Human serine threonine kinase-10 bound to bosutinib-like compound

Target ID:

STK10A

Aliases:

LOKPRO2729

Deposition Date:

Wednesday 31st August 2011

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

protein-ligand

Download Coordinates:

4BC6

Authors:

M.Vollmar,M.Szklarz,A.Chaikuad,J.Elkins, P.Savitsky,K.Abdul Azeez,E.Salah,T.Krojer,P.Canning,J.R.C.Muniz,F.Von Delft,C.Bountra,C.H.Arrowsmith,J.Weigelt,A.Edwards,S.Knapp

Site:

Oxford

4BC6

tabs

Structure Details

STK10 is a member of the STE20 family of serine/threonine kinases and the human homolog of the murine kinase LOK, a serine/threonine kinase highly expressed in lymphocytes. STK10 is closely related to the polo-like kinase kinase SLK and has been shown to associate with Plk1 and phosphorylate it in vitro. STK10 knockout mice do not exhibit any obvious histopathologic abnormalities and showed normal distribution of B cells, T cells and their precursors in the bone marrow, thymus, spleen and lymph nodes as well as normal lymphocytes development. STK10 has also been shown to play a role in modifying TcR/CD28 signals for activation of cytokine transcription and it is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion. STK10 has been found to be over-expressed in a number of tumor cells and a heterozygous somatic missense mutation (K277E) within the kinase domain has been found in human testicular germ-cell tumors. STK10 has been co-crystallized with a bosutinib-like compound. Bosutinib, which differs from this compound in the position of one chloride atom, is a 7-alkoxy-3-quinolinecarbonitrile that functions as a dual inhibitor of SRC and ABL kinases.

Materials and Methods

Entry Clone Source: TKC

Entry Clone Accession: n/a

SGC Construct ID: STK10A-c013

GenBank GI number: gi|5174701

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

Tags and additions: Tag sequence:
mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Amplified construct sequence:
ATGCACCATCATCATCATCATTCTTC
TGGTGTAGATCTGGGTACCGAGAACC
TGTACTTCCAATCCATGAGAAAGTCC
CGCGAATATGAGCACGTCCGCCGCGA
CCTGGACCCCAACGAGGTGTGGGAGA
TCGTGGGCGAGCTGGGCGACGGCGCC
TTCGGCAAGGTTTACAAGGCCAAGAA
TAAGGAGACGGGTGCTTTGGCTGCGG
CCAAAGTCATTGAAACCAAGAGTGAG
GAGGAGCTGGAGGACTACATCGTGGA
GATTGAGATCCTGGCCACCTGCGACC
ACCCCTACATTGTGAAGCTCCTGGGA
GCCTACTATCACGACGGGAAGCTGTG
GATCATGATTGAGTTCTGTCCAGGGG
GAGCCGTGGACGCCATCATGCTGGAG
CTGGACAGAGGCCTCACGGAGCCCCA
GATACAGGTGGTTTGCCGCCAGATGC
TAGAAGCCCTCAACTTCCTGCACAGC
AAGAGGATCATCCACCGAGATCTGAA
AGCTGGCAACGTGCTGATGACCCTCG
AGGGAGACATCAGGCTGGCTGACTTT
GGTGTGTCTGCCAAGAATCTGAAGAC
TCTACAGAAACGAGATTCCTTCATCG
GCACGCCTTACTGGATGGCCCCCGAG
GTGGTCATGTGTGAGACCATGAAAGA
CACGCCCTACGACTACAAAGCCGACA
TCTGGTCCCTGGGCATCACGCTGATT
GAGATGGCCCAGATCGAGCCGCCACA
CCACGAGCTCAACCCCATGCGGGTCC
TGCTAAAGATCGCCAAGTCAGACCCT
CCCACGCTGCTCACGCCCTCCAAGTG
GTCTGTAGAGTTCCGTGACTTCCTGA
AGATAGCCCTGGATAAGAACCCAGAA
ACCCGACCCAGTGCCGCGCAGCTGCT
GGAGCATCCCTTCGTCAGCAGCATCA
CCAGTAACAAGGCTCTGCGGGAGCTG
GTGGCTGAGGCCAAGGCCGAGGTGAT
GGAAGAGTGA

Final protein sequence:

The following mutations have been detected in that clone with respect to the reference sequence: V62A, E136V, G317E. The mutations have been confirmed by electron density and ESI-MS.

smRKSREYEHVRRDLDPNEVWEIVGE
LGDGAFGKVYKAKNKETGALAAAKVI
ETKSEEELEDYIVEIEILATCDHPYI
VKLLGAYYHDGKLWIMIEFCPGGAVD
AIMLELDRGLTEPQIQVVCRQMLEAL
NFLHSKRIIHRDLKAGNVLMTLEGDI
RLADFGVSAKNLKTLQKRDSFIGTPY
WMAPEVVMCETMKDTPYDYKADIWSL
GITLIEMAQIEPPHHELNPMRVLLKI
AKSDPPTLLTPSKWSVEFRDFLKIAL
DKNPETRPSAAQLLEHPFVSSITSNK
ALRELVAEAKAEVMEE

