Human CLK2 (cdc2-like kinase 2)

Target ID:

CLK2A

Aliases:

CLK2hCLK2MGC61500

Deposition Date:

Wednesday 30th June 2010

Families:

Protein Kinase

Structure type:

protein-ligand

Download Coordinates:

3NR9

Authors:

Chaikuad, A., Savitsky, P., Krojer, T., Muniz, J.R.C., Filippakopoulos, P., Rellos, P., Keates, T., Fedorov, O., Pike, A.C.W., Eswaran, J., Berridge, G., Phillips, C., Zhang, Y., von Delft, F., Weigelt, J., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Knapp, S., Structural Genomics Consortium (SGC)

Site:

Oxford

3NR9

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 [1].

CLK2 is widely expressed and exists in two different isoforms: a longer isoform consisting of a non-conserved N-terminal domain and a highly conserved C-terminal kinase domain, and a shorter isoform lacking the kinase domain [1]. No knockout mice for CLK2 have been generated yet but inactivating mutation of the Lammer kinase DOA (darkener of apricot) in Drosophila alters 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 [2, 3].

CLK2 regulates the intranuclear distribution of SR proteins - serine/arginine-rich (SR) family of splicing factors - and influences pre-mRNA splicing [1, 4]. Recently, it has been shown that CLK2 expression is regulated by insulin via the insulin/Akt pathway and CLK2 itself suppresses hepatic gluconeogenesis and glucose output implicating CLK2 as regulator of hepatic insulin signaling and glucose metabolism [5]. Interestingly, the CLK2 gene is located in a region on chromosome1 that has been implicated to be associated with diabetes in humans although no direct link could be established [6].

CLK2 is potentially associated with several neurodegerative diseases. CLK2 regulates splicing of exon 10 of the microtubule-associated protein Tau. Inclusion of exon 10 is associated with frontotemporal dementia and parkinsonism. In affected areas of the brain of Alzheimer's patients the splicing patterns suggests misregulation of alternative splicing as one factor for sporadic Alzheimer's disease [7]. A fusion protein of PAFAH1B3 and CLK2 has been described in a female patient with mental retardation, ataxia and atrophy of the brain but the precise contribution of CLK2 is still unclear [8].

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:3837777

SGC Construct ID: CLK2A-c015

GenBank GI number: gi|4502883

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGCGCTCATCTTCGCACAGC
AGCCGGAGAGCCAAGAGTGTAGAGGACGAC
GCTGAGGGCCACCTCATCTACCACGTCGGG
GACTGGCTACAAGAGCGATATGAAATCGTT
AGCACCTTAGGAGAGGGGACCTTCGGCCGA
GTTGTACAATGTGTTGACCATCGCAGGGGT
GGGGCTCGAGTTGCCCTGAAGATCATTAAG
AATGTGGAGAAGTACAAGGAAGCAGCTCGA
CTTGAGATCAACGTGCTAGAGAAAATCAAT
GAGAAAGACCCTGACAACAAGAACCTCTGT
GTCCAGATGTTTGACTGGTTTGACTACCAT
GGCCACATGTGTATCTCCTTTGAGCTTCTG
GGCCTTAGCACCTTCGATTTCCTCAAAGAC
AACAACTACCTGCCCTACCCCATCCACCAA
GTGCGCCACATGGCCTTCCAGCTGTGCCAG
GCTGTCAAGTTCCTCCATGATAACAAGCTG
ACACATACAGACCTCAAGCCTGAAAATATT
CTGTTTGTGAATTCAGACTATGAGCTCACC
TACAACCTAGAGAAGAAGCGAGATGAGCGC
AGTGTGAAGAGCACAGCTGTGCGGGTGGTA
GACTTTGGCAGTGCCACCTTTGACCATGAG
CACCATAGCACCATTGTCTCCACTCGCCAT
TACCGAGCACCAGAAGTCATCCTTGAGTTG
GGCTGGTCACAGCCTTGTGATGTGTGGAGT
ATAGGCTGCATCATCTTTGAATACTATGTG
GGATTCACCCTCTTCCAGACCCATGACAAC
AGAGAGCATCTAGCCATGATGGAAAGGATC
TTGGGTCCTATCCCTTCCCGGATGATCCGA
AAGACAAGAAAGCAGAAATATTTTTACCGG
GGTCGCCTGGATTGGGATGAGAACACATCA
GCTGGGCGCTATGTTCGTGAGAACTGCAAA
CCGCTGCGGCGGTATCTGACCTCAGAGGCA
GAGGAACACCACCAGCTCTTCGATCTGATT
GAAAGCATGCTAGAGTATGAACCAGCTAAG
CGGCTGACCTTGGGTGAAGCCCTTCAGCAT
CCTTTCTTCGCCCGCCTTCGGGCTGAGCCG
CCCAACAAGTTGTGGGACTCCAGTCGGGAT
TGACAGTAAAGGTGGATACGGATCCGAA

