Human SRPK2 (serine/arginine-rich protein-specific kinase 2)

Target ID:

SRPK2A

Deposition Date:

Friday 26th February 2010

Families:

Protein Kinase

Structure type:

protein-ligand

Download Coordinates:

2X7G

Authors:

A.C.W.PIKE,P.SAVITSKY,O.FEDOROV,T.KROJER,E.UGOCHUKWU,F.VON DELFT,O.GILEADI,A.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,C.BOUNTRA,S.KNAPP

Site:

Oxford

2X7G

tabs

Structure Details

The serine/arginine-rich protein-specific kinase 2, SRPK2, forms together with SRPK1 and SRPK3 a family of cell-cycle regulated Ser/Thr kinases. The substrates of SRPKs are splicing factors such as serine/arginine (SR) domain-containing proteins that are localized in nuclear speckles and mediate pre-mRNA splicing (Nakagawa et al., 2005). SRPKs are characterised by a bipartite kinase domain separated by a spacer region. The spacer region of SRPK2 comprises a proline rich region and a basic region which may act as nuclear localization signal, as well as an acidic domain, not present in SRPK1.

SRPK2 is highly expressed in brain as well as at a lower level in other organs (Wang et al., 1998). Although primarily located in the cytoplasm, SRPK2 can translocate to the nucleus, where it has been shown to be involved in regulation of cyclins (Jang et al., 2008) (Jang et al., 2009). In neurons ischemic conditions trigger phosphorylation of the SRPK2 residue Thr492 by AKT resulting in nuclear translocation, in cell cycle re-entry and neuronal apoptosis (Jang et al., 2009). In addition, knockdown experiments of SRPK2 in HeLa cells point to an essential role of SRPK2 for cell viability and splicosomal B complex formation (Mathew et al., 2008).

SRPK2 has been reported to phosphorylate the SR containing hepatitis B virus core protein, a prerequisite for pregenomic RNA encapsidation into viral capsids and thus presents a potential target for therapeutic intervention in HBV infection (Daub et al., 2002). High expression of SRPK2 has been found in some human AML patients and correlate with the oncogenic activity of cyclin A1 in leukemia and leukemia cell proliferation (Jang et al., 2008). Here we report the structure of the SRPK2 kinase domain at 2.5 Å resolution in complex with the ATP competitive inhibitor Purvalanol B.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: SRPK2A-c003

GenBank GI number: gi|33188449

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

Amplified construct sequence:
TACTTCCAATCCATGGGTAGCGATGACGAG
GAGCAGGAAGATCCCGCCGATTACTGTAAA
GGCGGTTATCATCCAGTGAAGATTGGCGAC
TTGTTCAACGGTCGTTACCACGTTATCCGC
AAACTGGGCTGGGGTCACTTTTCCACTGTC
TGGTTGTGCTGGGACATGCAAGGCAAGCGT
TTCGTAGCTATGAAAGTGGTAAAATCTGCG
CAGCACTATACAGAAACCGCACTGGATGAA
ATTAAGTTGCTGAAATGTGTCCGCGAGTCA
GATCCCTCGGACCCGAATAAGGACATGGTA
GTGCAACTGATCGATGACTTTAAGATTTCG
GGTATGAACGGCATCCATGTTTGCATGGTA
TTCGAAGTGTTGGGTCACCATCTGTTGAAA
TGGATTATCAAGAGCAATTACCAGGGCCTG
CCCGTACGTTGCGTGAAATCCATTATCCGC
CAAGTTTTGCAGGGCCTGGATTATCTGCAC
TCTAAGTGCAAAATCATTCATACTGACATC
AAACCAGAGAACATTTTGATGTGCGTCGAT
GACGCCTACGTACGTCGCATGGCTGCGGAA
GCAACAGAGTGGCAGAAGGCCGGCGCTCCT
CCGCCCTCAGGTAGTGCGGTGTCGACCGCC
CCAGCAGCTGATCTGTTGGTTAATCCTCTG
GACCCGCGTAACGCCGACAAAATCCGCGTC
AAGATTGCGGACCTGGGCAACGCATGCTGG
GTGCACAAACACTTTACTGAAGATATCCAA
ACACGTCAGTATCGCAGCATTGAGGTATTG
ATCGGTGCTGGCTACTCCACCCCAGCCGAC
ATCTGGTCTACTGCGTGTATGGCTTTCGAA
CTGGCAACCGGTGATTATTTGTTCGAACCC
CACTCAGGCGAGGACTACAGTCGTGATGAG
GACCATATTGCGCACATCATTGAGCTGTTG
GGTTCGATCCCTCGCCATTTTGCGCTGAGC
GGCAAATACTCCCGTGAGTTCTTTAATCGC
CGTGGTGAGCTGCGCCACATTACAAAGTTG
AAACCGTGGTCGCTGTTCGATGTTTTGGTC
GAAAAGTATGGCTGGCCACATGAGGACGCT
GCCCAGTTTACCGATTTCCTGATCCCCATG
CTGGAAATGGTGCCTGAGAAACGTGCATCA
GCCGGTGAATGCTTGCGTCACCCGTGGCTG
AACAGTTGACAGTAAAGGTGGATA

