F-BAR domain of human FES tyrosine kinase

Target ID:

FESA

Aliases:

FESFPS

Deposition Date:

Wednesday 29th February 2012

Families:

Non-protein KinaseNucleotide metabolism

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

4DYL

Authors:

Ugochukwu E., Salah E., Elkins J., Barr A., Krojer T., Filippakopoulos P., Weigelt J., Arrowsmith C.H., Edwards A., Bountra C., von Delft F., Knapp S.

Site:

Oxford

4DYL

tabs

Structure Details

Fes forms with Fer a separate family of the non-receptor protein tyrosine kinases. 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.

Here we have solved the structure of the N-terminal FCH domain, revealing the expected dimerization of the helical regions, but unexpectedly revealing a linear structure, in contrast to the curved structure of most FBAR (FCH-BAR) domains which is a feature of their ability to bind at the membrane and influence membrane curvature.

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.

Materials and Methods

FES

PDB:4DYL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Mammalian Gene Collection (IMAGE consortium clone ID 5170548).
SGC Clone Accession:
Tag:N-terminal, TEV cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMGFSSELCSPQGHGVLQQMQEAELRLLEGMRKWMAQRVKSDREYAGLLHHMSLQDSGGQSRAISPDSPISQSWAEITSQTEGLSRLLRQHAEDLNSGPLSKLSLLIRERQQLRKTYSEQWQQLQQELTKTHSQDIEKLKSQYRALARDSAQAKRKYQEASKDKDRDKAKDKYVRSLWKLFAHHNRYVLGVRAAQLHHQHHHQLLLPGLLRSLQDLHEEMACILKEILQEYLEISSLVQDEVVAIHREMAAAAARIQPEAEYQGFLRQYGSAPDVPPCVTFDESLLEEGEPLEPGELQLNELTVESVQHTLTSVTDELAVATEMVFRRQEMVTQLQQELRNEEENTHPRERVQLLGKRQVLQEALQGLQVALCSQAKLQAQQELLQTKLEHLGPGEPPPVLLLQDD

Met1 to Asp405
The N-terminal residues, MHHHHHHSSGVDLGTENLYFQS, derive from the vector
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Procedure
Cell Lysis: The resuspended cell pellet was thawed and lysed using a high pressure homogeniser. PEI (polyethyleneimine) was added to a final concentration of 0.15 %. The cell debris and precipitated DNA were spun down.Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5 mM Imidazole, 5% Glycerol, 0.5 mM TCEP, Protease inhibitor cocktail (Roche) and 15 U/mL benzonase. Purification: Column 1: 5 ml of Ni-Sepharose in a 2 cm diameter gravity flow column. Column 1 Buffers: Binding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5 mM Imidazole, 5% Glycerol, 0.5 mM TCEP. Wash Buffer: As Binding Buffer except 25 mM imidazole.Elution Buffer: As Binding Buffer except 250 mM imidazole. Column 1 Procedure: The clarified supernatant was passed through the column. The column was washed with 50 ml of Binding Buffer and Wash Buffer and 25 ml of Elute Buffer was passed through to elute the protein. Column 2: S75 16/60 Gel Filtration (GE Healthcare) Column 2 Buffers: GF Buffer: 20 mM Tris pH 7.4, 200 mM NaCl, 2 mM DTT Column 2 Procedure: The eluted protein was concentrated to 5 ml volume and injected onto the column.

Extraction

Procedure
Expression strain: BL-21(DE3)-R3-pRARE2 (A homemade phage resistant version of BL21(DE3)). Transformation: The construct DNA was transformed into homemade chemically competent cells of the expression strain by a standard heat shock procedure. Expression: Colonies were used to inoculate 2x 50 mL of TB media containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol in a 250 ml baffled shaker flask, which was placed in a 37°C shaker overnight. The next day 6x 10 mL of this starter culture was used to inoculate 6x 1L of TB media containing 50 µg/ml kanamycin in 2L baffled shaker flasks. When the OD600 was approximately 1.5, the temperature was reduced to 18°C and the cells were induced by the addition of 1 mM IPTG. The expression was continued overnight. Cell harvest: Cells were spun at 6000rpm for 10 mins and the pellets resuspended in Lysis Buffer and then frozen at -20°C
Concentration:The FES FBAR domain was concentrated to 19.8 mg/ml (measured by 280 nm absorbance).
Ligand
MassSpec:Expected: 46418Observed: 46419
Crystallization:The compound Phosphoinositol-3,4,5-triphosphate (PI(3,4.5)P3) was added to the concentrated protein sample to a concentration of 1 mM, although the compound was not visible in the resulting structure. Crystals grew from a 2:1 ratio of protein and precipitant solution (0.2M Sodium malonate, 0.1M BisTrisPropane pH 6.5, 20% PEG 3350, 10% Ethylene glycol), using the vapour diffusion method.

Selenomethione derivative preparation
Crystals of selenomethionine derivatised protein were used to solve the structure. This protein was expressed using a similar protocol as for the native protein except with M9 minimal media and selenomethionine added at the time of induction.The selenomethionine derivatised protein was purified similarly except with a gel filtration buffer of 20 mM Tris pH 7.4, 150 mM NaCl, 5% glycerol and 2 mM DTT.The observed MW indicated that 9x Se were incorporated into the protein.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected by equilibration into precipitant solution containing 20% ethylene glycol, and then flash frozen in liquid nitrogen. Data was collected at Diamond, beamline I24.
Data Processing:

References

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.