Biol. 11 :568-572.

Myers MP, Murphy MB , Landreth G. (1994). The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells. Mol Cell Biol. 14 :6954-6961.

Colwill K, Pawson T, Andrews B, Prasad J, Manley JL, Bell JC, Duncan PI. (1996). The Clk/Sty protein kinase phosphorylates SR splicing factors and regulates their intranuclear distribution. EMBO J . 15 :265-275.

Hartmann AM, Rujescu D, Giannakouros T, Nikolakaki E, Goedert M, Mandelkow EM, Gao QS, Andreadis A, Stamm S. Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors. (2001). Mol Cell Neurosci. 18 :80-90.

Yun B, Farkas R, Lee K, Rabinow L. (1994). The Doa locus encodes a member of a new protein kinase family and is essential for eye and embryonic development in Drosophila melanogaster. Genes Dev. 8 :1160-1073.