Human dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 + indirubin

Target ID:

DYRK2A

Aliases:

DYRK2FLJ21217FLJ21365

Deposition Date:

Monday 30th November 2009

Families:

Protein Kinase

Related Diseases:

CancerMetabolismSignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

3KVW

Authors:

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

Site:

Oxford

3KVW

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 in complex with an indirubin type inhibitor. Indirubins are potent kinase inhibitors that have been originally developed for cyclin dependent kinases. We show here that a variant of the classical indirubin scaffold binds in a non-canonical binding mode to DYRK2 involving 180 degree flip in inhibitor orientation. The inhibitor is highly selective for DYRK kinases and together with the observed binding mode the structure of the complex offers an interesting strategy for the design selective inhibitors for DYRK kinases.

Structure of the co-crystalized indirubin. The SGC is grateful to the group of Dr. E. Mikros (University of Athens) for making this inhibitor available to us.

Materials and Methods

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 ]

Coding DNA sequence:
CATATGCACCATCATCATCATCATTCTTC
TGGTGTAGATCTGGGTACCGAGAACCTGT
ACTTCCAATCCATGGGGAAGGTGAAAGCC
ACCCCCATGACACCTGAACAAGCAATGAA
GCAATACATGCAAAAACTCACAGCCTTCG
AACACCATGAGATTTTCAGCTACCCTGAA
ATATATTTCTTGGGTCTAAATGCTAAGAA
GCGCCAGGGCATGACAGGTGGGCCCAACA
ATGGTGGCTATGATGATGACCAGGGATCA
TATGTGCAGGTGCCCCACGATCACGTGGC
TTACAGGTATGAGGTCCTCAAGGTCATTG
GGAAGGGGAGCTTTGGGCAGGTGGTCAAG
GCCTACGATCACAAAGTCCACCAGCACGT
GGCCCTAAAGATGGTGCGGAATGAGAAGC
GCTTCCACCGGCAAGCAGCGGAGGAGATC
CGAATCCTGGAACACCTGCGGAAGCAGGA
CAAGGATAACACAATGAATGTCATCCATA
TGCTGGAGAATTTCACCTTCCGCAACCAC
ATCTGCATGACGTTTGAGCTGCTGAGCAT
GAACCTCTATGAGCTCATCAAGAAGAATA
AATTCCAGGGCTTCAGTCTGCCTTTGGTT
CGCAAGTTTGCCCACTCGATTCTGCAGTG
CTTGGATGCTTTGCACAAAAACAGAATAA
TTCACTGTGACCTTAAGCCCGAGAACATT
TTGTTAAAGCAGCAGGGTAGAAGCGGTAT
TAAAGTAATTGATTTTGGCTCCAGTTGTT
ACGAGCATCAGCGTGTCTACACGTACATC
CAGTCGCGTTTTTACCGGGCTCCAGAAGT
GATCCTTGGGGCCAGGTATGGCATGCCCA
TTGATATGTGGAGCCTGGGCTGCATTTTA
GCAGAGCTCCTGACGGGTTACCCCCTCTT
GCCTGGGGAAGATGAAGGGGACCAGCTGG
CCTGTATGATTGAACTGTTGGGCATGCCC
TCACAGAAACTGCTGGATGCATCCAAACG
AGCCAAAAATTTTGTGAGCTCCAAGGGTT
ATCCCCGTTACTGCACTGTCACGACTCTC
TCAGATGGCTCTGTGGTCCTAAACGGAGG
CCGTTCCCGGAGGGGGAAACTGAGGGGCC
CACCGGAGAGCAGAGAGTGGGGGAACGCG
CTGAAGGGGTGTGATGATCCCCTTTTCCT
TGACTTCTTAAAACAGTGTTTAGAGTGGG
ATCCTGCAGTGCGCATGACCCCAGGCCAG
GCTTTGCGGCACCCCTGGCTGAGGAGGCG
GTTGCCAAAGCCTCCCACCGGGGAGAAAA
CGTCAGTGAAAAGGTGACAGTAAAGGTGG
ATACGGATCCGAA

Tags and additions: Cleavable N-terminal His6 tag.

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

Expressed protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^sMGKVKA
TPMTPEQAMKQYMQKLTAFEHHEIFSYPE
IYFLGLNAKKRQGMTGGPNNGGYDDDQGS
YVQVPHDHVAYRYEVLKVIGKGSFGQVVK
AYDHKVHQHVALKMVRNEKRFHRQAAEEI
RILEHLRKQDKDNTMNVIHMLENFTFRNH
ICMTFELLSMNLYELIKKNKFQGFSLPLV
RKFAHSILQCLDALHKNRIIHCDLKPENI
LLKQQGRSGIKVIDFGSSCYEHQRVYTYI
QSRFYRAPEVILGARYGMPIDMWSLGCIL
AELLTGYPLLPGEDEGDQLACMIELLGMP
SQKLLDASKRAKNFVSSKGYPRYCTVTTL
SDGSVVLNGGRSRRGKLRGPPESREWGNA
LKGCDDPLFLDFLKQCLEWDPAVRMTPGQ
ALRHPWLRRRLPKPPTGEKTSVKR

^ TEV cleave site

Growth medium, induction protocol: 5 ml from a 50 ml overnight culture containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol were used to inoculate each of two 1 litre cultures of LB containing 50 μg/ml kanamycin and 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.

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).

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

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

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 spectrometry 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 0.2M Na(ac); 0.1 M cacodylate pH 6.5; 30% PEG 8K.

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 2.2 Å.
Phasing: The structure was solved by molecular replacement using the structure of human DYRK2 (PDB ID 3K2L) as a starting model.

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. Nat Cell Biol 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 Res 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. Mol 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 Res 29, 2753-2757.
Yoshida, K. (2008). Role for DYRK family kinases on regulation of apoptosis. Biochem Pharmacol 76, 1389-1394.