Human PCTAIRE protein kinase 1 in complex with Indirubin E804

Target ID:

PCTK1A

Aliases:

FLJ16665PCTAIREPCTAIRE1PCTGAIREPCTK1

Deposition Date:

Friday 30th April 2010

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

3MTL

Authors:

Krojer, T., Sharpe, T. D., Roos, A., Savitsky. P., Amos, A., Ayinampudi, V., Berridge, G., Fedorov, O., Keates, T., Phillips, C., Burgess-Brown, N., Zhang, Y., Pike, A. C. W., Muniz, J., Vollmar, M., Thangaratnarajah, C., Rellos, P., Ugochukwu, E., Filippakopoulos, P., Yue, W., Das, S., von Delft, F., Edwards, A., Arrowsmith, C.H., Weigelt, J., Bountra, C., Knapp, S., Bullock, A.

Site:

Oxford

ICM Datapack:

ICM Datapack

3MTL

tabs

Structure Details

PCTAIRE1, also known as PCTK1 or CDK16, belongs to the CDK family of protein kinases. The PCTAIRE protein kinases comprise a distinct subfamily and are so named for the presence of a serine to cysteine substitution in the conserved PSTAIRE amino acid motif found in prototypic CDK kinases. Three members of this kinase subfamily, PCTAIRE1-3, have been identified in humans. The three PCTAIRE kinases are 65% identical to one another (80% within the catalytic domain) (Meyerson et al., 1992), (Okuda et al., 1992). So far no cyclin interaction partners have been identified for any of the PCTAIREs and it has been speculated that conserved sequences in the N-terminus provide the activation that cyclins normally provide to the rest of the CDK family (Graeser et al., 2002; Cole 2009).

The human PCTAIRE1 gene maps to the X chromosome (Xp11.3-p11.23), a chromosomal region associated with a growing number of diseases with a genetic basis. PCTAIRE1 is widely expressed with high levels in brain and testis (Besset et al., 1999; Rhee and Wolgemuth, 1995). Little is known about the function of this kinase and diverse roles have been suggested including as a negative regulator of insulin-responsive glucose transport (Tang et al., 2006), a modulator of exocytosis, (Liu et al., 2006), a control factor for neurite outgrowth (Graeser et al., 2002) and for membrane traffic through the early secretory pathway (Palmer et al., 2005). So far no mouse model has been established to confirm any of the proposed functions. ES cells lacking PCTAIRE1 have been generated but no germline transmission could be achieved, which could point to an essential role of PCTAIRE 1 in the formation of viable sperm (Graeser et al., 2002).

Here we present the structure of the kinase domain of PCTAIRE1 refined at 2.4 Å resolution.

Materials and Methods

Entry Clone Source: Site-directed mutagenesis

Entry Clone Accession: n/a

SGC Construct ID: PCTK1A-c031

GenBank GI number: gi|5453860

Vector: pNIC-CH. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
TTAAGAAGGAGATATACTATGGAGACCTAC
ATTAAGCTGGACAAACTGGGCGAGGGTACC
TATGCCACCGTCTACAAAGGCAAAAGCAAG
CTCACAGACAACCTTGTGGCACTCAAGGAG
ATCAGACTGGAACATGAAGAGGGGGCACCC
TGCACCGCCATCCGGGAAGTGTCCCTGCTC
AAGGACCTCAAACACGCCAACATCGTTACG
CTACATGACATTATCCACACGGAGAAGTCC
CTCACCCTTGTCTTTGAGTACCTGGACAAG
GACCTGAAGCAGTACCTGGATGACTGTGGG
AACATCATCAACATGCACAACGTGAAACTG
TTCCTGTTCCAGCTGCTCCGTGGCCTGGCC
TACTGCCACCGGCAGAAGGTGCTACACCGA
GACCTCAAGCCCCAGAACCTGCTCATCAAC
GAGAGGGGAGAGCTCAAGCTGGCTGACTTT
GGCCTGGCCCGAGCCAAGTCAATCCCAACA
AAGACATACGACAATGAGGTGGTGACACTG
TGGTACCGGCCCCCTGACATCCTGCTTGGG
TCCACGGACTACTCCACTCAGATTGACATG
TGGGGTGTGGGCTGCATCTTCTATGAGATG
GCCACAGGCCGTCCCCTCTTTCCGGGCTCC
ACGGTGGAGGAACAGCTACACTTCATCTTC
CGTATCTTAGGAACCCCAACTGAGGAGACG
TGGCCAGGCATCCTGTCCAACGAGGAGTTC
AAGACATACAACTACCCCAAGTACCGAGCC
GAGGCCCTTTTGAGCCACGCACCCCGACTT
GATAGCGACGGGGCCGACCTCCTCACCAAG
CTGTTGCAGTTTGAGGGTCGAAATCGGATC
TCCGCAGAGGATGCCATGAAACATCCATTC
TTCCTCAGTCTGGGGGAGCGGATCCACAAA
CTTCCTGACACTACTTCCATATTTGCACTA
AAGGAGATTCAGCTACAAAAGGAGGCCAGC
CTTCGGTCTGCGCACCATCATCACCACCAT
T

