Human dual-specificity tyrosine phosphorylation regulated kinase 2

Target ID:

DYRK2A

Aliases:

DYRK2FLJ21217FLJ21365

Deposition Date:

Wednesday 30th September 2009

Families:

Protein Kinase

Structure type:

apo

Download Coordinates:

3K2L

Authors:

Filippakopoulos, P., Myrianthopoulos, V., Soundararajan, M., Krojer, T., Hapka, E., Fedorov, O., Berridge, G., Wang, J., Shrestha, L., Pike, A.C.W., Ugochukwu, E., von Delft, F., Arrowsmith, C.H., Edwards, A., Weigelt, J., Bountra, C., Mikros, E., Knapp, S.

Site:

Oxford

3K2L

tabs

Structure Details

DYRK2 belongs to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK)/Minibrain family of protein kinases. Members of this family share a high degree of conservation in the catalytic domain and in the adjacent N-terminal DH box (DYRK homology), but are very divergent in their N- and C-terminal extensions. The DYRK family is distinguished from other kinases by its activation mechanism. DYRK kinase activity depends on tyrosine autophosphorylation at a conserved YxY site in the catalytic domain. This activation domain has been shown to be intramolecularly phosphorylated during translation while mature (phosphorylated) members of the DYRK family have only serine/threonine kinase activity (Lochhead et al., 2005).

DYRK2 is mainly expressed in during development and in adult testis but has been found to be overexpressed in adenocarcinomas of the oesophagus and lung (Miller et al., 2003). Interestingly, in patients suffering from recurrent non-small cell lung cancer, high DYRK2 expression is an indicator for their response to chemotherapy (Yamashita et al., 2009). The precise role of DYRK2 in cancer however needs further investigation.
DYRK2 has been implicated in many key developmental and cellular processes such as regulation of protein and glycogen synthesis, cell proliferation, cytokinesis and cellular differentiation (Yoshida, 2008). DYRK2 also regulates p53 to induce apoptosis in response to DNA damage (Taira et al., 2007). More recently a role as a scaffold for an E3 ubiquitin ligase complex has been reported. The scaffolding function is independent of its kinase activity but the subsequent phosphorylation and degradation of the substrate katanin p60 is dependent on the catalytic activity of DYRK2. (Maddika and Chen, 2009). Here we report the structure of the DYRK2 kinase domain at 2.36 Å resolution.

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:4139392

SGC Construct ID: DYRK2A-c022

GenBank GI number: gi|4503427

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGGGGAAGGTGAAAGCCACC
CCCATGACACCTGAACAAGCAATGAAGCAA
TACATGCAAAAACTCACAGCCTTCGAACAC
CATGAGATTTTCAGCTACCCTGAAATATAT
TTCTTGGGTCTAAATGCTAAGAAGCGCCAG
GGCATGACAGGTGGGCCCAACAATGGTGGC
TATGATGATGACCAGGGATCATATGTGCAG
GTGCCCCACGATCACGTGGCTTACAGGTAT
GAGGTCCTCAAGGTCATTGGGAAGGGGAGC
TTTGGGCAGGTGGTCAAGGCCTACGATCAC
AAAGTCCACCAGCACGTGGCCCTAAAGATG
GTGCGGAATGAGAAGCGCTTCCACCGGCAA
GCAGCGGAGGAGATCCGAATCCTGGAACAC
CTGCGGAAGCAGGACAAGGATAACACAATG
AATGTCATCCATATGCTGGAGAATTTCACC
TTCCGCAACCACATCTGCATGACGTTTGAG
CTGCTGAGCATGAACCTCTATGAGCTCATC
AAGAAGAATAAATTCCAGGGCTTCAGTCTG
CCTTTGGTTCGCAAGTTTGCCCACTCGATT
CTGCAGTGCTTGGATGCTTTGCACAAAAAC
AGAATAATTCACTGTGACCTTAAGCCCGAG
AACATTTTGTTAAAGCAGCAGGGTAGAAGC
GGTATTAAAGTAATTGATTTTGGCTCCAGT
TGTTACGAGCATCAGCGTGTCTACACGTAC
ATCCAGTCGCGTTTTTACCGGGCTCCAGAA
GTGATCCTTGGGGCCAGGTATGGCATGCCC
ATTGATATGTGGAGCCTGGGCTGCATTTTA
GCAGAGCTCCTGACGGGTTACCCCCTCTTG
CCTGGGGAAGATGAAGGGGACCAGCTGGCC
TGTATGATTGAACTGTTGGGCATGCCCTCA
CAGAAACTGCTGGATGCATCCAAACGAGCC
AAAAATTTTGTGAGCTCCAAGGGTTATCCC
CGTTACTGCACTGTCACGACTCTCTCAGAT
GGCTCTGTGGTCCTAAACGGAGGCCGTTCC
CGGAGGGGGAAACTGAGGGGCCCACCGGAG
AGCAGAGAGTGGGGGAACGCGCTGAAGGGG
TGTGATGATCCCCTTTTCCTTGACTTCTTA
AAACAGTGTTTAGAGTGGGATCCTGCAGTG
CGCATGACCCCAGGCCAGGCTTTGCGGCAC
CCCTGGCTGAGGAGGCGGTTGCCAAAGCCT
CCCACCGGGGAGAAAACGTCAGTGAAAAGG
TGACAGTAAAGGTGGATACGGATCCGAA

