Human feline sarcoma oncogene in complex with Staurosporine and a Consensus Peptide

Target ID:

FESA

Aliases:

FESFPS

Deposition Date:

Friday 22nd February 2008

Families:

Protein Kinase

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

3CBL

Authors:

Filippakopoulos, P., Salah, E., Cooper, C., Elkins, J. M., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S.

Site:

Oxford

3CBL

tabs

Structure Details

Fes forms with Fer a separate family of the non-receptor protein tyrosine kinase. Both proteins share a similar domain organization comprising a unique N-terminal Fps/Fes/Fer/CIP4 homology (FCH) domain, regions of predicted coiled coils (CC), a central Src homology 2 (SH2) domain, and a C-terminal tyrosine kinase domain. The coiled-coil motif in Fps/Fes is thought to have an auto-regulatory function and a model has been proposed in which the coiled-coil domains regulate interconversion between inactive monomers and (auto)-activated oligomers.

Fes is involved in several processes in the function of hematopioetic cells including regulation of innate immune response, hematopoiesis, regulation of angiogenesis and it may play a general role in the regulation of vesicular trafficking and microtubule dynamics including microtubule nucleation and bundling. In addition, Fes is a regulator of inflammation and plays a role in leucocyte recruitment to areas of inflammation.

Interestingly, a proto-oncogene as well as a tumor suppressor function has been proposed. For instance, in acute promyelocytic leukemia the chromosomal location of Fes has been linked to a specific translocation event and activation of Fes can mediate cellular transformation. In addition, elevated expression levels of Fes are found in adult human myeloid leukemia cells and transgenic mice, expression of Gag–Fps/Fes under the control of a heterologous promoter, develop tumors in lymphoid and mesencymal tissues.

On the other hand, reduced expression of Fes has been described in anaplastic thyroid cancers and colorectal cancers. Introduction of Fes into these lines with a recombinant retrovirus suppresses cell growth in soft agar and missense mutations in sequences encoding the kinase domain of Fes have been described in a screen of human colorectal cancer that lead to Fes inactivation.

In contrast to Src, Hck and Abl, Fes/Fer does not posses an SH3 domain and can therefore not assume an auto-inhibited state in which the SH2/SH3 unit suppresses kinase activity. In order to shed light on the regulation of FES we determined the structure of the SH2-kinase domain at high resolution of the apo-protein (3BKB), mono-phosphorylated FES (3CD3) as well as inactive FES (3CBL) in complex with a consensus kinase substrate peptide.

Materials and Methods

FES + staurosporin + peptide

PDB:3CBL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:5170548
Entry Clone Source:MGC
SGC Clone Accession:
Tag:Tag sequence:mhhhhhhssgvdlgtenlyfq*sm, cleaved at the * with TEV protease.
Host:BL21(DE3)-R3-Yop-PPase: a BL21(DE3) derivative expressing Yersinia phosphatase YopJ.The co-expression plasmid has been described in: Seeliger MA, Young M, Henderson MN, Pellicena P, King DS, Falick AM, Kuriyan J. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 2005 Dec;14(12):3135-9 and has been kindly provided by the Kuriyan laboratory.

Construct

Prelude:
Sequence:lowercase refers to tag sequencemhhhhhhssgvdlgtenlyfq*smIPEVQKPLHEQLWYHGAIPRAEVAELLVHSGDFLVRESQGKQEYVLSVLWDGLPRHFIIQSLDNLYRLEGEGFPSIPLLIDHLLSTQQPLTKKSGVVLHRAVPKDKWVLNHEDLVLGEQIGRGNFGEVFSGRLRADNTLVAVKSCRETLPPDLKAKFLQEARILKQYSHPNIVRLIGVCTQKQPIYIVMELVQGGDFLTFLRTEGARLRVKTLLQMVGDAAAGMEYLESKCCIHRDLAARNCLVTEKNVLKISDFGMSREEADGVYAASGGLRQVPVKWTAPEALNYGRYSSESDVWSFGILLWETFSLGASPYPNLSNQQTREFVEKGGRLPCPELCPDAVFRLMEQCWAYEPGQRPSFSTIYQELQSIRKRHR
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Host cells transformed with the expression plasmids were plated out onto LB-agar plates containing 50 µg/ml kanamycin + 50 µg/ml streptomycin. The next day several colonies were combined into 1 ml TB (Terrific Broth), 50 µg/ml kanamycin + 50 µg/ml streptomycin, which was then grown overnight and stored as glycerol stocks at -80°C.The glycerol stock was used to inoculate a 10-ml starter culture in TB + kanamycin and streptomycin (50 µg/ml each). This starter culture was grown overnight at 37°C and used to inoculate a 1 liter culture in the same medium. The culture was grown in baffled flasks at 37°C until the OD600reached ~3.5. After that the temperature was lowered to 18°C. Protein production was induced with 0.1mM IPTG and recombinant FESA was incubation continued at 18°C overnight. The next day cells were harvested by centrifugation at 4000 rpm for 30 minutes. The cell pellet was stored at -80°C.