Expressed protein sequence:
METYIKLDKLGEGTYATVYKGKSKLTDNLV
ALKEIRLEHEEGAPCTAIREVSLLKDLKHA
NIVTLHDIIHTEKSLTLVFEYLDKDLKQYL
DDCGNIINMHNVKLFLFQLLRGLAYCHRQK
VLHRDLKPQNLLINERGELKLADFGLARAK
SIPTKTYDNEVVTLWYRPPDILLGSTDYST
QIDMWGVGCIFYEMATGRPLFPGSTVEEQL
HFIFRILGTPTEETWPGILSNEEFKTYNYP
KYRAEALLSHAPRLDSDGADLLTKLLQFEG
RNRISAEDAMKHPFFLSLGERIHKLPDTTS
IFALKEIQLQKEASLRSAHHHHHH
Engineered S319D phospho-mimetic mutation shown bold and underlined.

Host: BL21(DE3)-R3-pRARE2

Growth medium, induction protocol: A glycerol stock was used to inoculate a 10ml starter culture containing LB media with 50µg/ml Kanamycin and 34 µg/ml chloramphenicol. The starter culture was grown overnight at 37°C with shaking at 200 rpm. The following morning, four flasks containing 1 L LB/kanamycin/chloramphenicol were each inoculated with 5 ml of the starter culture. Cultures were incubated at 37°C with shaking at 180 rpm until an OD600nm ≥ 0.7 was reached. The flasks were then cooled down to 18°C and 0.5mM IPTG added to induce protein expression overnight. Cells were harvested by centrifugation at 4500 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 30ml binding buffer (50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole), transferred to 50 ml tubes, and stored at -20°C.

Extraction buffer, extraction method: The frozen cells were thawed and 0.5mM TCEP, 1mM PMSF added to the cell suspension. The cells were lysed by ultrasonication over 12 min with the sonicator pulsing ON for 5 sec and OFF for 15 sec. The cell lysate was spun down by centrifugation at 17000 rpm at 4°C for 1 h. The supernatant was recovered for purification.

Column 1: Ni-Affinity Chromatography: 5ml of 50 % Ni-sepharose slurry (Amersham) was applied onto a 1.5 x 10 cm column. The column was first washed with deionised distilled H₂O, and then equilibrated with binding buffer.

Column 1 Buffers:
Binding buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole
Wash buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 30mM Imidazole
Elution buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 50 to 250mM Imidazole

Column 1 Procedure: The supernatant was applied by gravity flow onto the Ni-sepharose column. The bound protein was eluted by applying a step gradient of imidazole (5 ml fractions of elution buffer supplemented with 50mM, 100mM, 150mM and 250mM imidazole). 10mM DTT was added to each fraction collected for overnight storage at 4°C.

Enzymatic treatment:: N/A

Column 2: Size Exclusion Chromatography - S200 HiLoad 16/60 Superdex run on ÄKTA-Express.

Column 2 Buffers: Gel Filtration buffer: 300mM NaCl, 50mM HEPES pH 7.5, 0.5mM TCEP

Column 2 Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. Eluted protein from the Ni-sepharose column was pooled and concentrated to 3ml using an Amicon Ultra-15 filter with a 10kDa cut-off. The concentrated protein was directly applied onto the equilibrated S200 16/60 column, and run at a flow-rate of 1 ml/min. Fractions containing the protein were pooled together, and 10mM DTT was added for overnight storage at 4°C.