Final protein sequence (tag sequence in lowercase)
mhhhhhhssgvdlgtenlyfqs^MRSSSHS
SRRAKSVEDDAEGHLIYHVGDWLQERYEIV
STLGEGTFGRVVQCVDHRRGGARVALKIIK
NVEKYKEAARLEINVLEKINEKDPDNKNLC
VQMFDWFDYHGHMCISFELLGLSTFDFLKD
NNYLPYPIHQVRHMAFQLCQAVKFLHDNKL
THTDLKPENILFVNSDYELTYNLEKKRDER
SVKSTAVRVVDFGSATFDHEHHSTIVSTRH
YRAPEVILELGWSQPCDVWSIGCIIFEYYV
GFTLFQTHDNREHLAMMERILGPIPSRMIR
KTRKQKYFYRGRLDWDENTSAGRYVRENCK
PLRRYLTSEAEEHHQLFDLIESMLEYEPAK
RLTLGEALQHPFFARLRAEPPNKLWDSSRD
^ TEV cleave site

Tags and additions: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Host: BL21(DE3)-R3-lambda-PPase

Growth medium, induction protocol: The expression plasmid was transformed into the host strain and plated on LB-agar containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol. Several colonies were combined to inoculate a 1-ml culture in TB (+ 50 µg/ml kanamycin, 35 µg/ml chloramphenicol). The culture was grown overnight, glycerol was added to 15% v/v (from a 60% stock), and the resulting glycerol stock was frozen at -80°C in 100 µl aliquots. A loopful of cells from the glycerol stock was inoculated into 5-ml of LB medium containing 100 µg/ml kanamycin and 35 µg/ml chloramphenicol and grown overnight at 37°C. Overnight culture was used to inoculate 1 liter of LB containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol. Culture was grown at 37 °C until the OD₆₀₀ reached ~3 and then cooled to 18°C for 1 hour. IPTG was added to 0.1 mM, and growth continued at 18°C overnight. The cells were collected by centrifugation then pellet re-suspended in 2x lysis buffer and frozen. Lysis buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, 1x Protease Inhibitors Cocktail Set VII (Calbiochem, 1/1000 dilution), and 15 units/ml Benzonase. 2x Lysis buffer contains the same components at double concentration.

Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5 mM TCEP added to the lysate. Cells were lysed by high pressure homogenization (20 kpsi). The lysate was centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity, HisTrap Crude FF, 5 ml (GE Healthcare).

Column 1 Buffers:
Affinity buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5% Glycerol, 0.5 mM TCEP.
Wash buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 30 mM imidazole, 5% Glycerol, 0.5 mM TCEP
Elution buffer: 50 mM HEPES buffer, pH 7.5, 500 mM NaCl, 300 mM imidazole, 5% Glycerol, 0.5 mM TCEP.