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smGKVKAT
PMTPEQAMKQYMQKLTAFEHHEIFSYPEIY
FLGLNAKKRQGMTGGPNNGGYDDDQGSYVQ
VPHDHVAYRYEVLKVIGKGSFGQVVKAYDH
KVHQHVALKMVRNEKRFHRQAAEEIRILEH
LRKQDKDNTMNVIHMLENFTFRNHICMTFE
LLSMNLYELIKKNKFQGFSLPLVRKFAHSI
LQCLDALHKNRIIHCDLKPENILLKQQGRS
GIKVIDFGSSCYEHQRVYTYIQSRFYRAPE
VILGARYGMPIDMWSLGCILAELLTGYPLL
PGEDEGDQLACMIELLGMPSQKLLDASKRA
KNFVSSKGYPRYCTVTTLSDGSVVLNGGRS
RRGKLRGPPESREWGNALKGCDDPLFLDFL
KQCLEWDPAVRMTPGQALRHPWLRRRLPKP
PTGEKTSVKR
^ TEV cleave site

Tags and additions: Cleavable N-terminal His6 tag.

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

Growth medium, induction protocol: 5 ml from a 50 ml overnight culture containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol were used to inoculate each of two 1 litre cultures of LB containing 50 µg/ml kanamycin & 34 µg/ml chloramphenicol. Cultures were grown at 37°C until the OD₆₀₀ reached ~0.5 then the temperature was adjusted to 18°C. Expression was induced overnight using 0.5 mM IPTG at an OD₆₀₀ of 0.9. The cells were collected by centrifugation and the pellet re-suspended in binding buffer and frozen. Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5 % glycerol.

Extraction buffer, extraction method: Frozen pellets were thawed and fresh 0.5 mM TCEP added to the lysate. Cells were lysed using sonication. The lysate was centrifuged at 16,500 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Ni-sepharose (Amersham), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Column 1 Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% Glycerol; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol (step elution).

Column 1 Procedure: The lysate supernatant was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 30 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.

Column 2: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad

Column 2 Buffers: 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP

Column 2 Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel filtration column equilibrated in 25 mM HEPES, pH 7.5; 500 mM NaCl, 0.5 mM TCEP using an ÄKTAexpress system.

Mass spec characterization: LC- ESI -MS TOF showed that the protein was herterogeneously phosphorylated at up to 4 sites in accordance with a mass of 49279 for this construct as predicted from the sequence of this protein.

Protein concentration: Protein was concentrated to 7.3 mg/ml using an Amicon 10 kDa cut-off concentrator.

Crystallization: Crystals were grown at 4°C in 300 nl sitting drops from a 2:1 ratio of protein to reservoir solution containing 1.26 M (NH₄)₂SO₄; 0.2 M Li₂SO₄; 0.1 M Tris pH 8.5.

Data Collection: Crystals were cryo-protected using the well solution supplemented with 2M Li₂SO₄ and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected from a single crystal on Diamond beamline IO3 at a single wavelength of 0.9763 Å and the structure was refined to 2.36 Å. Phasing: The structure was solved by molecular replacement using the structure of human DYRK1 (PDB ID 2VX3) as a starting model.

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

Lochhead, P.A., Sibbet, G., Morrice, N., and Cleghon, V. (2005). Activation-loop autophosphorylation is mediated by a novel transitional intermediate form of DYRKs. Cell 121, 925-936.
Maddika, S., and Chen, J. (2009). Protein kinase DYRK2 is a scaffold that facilitates assembly of an E3 ligase. Nature Cell Biology 11, 409-419.
Miller, C.T., Aggarwal, S., Lin, T.K., Dagenais, S.L., Contreras, J.I., Orringer, M.B., Glover, T.W., Beer, D.G., and Lin, L. (2003). Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas. Cancer Research 63, 4136-4143.
Taira, N., Nihira, K., Yamaguchi, T., Miki, Y., and Yoshida, K. (2007). DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage. Molecular Cell 25, 725-738.
Yamashita, S., Chujo, M., Moroga, T., Anami, K., Tokuishi, K., Miyawaki, M., Kawano, Y., Takeno, S., Yamamoto, S., and Kawahara, K. (2009). DYRK2 expression may be a predictive marker for chemotherapy in non-small cell lung cancer. Anticancer Research 29, 2753-2757.
Yoshida, K. (2008). Role for DYRK family kinases on regulation of apoptosis. Biochemical Pharmacology 76, 1389-1394.