Column 3: Anion Exchange Chromatography (monoQ)

Column 3 Buffers:
Buffer A: 50mM TRIS, pH 9.0
Buffer B: 50mM TRIS, pH 9.0; 1M NaCl

Column 3 Procedure: PCTK1 protein from gel filtration was buffer exchanged into 50mM Tris pH 9.0 and loaded onto a 1 ml monoQ anion exchange column equilibrated in the same buffer. A linear elution gradient was run from 0-1M NaCl. PCTK1 containing fractions were pooled and the buffer adjusted to 25 mM HEPES, pH 7.5, 250 mM NaCl, 5 % Glycerol, 10 mM DTT during concentration in a 5 kD MWCO Amicon Ultra concentrator.

Concentration: The protein was concentrated in a 5 kD MWCO Amicon Ultra concentrator to 15 mg/ml using an estimated extinction coefficient of 35870 and MW of 37252.

Mass spectrometry characterization: The purified native protein was homogeneous and had an experimental mass of 37252.74 (expected MW = 37252). Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser.

Crystallisation: Protein was buffered in 25 mM HEPES, pH 7.5, 250 mM NaCl, 5 % Glycerol, 10 mM DTT. Protein was concentrated to 15 mg/ml in the presence of indirubin E804 (final inhibitor concentration of 1 mM). Crystals were grown by micro-seeding at 20°C in 130 nl sitting drops mixing 96 nl protein solution with 10 nl micro-seed solution (crystals prepared from the same precipitant) and 24 nl of a reservoir solution containing 2.1M Na-formate pH 7.0, 0.1 M Bis-Tris pH 7.0. On mounting crystals were cryo-protected with reservoir solution mixed with 25% glycerol.

Data Collection: Resolution: Resolution: 2.4 Å, X-ray source: Diamond I03. The native crystal diffracted to a resolution of 2.4 Å.

References

Besset, V., Rhee, K., and Wolgemuth, D.J. (1999). The cellular distribution and kinase activity of the Cdk family member Pctaire1 in the adult mouse brain and testis suggest functions in differentiation. Cell Growth Differ 10, 173-181. PubMed 10099831
Cole, A.R. (2009). PCTK proteins: the forgotten brain kinases? Neurosignals. 17, 288-97. PubMed 19816065
Graeser, R., Gannon, J., Poon, R.Y., Dubois, T., Aitken, A., and Hunt, T. (2002). Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells. J. Cell Science 115, 3479-3490. PubMed 12154078
Liu, Y., Cheng, K., Gong, K., Fu, A.K., and Ip, N.Y. (2006). Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis. J. Biol. Chem. 281, 9852-9858. PubMed 16461345
Meyerson, M., Enders, G.H., Wu, C.L., Su, L.K., Gorka, C., Nelson, C., Harlow, E., and Tsai, L.H. (1992). A family of human cdc2-related protein kinases. EMBO J. 11, 2909-2917. PubMed 1639063
Okuda, T., Cleveland, J.L., and Downing, J.R. (1992). PCTAIRE-1 and PCTAIRE-3, two members of a novel cdc2/CDC28-related protein kinase gene family. Oncogene 7, 2249-2258. PubMed 1437147
Palmer, K.J., Konkel, J.E., and Stephens, D.J. (2005). PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell Science 118, 3839-3847. PubMed 16091426
Rhee, K., and Wolgemuth, D.J. (1995). Cdk family genes are expressed not only in dividing but also in terminally differentiated mouse germ cells, suggesting their possible function during both cell division and differentiation. Dev Dyn 204, 406-420. PubMed 8601034
Tang, X., Guilherme, A., Chakladar, A., Powelka, A.M., Konda, S., Virbasius, J.V., Nicoloro, S.M., Straubhaar, J., and Czech, M.P. (2006). An RNA interference-based screen identifies MAP4K4/NIK as a negative regulator of PPARgamma, adipogenesis, and insulin-responsive hexose transport.Proc. Natl. Acad. Sci. USA 103, 2087-2092. PubMed 16461467