Column 1 Procedure: The cell extract was loaded on the column at 4 ml/minute on an ÄKTA-express system (GE Healthcare). The column was washed with 10 volumes of lysis buffer, 10 volumes of wash buffer, and then eluted with elution buffer at 4 ml/min. The eluted peak of A280 was automatically collected.

Column 2: Gel filtration, Hiload 16/60 Superdex S75 prep grade, 120 ml (GE Healthcare)

Column 2 Buffers: 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% Glycerol, 0.5 mM TCEP.

Column 2 Procedure: The eluted fractions from the Ni-affinity Histrap column were loaded on the gel filtration column in GF buffer at 1.2 ml/min. Eluted proteins were collected in 2-ml fractions and analyzed on SDS-PAGE.

TEV protease digestion: Peak fractions containing CLK2 were pooled and TEV protease was added at a molar ratio of 1:30. The digestion was left overnight at 4 °C. His-TEV and contaminating proteins were removed by passing to 300µl Ni resin pre-equilibrated in GF buffer. The flow through containing TEV-cleaved protein was collected, concentrated to 30 mg/ml using a centricon centrifugal device with a 30kDa MWCO, frozen at -80°C in 500µl aliquots.

Mass spectrometry characterization: Measured: 43319; Expected: 43318

Protein concentration: 2.5 mg of CLK2 was diluted into 15 ml of low salt buffer (5mM HEPES. pH 7.5, 200 mM NaCl, 1% glycerol). 2 molar excess of ligand was added to diluted CLK2 and protein-ligand complex was concentrated to 4 mg/ml using an Amicon 30 kDa cut-off concentrator.

Crystallisation: Prior to crystallization, pure protein at 4 mg/ml was incubated with 1mM K00750 compound. Optimized condition contained 25% MPD and 0.1M Bicine, pH 9.3. Viable crystals were obtained at 4 degree in a sitting drop mixing protein and reservoir solution at 2:1 volume ratio.

Data collection:
Resolution: The crystals were cryo-protected with the mother liquor containing 15% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at Diamond Beamline I24 using monochromatic radiation at wavelength 0.9790 Å .
Phasing: Structure of CLK2 was solved by molecular replacement method using the human CLK3 model as a search model.

References

Duncan, P.I., et al., The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing. Exp Cell Res, 1998. 241(2): p. 300-8. PubMed 9637771
Yun, B., et al., The LAMMER protein kinase encoded by the Doa locus of Drosophila is required in both somatic and germline cells and is expressed as both nuclear and cytoplasmic isoforms throughout development. Genetics, 2000. 156(2): p. 749-61. PubMed 11014821
Du, C., et al., Protein phosphorylation plays an essential role in the regulation of alternative splicing and sex determination in Drosophila. Mol Cell, 1998. 2(6): p. 741-50. PubMed 9885562
Nayler, O., et al., The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation. J Biol Chem, 1998. 273(51): p. 34341-8. PubMed 9852100
Rodgers, J.T., et al., Cdc2-like kinase 2 is an insulin-regulated suppressor of hepatic gluconeogenesis. Cell Metab. 11(1): p. 23-34. PubMed 20074525
Wang, H., et al., Phenotypic and molecular evaluation of a chromosome 1q region with linkage and association to type 2 diabetes in humans. J Clin Endocrinol Metab, 2009. 94(4): p. 1401-8. PubMed 19141583
Glatz, D.C., et al., The alternative splicing of tau exon 10 and its regulatory proteins CLK2 and TRA2-BETA1 changes in sporadic Alzheimer's disease. J Neurochem, 2006. 96(3): p. 635-44. PubMed 16371011
Nothwang, H.G., et al., Functional hemizygosity of PAFAH1B3 due to a PAFAH1B3-CLK2 fusion gene in a female with mental retardation, ataxia and atrophy of the brain. Hum Mol Genet, 2001. 10(8): p. 797-806. PubMed 11285245