Tags and additions: Cleavable N-terminal His6 tag.

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqs^MGSDDEE
QEDPADYCKGGYHPVKIGDLFNGRYHVIRK
LGWGHFSTVWLCWDMQGKRFVAMKVVKSAQ
HYTETALDEIKLLKCVRESDPSDPNKDMVV
QLIDDFKISGMNGIHVCMVFEVLGHHLLKW
IIKSNYQGLPVRCVKSIIRQVLQGLDYLHS
KCKIIHTDIKPENILMCVDDAYVRRMAAEA
TEWQKAGAPPPSGSAVSTAPAADLLVNPLD
PRNADKIRVKIADLGNACWVHKHFTEDIQT
RQYRSIEVLIGAGYSTPADIWSTACMAFEL
ATGDYLFEPHSGEDYSRDEDHIAHIIELLG
SIPRHFALSGKYSREFFNRRGELRHITKLK
PWSLFDVLVEKYGWPHEDAAQFTDFLIPML
EMVPEKRASAGECLRHPWLNS
^ TEV cleave site

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

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 litre 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 Buffer: 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 SRPK2 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: 44237.55; Expected: 44238

Protein Concentration: 4.5 mg of SRPK2 was diluted into 15 ml of low salt buffer (5mM HEPES. pH 7.5, 150 mM NaCl, 1% glycerol and 20 mM DTT). 2 molar excess of ligand was added to diluted SRPK2 and protein-ligand complex was concentrated to 30 mg/ml using an Amicon 30 kDa cut-off concentrator.

Crystallization: Crystals in complex with the ATP competitive inhibitor Purvalanol B (Sigma catalogue number "P5234") were grown at 4°C in 150nl sitting drops from a 1:1 ratio of reservoir solution (0.5M ammonium sulphate, 0.1M sodium acetate pH4.75) and protein (23mg/ml). Thin rod-shaped crystals appeared after 7-14days.

Data Collection: Resolution: Crystals were cryo-protected using the well solution supplemented with 35 % ethylene glycol and flash frozen in liquid nitrogen.
X-ray source: Diffraction data were collected from a single crystal to a resolution of 2.5Å on the I24 microfocus beamline at the DIAMOND Light Source.

References

Daub, H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A., and Cotten, M. (2002). Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein. J Virol 76, 8124-8137. PubMed 12134018
Jang, S.W., Liu, X., Fu, H., Rees, H., Yepes, M., Levey, A., and Ye, K. (2009). Interaction of Akt-phosphorylated SRPK2 with 14-3-3 mediates cell cycle and cell death in neurons. J Biol Chem 284, 24512-24525. PubMed 19592491
Jang, S.W., Yang, S.J., Ehlen, A., Dong, S., Khoury, H., Chen, J., Persson, J.L., and Ye, K. (2008). Serine/arginine protein-specific kinase 2 promotes leukemia cell proliferation by phosphorylating acinus and regulating cyclin A1. Cancer Res 68, 4559-4570. PubMed 18559500
Mathew, R., Hartmuth, K., Mohlmann, S., Urlaub, H., Ficner, R., and Luhrmann, R. (2008). Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome. Nat Struct Mol Biol 15, 435-443. PubMed 18425142
Nakagawa, O., Arnold, M., Nakagawa, M., Hamada, H., Shelton, J.M., Kusano, H., Harris, T.M., Childs, G., Campbell, K.P., Richardson, J.A., et al. (2005). Centronuclear myopathy in mice lacking a novel muscle-specific protein kinase transcriptionally regulated by MEF2. Genes Dev 19, 2066-2077. PubMed 16140986
Wang, H.Y., Lin, W., Dyck, J.A., Yeakley, J.M., Songyang, Z., Cantley, L.C., and Fu, X.D. (1998). SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells. J Cell Biol 140, 737-750. PubMed 9472028