Purification

Procedure
Column 1: Ni-affinity chromatography: HisTrap FF Crude, 5 ml (GE Healthcare).Buffers: Binding Buffer: 50mM NaH2PO4, 500mM NaCl, 10mM Imidazole, pH 8.0, 5% glycerol, 0.5mM TCEP; Wash Buffer: 50mM NaH2PO4, 500mM NaCl, 20mM Imidazole, pH 8.0, 5% glycerol. 0.5mM TCEP; Elution Buffer: 50mM NaH2PO4, 500mM NaCl, 300mM Imidazole, pH 8.0,, 5% glycerol, 0.5mM TCEP.Procedure: All purification steps were carried out using an AKTAexpress system (GE Healthcare) at 7ºC. The lysate was loaded on a pre-equilibrated His-trap column at 0.8 ml/min. After loading, the column was washed at 0.8 ml/min with 50 ml binding buffer, then 100 ml wash buffer, and the protein was eluted with 25 ml of elution buffer. The peak fraction was collected automatically according to A280.Enzymatic treatment and Tag removal: TEV protease (1:20 w/w), was added to the sample after gel filtration. The sample was incubated at 4°C overnight. The sample was then passed over a column of Ni-sepharose (0.5 ml) to trap the cleaved tag and other Ni-binding proteins; FES was found in the flow-through fraction.Column 2: Size exclusion chromatography HiLoad 16/60 Superdex 75SEC-Buffers: 10mM Hepes, pH 7.5, 500 mM NaCl, 5% glycerol,10 mM DTTProcedure: The FES fractions were collected after his6-tag cleavage and were loaded on a SEC column at 1.0 ml/min. FES eluted at a retention time corresponding to the monomeric protein. Eluted fractions were >95% pure as judged by SDS-PAGE

Extraction

Procedure
2xExtraction buffer: 10mM imidazole, 1.0 M NaCl, 100mM NaH2PO4, pH8.0, 5 mM TCEP, 5% glycerol, 1x protease inhibitor cocktail (Complete PI EDTA-free tablets); Benzonase Nuclease HC, (3 µl per 30ml).Extraction buffer: 10mM imidazole, 500mM NaCl, 50mM NaH2PO4, pH8.0, 0.5mM TCEP, 5% glycerol, 1x protease inhibitor cocktail (Complete PI EDTA-free tablets); Benzonase Nuclease HC, (3 µl per 60ml extraction buffer).Procedure: The cell pellet (40g) from 4 L culture was re-suspended in one volume (40 ml) of 2x extraction buffer. The re-suspended cells were lysed by one passage through a Constant Systems cell breaker and subsequent sonication; the cell breaker was washed with 1x extraction buffer, bringing the total volume to 120 ml. DNA was precipitation by addition of polyethyleneimine (PEI, pH 7.5) to a final concentration of 0.15 % during an incubation time of 30 min on ice, followed by a centrifugation at 17,000 rpm (4°C); The supernatant was further cleared by filtration through a 0.2 µm serum Acrodisc filter.
Concentration:The protein was concentrated to 11 mg/ml in SEC buffer using a centricon device with a 10kDa cut off.
Ligand
staurosporin and Ac-IYESL (peptide)MassSpec:(after tag cleavage): Expected MW: 42704; Measured MW: 42705.
Crystallization:The protein (15 mg/ml) in SEC buffer was incubated with staurosporin and the peptide Ac-IYESL, mixed with an equal volume (300 nl) of reservoir solution (20% PEG 3350, 0.1M NaMalate) and equilibrated as a sitting drop at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were flash frozen in liquid nitrogen using the crystallization condition supplemented with 20% ethylene glycol. Diffraction data were collected to 1.75 Å at a RIGAKU FR-E+ SuperBright light source at a single wavelength of 1.54128 Å.
Data Processing:

References

Fang, F., Ahmad, S., Lei, J., Klecker, R.W., Trepel, J.B., Smithgall, T.E., and Glazer, R.I. (1993). Effect of the mutation of tyrosine 713 in p93c-fes on its catalytic activity and ability to promote myeloid differentiation in K562 cells. Biochemistry 32, 6995-7001.

Groffen, J., Heisterkamp, N., Grosveld, F., Van de Ven, W., and Stephenson, J.R. (1982). Isolation of human oncogene sequences (v-fes homolog) from a cosmid library. Science (New York, NY 216, 1136-1138.

He, Y., Borellini, F., Koch, W.H., Huang, K.X., and Glazer, R.I. (1996). Transcriptional regulation of c-Fes in myeloid leukemia cells. Biochimica et biophysica acta 1306, 179-186.

Pawson, T., Letwin, K., Lee, T., Hao, Q.L., Heisterkamp, N., and Groffen, J. (1989). The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family. Molecular and cellular biology 9, 5722-5725.

Rogers, J.A., Cheng, H.Y., and Smithgall, T.E. (2000). Src homology 2 domain substitution modulates the kinase and transforming activities of the Fes protein-tyrosine kinase. Cell Growth Differ 11, 581-592.

Smithgall, T.E., Rogers, J.A., Peters, K.L., Li, J., Briggs, S.D., Lionberger, J.M., Cheng, H., Shibata, A., Scholtz, B., Schreiner, S., et al. (1998). The c-Fes family of protein-tyrosine kinases. Critical reviews in oncogenesis 9, 43-62.