Human vaccinia related kinase 1

Target ID:

VRK1A

Aliases:

MGC117401MGC138280MGC142070VRK1

Deposition Date:

Tuesday 31st August 2010

Families:

Protein Kinase

Structure type:

protein-ligand

Download Coordinates:

3OP5

Authors:

C.K.Allerston,S.Uttarkar,P.Savitsky,J.M.Elkins,P.Filippakopoulos,T.Krojer,E.Ugochukwu,P.Rellos,O.Federov,J.Eswaran,B.Brenner,O.King,R.Chalk,G.Berridge,O.Gileadi,F.vonDelft,C.H.Arrowsmith,A.M.Edwards,J.Weigelt,C.Bountra,S.Knapp

Site:

Oxford

3OP5

tabs

Structure Details

VRK1 belongs together with VRK2 and VRK3 to the vaccinia related kinase (VRK) protein family, which is characterized by notable sequence homology to the vaccinia virus-encoded B1 kinase (vvB1). Whereas VRK1 and VRK2 show kinase activity VRK3 has key amino acid substitutions that disrupt invariant motifs required for catalytic activity (Scheeff et al., 2009). VRK1 contains an N-terminal ATP-binding site, a serine/threonine kinase domain, endosomal and lysosomal targeting sequence, and C-terminal nuclear localization signal and BAB motif (Nichols and Traktman, 2004). Accordingly, VRK1 can be found not only in the nucleus but also in the cytosol and Golgi (Valbuena et al., 2007a). VRK1 is widely expressed with particular high levels in actively dividing cells, such as those in testis, thymus, fetal liver and carcinomas (Nezu et al., 1997). Overexpression of VRK1 has been found in several cancer types and the autoregulatory loop of VRK1 and the tumor suppressor p53 has been suggested to be dysfunctional in different tumors (Valbuena et al., 2007b; Valbuena et al., 2006).

VRK1 has been implicated in a variety of different cellular functions like cell proliferation, Golgi fragmentation and formation of the nuclear envelop (Valbuena et al., 2008), (Lopez-Sanchez et al., 2009), (Renbaum et al., 2009). VRK1 also regulates gametogenesis demonstrated by the phenotype of hypomorphic Vrk1 mice expressing VRK1 at ~15% of wild type level. Both male and female mice are infertile. In male mice this is due to a defect in spermatogonial proliferation or differentiation and lack of mitotic and meiotic cells in adult testis (Wiebe et al., 2010).

Recently, a role for VRK1 in neuronal development and maintenance has become apparent. A nonsense mutation in VRK1 has been identified in Spinal Muscular Atrophy with Pontocerebellar Hypoplasia (SMA-PCH) - a rare infantile variant of spinal muscular atrophy characterized by degeneration and loss of anterior horn cells in the spinal cord (Renbaum et al., 2009). Here we report the crystal structure of the kinase domain of VRK1 at 2.4 Å.

Materials and Methods

Entry Clone Source: Site directed mutagenesis

Entry Clone Accession: n/a

SGC Construct ID: VRK1A-c510

GenBank GI number: gi|4507903

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

Amplified construct sequence:

Nucleotides in bold and underlined were multated from refseq in an attempt to reduce surface entropy in the translated protein.

CATATGCACCATCATCATCATCATTC
TTCTGGTGTAGATCTGGGTACCGAGA
ACCTGTACTTCCAATCCATGCGTGTA
AAAGCAGCTCAAGCTGGAAGACAGAG
CTCTGCAAAGAGACATCTTGCAGAAC
AATTTGCAGTTGGAGAGATAATAACT
GACATGGCAAAAAAGGAATGGAAAGT
AGGATTACCCATTGGCCAAGGAGGCT
TTGGCTGTATATATCTTGCTGATATG
AATTCTTCAGAGTCAGTTGGCAGTGA
TGCACCTTGTGTTGTAAAAGTGGAAC
CCAGTGACAATGGACCTCTTTTTACT
GAATTAAAGTTCTACCAACGAGCTGC
AAAACCAGAGCAAATTCAGAAATGGA
TTCGTACCCGTAAGCTGAAGTACCTG
GGTGTTCCTAAGTATTGGGGGTCTGG
TCTACATGACAAAAATGGAAAAAGTT
ACAGGTTTATGATAATGGATCGCTTT
GGGAGTGACCTTCAGAAAATATATGA
AGCAAATGCCAAAAGGTTTTCTCGGA
AAACTGTCTTGCAGCTAAGCTTAAGA
ATTCTGGATATTCTGGAATATATTCA
CGAGCATGAGTATGTGCATGGAGATA
TCAAGGCCTCAAATCTTCTTCTGAAC
TACAAGAATCCTGACCAGGTGTACTT
GGTAGATTATGGCCTTGCTTATCGGT
ACTGCCCAGAAGGAGTTCATAAAGAA
TACAAAGAAGACCCCAAAAGATGTCA
CGATGGCACTATTGAATTCACGAGCA
TCGATGCACACAATGGCGTGGCCCCA
TCAAGACGTGGTGATTTGGAAATACT
TGGTTATTGCATGATCCAATGGCTTA
CTGGCCATCTTCCTTGGGAGGATAAT
TTGAAAGATCCTAAATATGTTAGAGA
TTCCAAAATTAGATACAGAGAAAATA
TTGCAAGTTTGATGGACAAATGTTTT
CCTGAGAAAAACAAACCAGGTGAAAT
TGCCAAATACATGGAAACAGTGAAAT
TACTAGACTACACTGAAAAACCTCTT
TATGAAAATTTACGTGACATTCTTTT
GCAAGGACTAAAAGCTATAGGAAGTA
AGGATGATGGCAAATTGGACCTCAGT
GTTGTGGAGAATGGAGGTTTGAAAGC
AAAAACAATAACAAAGAAGCGAAAGA
AAGAAATTGAAGAATGACAGTAAAGG
TGGATACGGATCCGAA