Host: BL21 (DE3)-R3-pRARE2

Growth medium, induction protocol: 4x 10 ml from a 50 ml overnight culture in LB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol were used to inoculate 4x 1 L of LB media containing 35 µg/ml kanamycin (no chloramphenicol) in 2 L baffled shaker flasks. Cultures were grown at 37°C in a shaking incubator until the OD₆₀₀ reached ~0.4. After that the temperature was adjusted to 20°C. Expression was induced using 0.5 mM IPTG at an OD₆₀₀ of 0.6, and the expression left overnight. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5, 200 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 0.2 mM PMSF.

Extraction buffer, extraction method: Cell pellets were lysed by sonication. The lysate was clarified by centrifugation and the supernatant collected for purification.

Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: 50 mM HEPES pH 7.4, 200mM NaCl, 20 mM Imidazole, 0.5 mM TCEP. Wash buffer 1: As Binding buffer except 40mM Imidazole. Wash buffer 2: As Binding buffer except 60 mM Imidazole. Elution buffer: As Binding buffer except 250 mM Imidazole.

Procedure: 10 ml of 50% Ni-Sepharose slurry (GE Healthcare) (5 ml of resin) was applied to a 2 cm diameter gravity column. The column was equilibrated with binding buffer. The lysate was applied to the column which was subsequently washed with binding buffer, wash buffer 1 and 2. STK10 was eluted with 25 ml of elution buffer. The N-terminal His6 tag was cleaved by incubating the protein overnight with TEV protease.

Column 2: Size exclusion chromatography (Superdex S200, 16/60)

SEC-Buffers: 20 mM Hepes, pH 7.4, 300 mM NaCl, 0.5 mM TCEP.

Procedure: The fractions eluted from the Ni-affinity chromatography were concentrated to about 10 ml by ultrafiltration. The concentrated protein was applied to the column (pre-equilibrated in SEC buffer) at a flow rate of 0.7 ml/min.

Column 3: Ni-affinity chromatography (reverse purification).

Procedure: Fractions from the gel filtration containing STK10 were pooled and passed through a column of Ni-Sepharose (2 ml, pre-equilibrated in SEC Buffer). The resin was eluted with SEC Buffer containing 10, 20, 30, 40 mM imidazole. STK10 was present in the flow-through and 10 mM imidazole elution.

Crystallization: STK10 was concentrated to 13.6 mg/ml. Crystals were obtained using the vapor diffusion method using this protein mixed with 1 mM Bosutinib-like compound, mixed 1:1 with a well solution containing 0.1M SPG pH 7.0, 60% MPD. Crystals appeared after a couple of days at 4°C.

Data Collection: Crystals were directly flash frozen in liquid nitrogen. Diffraction data were collected at the Diamond beam line I04.

References

Berthou, S., Aebersold, D. M., Schmidt, L. S., Stroka, D., Heigl, C., Streit, B., Stalder, D., Gruber, G., Liang, C., Howlett, A. R. , et al. (2004). The Met kinase inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants. Oncogene 23 , 5387-5393.
Endo, J., Toyama-Sorimachi, N., Taya, C., Kuramochi-Miyagawa, S., Nagata, K., Kuida, K., Takashi, T., Yonekawa, H., Yoshizawa, Y., Miyasaka, N., and Karasuyama, H. (2000). Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes. FEBS Lett 468 , 234-238.
Kuramochi, S., Matsuda, Y., Okamoto, M., Kitamura, F., Yonekawa, H., and Karasuyama, H. (1999). Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues. Immunogenetics 49 , 369-375.
Kuramochi, S., Moriguchi, T., Kuida, K., Endo, J., Semba, K., Nishida, E., and Karasuyama, H. (1997). LOK is a novel mouse STE20-like protein kinase that is expressed predominantly in lymphocytes. J Biol Chem 272 , 22679-22684.
Ma, P. C., Jagadeeswaran, R., Jagadeesh, S., Tretiakova, M. S., Nallasura, V., Fox, E. A., Hansen, M., Schaefer, E., Naoki, K., Lader, A. , et al. (2005). Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res 65 , 1479-1488.
Schreiner, B., Baur, D. M., Fingerle, A. A., Zechner, U., Greten, F. R., Adler, G., Sipos, B., Kloppel, G., Hameister, H., and Schmid, R. M. (2003). Pattern of secondary genomic changes in pancreatic tumors of Tgf alpha/Trp53+/- transgenic mice. Genes Chromosomes Cancer 38 , 240-248.
Walter, S. A., Cutler, R. E., Jr., Martinez , R., Gishizky, M., and Hill, R. J. (2003). Stk10, a new member of the polo-like kinase kinase family highly expressed in hematopoietic tissue. J Biol Chem 278 , 18221-18228.