Human cdc-2 like kinase 3 in complex with V25

Target ID:

CLK3A

Aliases:

FLJ22858PHCLK3PHCLK3/152

Deposition Date:

Wednesday 30th September 2009

Families:

Protein Kinase

Related Diseases:

SignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2WU7

Authors:

Muniz, J.R.C., Fedorov, O., King, O., Filippakopoulos, P., Bullock, A., Heightman, T., Ugochukwu, E., von Delft, F., Arrowsmith, C.H., Bracher, F., Huber, K., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

2WU7

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 \'EHLAMMERILG\', giving rise to the name of these kinases: \'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. Here we describe two structures of CLK3 in complex with ATP mimetic inhibitors that can be used as chemical probes to study the function of CLK kinases in a cellular environment.

Figure 1: Structures of the two ATP competitive inhibitors that were co-crystallized with CLK3. The SGC would like to thank the laboratory of Prof. F. Bracher and Dr. Kilian Huber (Ludwig-Maximilians-Universität München) for providing us with the dichloroindole inhibitor (right panel).

Materials and Methods

Materials and Method
Note: To our best knowledge, this should represent an accurate description of the materials and methods required to reproduce our work. If any of the content on this page is difficult to interpret or should you have trouble repeating our work, do not hesitate to contact us as soon as possible in order for us to provide additional information and advice.

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:5114635

SGC Construct ID: CLK3A-c005

GenBank GI number: gi|4502885

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
TACTTCCAATCCATGCAGAGCAGTAAGCGC
AGCAGCCGGAGTGTGGAAGATGACAAGGAG
GGTCACCTGGTGTGCCGGATCGGCGATTGG
CTCCAAGAGCGATATGAGATTGTGGGGAAC
CTGGGTGAAGGCACCTTTGGCAAGGTGGTG
GAGTGCTTGGACCATGCCAGAGGGAAGTCT
CAGGTTGCCCTGAAGATCATCCGCAACGTG
GGCAAGTACCGGGAGGCTGCCCGGCTAGAA
ATCAACGTGCTCAAAAAAATCAAGGAGAAG
GACAAAGAAAACAAGTTCCTGTGTGTCTTG
ATGTCTGACTGGTTCAACTTCCACGGTCAC
ATGTGCATCGCCTTTGAGCTCCTGGGCAAG
AACACCTTTGAGTTCCTGAAGGAGAATAAC
TTCCAGCCTTACCCCCTACCACATGTCCGG
CACATGGCCTACCAGCTCTGCCACGCCCTT
AGATTTCTGCATGAGAATCAGCTGACCCAT
ACAGACTTGAAACCAGAGAACATCCTGTTT
GTGAATTCTGAGTTTGAAACCCTCTACAAT
GAGCACAAGAGCTGTGAGGAGAAGTCAGTG
AAGAACACCAGCATCCGAGTGGCTGACTTT
GGCAGTGCCACATTTGACCATGAGCACCAC
ACCACCATTGTGGCCACCCGTCACTATCGC
CCGCCTGAGGTGATCCTTGAGCTGGGCTGG
GCACAGCCCTGTGACGTCTGGAGCATTGGC
TGCATTCTCTTTGAGTACTACCGGGGCTTC
ACACTCTTCCAGACCCACGAAAACCGAGAG
CACCTGGTGATGATGGAGAAGATCCTAGGG
CCCATCCCATCACACATGATCCACCGTACC
AGGAAGCAGAAATATTTCTACAAAGGGGGC
CTAGTTTGGGATGAGAACAGCTCTGACGGC
CGGTATGTGAAGGAGAACTGCAAACCTCTG
AAGAGTTACATGCTCCAAGACTCCCTGGAG
CACGTGCAGCTGTTTGACCTGATGAGGAGG
ATGTTAGAATTTGACCCTGCCCAGCGCATC
ACACTGGCCGAGGCCCTGCTGCACCCCTTC
TTTGCTGGCTTGACCCCTGAGGAGCGGTCC
TTCCACACCTAAGACAGTAAAGGTGGATA

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqSMQSSKRSS
RSVEDDKEGHLVCRIGDWLQERYEIVGNLG
EGTFGKVVECLDHARGKSQVALKIIRNVGK
YREAARLEINVLKKIKEKDKENKFLCVLMS
DWFNFHGHMCIAFELLGKNTFEFLKENNFQ
PYPLPHVRHMAYQLCHALRFLHENQLTHTD
LKPENILFVNSEFETLYNEHKSCEEKSVKN
TSIRVADFGSATFDHEHHTTIVATRHYRPP
EVILELGWAQPCDVWSIGCILFEYYRGFTL
FQTHENREHLVMMEKILGPIPSHMIHRTRK
QKYFYKGGLVWDENSSDGRYVKENCKPLKS
YMLQDSLEHVQLFDLMRRMLEFDPAQRITL
AEALLHPFFAGLTPEERSFHT

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m). TEV-cleavable (*) N-terminal hexaHis tag.

Host: BL21 (DE3)

Growth medium, induction protocol: 1 ml from a 10 ml overnight culture in LB, 50 µg/ml kanamycin was used to inoculate 1 litre of LB medium containing 50 µg/ml kanamycin. 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.

Extraction buffer, extraction method: 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.

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.

Column 1 Buffers: Binding buffer: 50 mM HEPES, 500 mM NaCl, 5% Glycerol, 50 mM L-Arg and L-Glu.

Column 1 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.

Column 2 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.

Column 2 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 MnCl₂. For crystallization of phosphorylated CLK3 the protein was only treated with TEV protease

Column 3: Size Exclusion Chromatography

Column 3 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.

Column 3 Procedure: AKTA-prime

Column 4: Anion Exchange Chromatography

Column 4 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.

Column 4 Procedure: AKTA-express

Mass spec characterization: LC- ESI -MS TOF confirmed the correct mass expected for this construct.

Intact Mass: Masses of purified proteins were confirmed by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid.

Crystallization: Crystals were grown at 4°C in 150nl sitting drops mixing 75 nl of CLK3 11.4mg/ml in 50mM Hepes pH 7.5, 200mM NaCl,10mM DTT, with 75 nl of a solution containing 0.2M (NH4)₂SO₄; 0.1M BIS-TRIS pH 5.5; 25% PEG 3350. The inhibitors were added to the concentrated protein solution (1 mM end concentration) using a DMSO stock solution.

Data Collection: Resolution: X-ray source: Synchrotron SLS -X10, single wavelength (2wu6) and using Brucker rotating anode (Copper target) generator equipped with a CCD detector (2wu7), respectively. Crystals were cryoprotected using the crystallization solution supplemented with 25% ethylene glycol.

Note: To our best knowledge, this should represent an accurate description of the materials and methods required to reproduce our work. If any of the content on this page is difficult to interpret or should you have trouble repeating our work, do not hesitate to contact us as soon as possible in order for us to provide additional information and advice.

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.