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqs^MMH
HHHHHSSGVDLGTENLYFQSMRVKAA
QAGRQSSAKRHLAEQFAVGEIITDMA
AAAWKVGLPIGQGGFGCIYLADMNSS
ESVGSDAPCVVKVEPSDNGPLFTELK
FYQRAAKPEQIQKWIRTRKLKYLGVP
KYWGSGLHDKNGKSYRFMIMDRFGSD
LQKIYEANAKRFSRKTVLQLSLRILD
ILEYIHEHEYVHGDIKASNLLLNYKN
PDQVYLVDYGLAYRYCPEGVHKAYAA
DPKRCHDGTIEFTSIDAHNGVAPSRR
GDLEILGYCMIQWLTGHLPWEDNLKD
PKYVRDSKIRYRENIASLMDKCFPAA
NAPGEIAKYMETVKLLDYTEKPLYEN
LRDILLQGLKAIGSKDDGKLDLSVVE
NGGLKAKTITKKRAAEIEE

^ TEV cleave site

Tags and additions: Cleavable N-terminal His6 tag.

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 50µg/ml kanamycin and 35µg/ml chloramphenicol. Several colonies were combined to inoculate a 1ml culture in TB (+ 50µg/ml kanamycin, 35µg/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 5ml of LB medium containing 100µg/ml kanamycin and 35µg/ml chloramphenicol and grown overnight at 37°C. Overnight culture was used to inoculate 1 liter of LB containing 50µg/ml kanamycin & 34µg/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 VRK1 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 30mg/ml using a centricon centrifugal device with a 30kDa MWCO, frozen at -80°C in 500µl aliquots.

Mass spectrometry characterization: Measured: 41078, 41163 (one phosphorylation site) and 41240 (2 phosphorylation sites). Expected mass of unmodified protein: 41076.2.

Protein concentration: 2.5 mg of VRK1 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 VRK1 and protein-ligand complex was concentrated to 20 mg/ml using an Amicon 30 kDa cut-off concentrator.

Crystallisation: Prior to crystallization, pure protein at 20 mg/ml was incubated with 1mM K00604a (ASC24, provided by the Shokat laboratory) compound. Optimized condition contained 0.2 M K/Na(tart) and 20% PEG 3350, pH 7.0. Viable crystals were obtained at 4°C in a sitting drop mixing protein and reservoir solution at 2:1 volume ratio.

Data Collection: The crystals were cryo-protected with the mother liquor containing 25% ethylene glycol and flash frozen in liquid nitrogen. Diffraction data were collected at Diamond Beamline I02 using monochromatic radiation at wavelength 0.9790 Å.
Phasing: Structure of VRK1 was solved by molecular replacement method using an ensemble of the following PDB codes as a search model; 2V62, 2JII, 1CKJ.

References

Lopez-Sanchez, I., Sanz-Garcia, M., and Lazo, P.A. (2009). Plk3 interacts with and specifically phosphorylates VRK1 in Ser342, a downstream target in a pathway that induces Golgi fragmentation. Mol Cell Biol 29, 1189-1201.
Nezu, J., Oku, A., Jones, M.H., and Shimane, M. (1997). Identification of two novel human putative serine/threonine kinases, VRK1 and VRK2, with structural similarity to vaccinia virus B1R kinase. Genomics 45, 327-331.
Nichols, R.J., and Traktman, P. (2004). Characterization of three paralogous members of the Mammalian vaccinia related kinase family. J Biol Chem 279, 7934-7946.
Renbaum, P., Kellerman, E., Jaron, R., Geiger, D., Segel, R., Lee, M., King, M.C., and Levy-Lahad, E. (2009). Spinal muscular atrophy with pontocerebellar hypoplasia is caused by a mutation in the VRK1 gene. Am J Hum Genet 85, 281-289.
Scheeff, E.D., Eswaran, J., Bunkoczi, G., Knapp, S., and Manning, G. (2009). Structure of the pseudokinase VRK3 reveals a degraded catalytic site, a highly conserved kinase fold, and a putative regulatory binding site. Structure 17, 128-138.
Valbuena, A., Lopez-Sanchez, I., and Lazo, P.A. (2008). Human VRK1 is an early response gene and its loss causes a block in cell cycle progression. PLoS One 3, e1642.
Valbuena, A., Lopez-Sanchez, I., Vega, F.M., Sevilla, A., Sanz-Garcia, M., Blanco, S., and Lazo, P.A. (2007a). Identification of a dominant epitope in human vaccinia-related kinase 1 (VRK1) and detection of different intracellular subpopulations. Arch Biochem Biophys 465, 219-226.
Valbuena, A., Suarez-Gauthier, A., Lopez-Rios, F., Lopez-Encuentra, A., Blanco, S., Fernandez, P.L., Sanchez-Cespedes, M., and Lazo, P.A. (2007b). Alteration of the VRK1-p53 autoregulatory loop in human lung carcinomas. Lung Cancer 58, 303-309.
Valbuena, A., Vega, F.M., Blanco, S., and Lazo, P.A. (2006). p53 downregulates its activating vaccinia-related kinase 1, forming a new autoregulatory loop. Mol Cell Biol 26, 4782-4793.
Wiebe, M.S., Nichols, R.J., Molitor, T.P., Lindgren, J.K., and Traktman, P. (2010). Mice deficient in the serine/threonine protein kinase VRK1 are infertile due to a progressive loss of spermatogonia. Biol Reprod 82, 182-193.