Human suppressor of cytokine signaling 6: SH2 complex with c-KIT phosphopeptide

Target ID:

SOCS6B

Aliases:

CIS4HSPC060SOCS4SOCS6SSI4STAI4STATI4

Deposition Date:

Friday 30th November 2007

Families:

SOCS-box-containingUbiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

MetabolismSignalling

Structure type:

apo

Download Coordinates:

2VIF

Authors:

A.BULLOCK,A.C.W.PIKE,P.SAVITSKY,T.KEATES,E.S.PILKA,F.VON DELFT,A.EDWARDS,J.WEIGELT,C.H.ARROWSMITH,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2VIF

tabs

Structure Details

The suppressor of cytokine signaling (SOCS) family comprises CIS1 and SOCS1-7. Members of this family are characterized by a SH2 substrate recognition domain and a C-terminal SOCS box which mediates assembly into ElonginBC-cullin ubiquitin ligase complexes. CIS1 and SOCS1-3 are well characterized as negative feedback inhibitors of cytokine receptor signaling via the JAK/STAT pathway, but the function of SOCS4-7 is less understood.

SOCS6 is ubiquitously expressed and is present in both the nuclear and cytoplasmic fractions. Transgenic mice overexpressing SOCS6 display improved glucose tolerance and insulin sensitivity similar to p85PI3K heterozygous mice (1). An insulin-dependent interaction between SOCS6 and the p85a subunit of the PI 3-Kinase was found, but its physiological importance is uncertain as SOCS6 knockout mice develop normally with the exception of a 10% reduction in body weight (2).

The SOCS6 SH2 domain preferentially binds to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions (2). The in vivo interaction between SOCS6 and c-KIT (SCF receptor) was mapped to the juxtamembrane region pY568 site of c-KIT which matches the determined consensus (NGNN(pY)VYIDPT). This interaction was dependent on SCF-stimulation and decreased cell proliferation by reduced activation of ERK1/2 and p38, but did not inhibit activation of AKT or STATs (3). In order to gain insight into the substrate recognition of SOCS6 we determined the structure of the SOCS6 SH2 domain in complex with the c-KIT juxtamembrane tyrosine phosphorylated peptide at 1.45Å resolution.

Materials and Methods

SOCS6 + phosphopeptide

PDB:2VIF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi| 21450784
Entry Clone Source:FivePrime
SGC Clone Accession:
Tag:Cleavable N-terminal His6 tag (*): mhhhhhhssgvdlgtenlyfq*SM
Host:Phage-resistant derivative of BL21(DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*SMVQSSGPM-D-VTSLTEELKKLAKQGWYWGPITRWEAEGKLANVPDGSFLVRDSSDDRYLLSLSFRSHGKTLHTRIEHSNGRFSFYEQPDVEGHTSIVDLIEHSI-G-DSENGAFCYSRSRLPGSATYPVRLTNPVSRFMQVRS

Two amino acids changes from gi| 21450784 originated from PCR and are highlighted.

Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:5ml from a 50 ml overnight TB culture containing 50 µg/ml kanamycin/34 µg/ml chloramphenicol was used to inoculate each of 4x 1 litre of TB containing 50 µg/ml kanamycin/34 µg/ml chloramphenicol. Cultures were grown at 37°C until the OD600 reached ~2.0 then the temperature was adjusted to 18°C. Expression was induced overnight using 0.5 mM IPTG. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol.

Purification

Procedure
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, washed with 20 ml binding buffer prior to loading the sample.Buffers: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerolProcedure: Supernatant was applied by gravity flow, followed by a wash with 50 ml binding buffer. The column flow-through was collected.Column 2: 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, 30 mM Imidazole, 5% glycerol; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole, 5% GlycerolProcedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-sepharose column. The column was then washed with 100 ml wash buffer under gravity flow. The protein was eluted by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM and 250 mM); fractions were collected until essentially all protein was eluted. 50 mM L-arginine/50 mM L-glutamate and 10 mM DTT were added for overnight storage together with TEV protease for cleavage of the N-terminal hexahistidine tag.Column 3: Size Exclusion Chromatography. Superdex S75 16/60 HiLoadBuffers : 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTTProcedure: The TEV-cleaved protein was concentrated to 3ml using an Amicon 5 kDa cut-off concentrator and ran on a S75 gel filtration column. Elution fractions containing SOCS6 were pooled and an additional 5 mM DTT was added for overnight storage.Column 4: Ion Exchange Chromatography. Source15QBuffers: Buffer A: 50 mM HEPES pH 7.5; Buffer B: 50 mM HEPES pH 7.5, 2M NaClProcedure: The gel filtration elution was diluted five fold with BufferA and loaded onto a Source15Q anion exchange column equilibrated in the same buffer. SOCS6 did not bind the column and was collected in the flow through. Contaminants bound the column and were eluted with BufferB.

Extraction

Procedure
Extraction method: Frozen pellets were thawed and fresh 1 mM PMSF and 0.5 mM TCEP were added. Cells were lysed by sonication. The lysate was centrifuged at 16,000 rpm for 60 minutes and the supernatant collected for purification.
Concentration:Protein concentration: The protein buffer was adjusted to 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM DTT, 50 mM L-arginine/50 mM L-glutamate and concentrated to 13.0 mg/ml using an Amicon 5 kDa cut-off concentrator. An excess of phosphopeptide (NGNN(pY)VYIDPT derived from c-KIT pY568) was present during concentration and a further 2mM peptide was added from dry powder to the final crystal sample.
Ligand
phosphopeptide (NGNN(pY)VYIDPT derived from c-KIT pY568)MassSpec:Mass spec characterization: LC- ESI -MS TOF. Expected mass (after TEV cleavage): 18363. The expected mass was observed.
Crystallization:Crystallization: Crystals were grown at 4°C in 150 nl sitting drops mixing 50 nl of protein with 100 nl of a solution containing 0.2M (NH4)2SO4; 0.1M MES pH 6.5; 30% mPEG 5K. The crystals were cryo-protected using 20% ethylene glycol which was added to the drop 20 seconds prior to mounting and flash freezing in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Data collection: Resolution: 1.45 Å. X-ray source: Diffraction data were collected at the SLS beamline X10SA at a single wavelength (0.98248 Å).
Data Processing:

References

1. Li L, Grønning LM, Anderson PO, Li S, Edvardsen K, Johnston J, Kioussis D, Shepherd PR, Wang P. (2004) Insulin induces SOCS-6 expression and its binding to the p85 monomer of phosphoinositide 3-kinase, resulting in improvement in glucose metabolism. J Biol Chem. 279, 34107-14.

2. Krebs DL, Uren RT, Metcalf D, Rakar S, Zhang JG, Starr R, De Souza DP, Hanzinikolas K, Eyles J, Connolly LM, Simpson RJ, Nicola NA, Nicholson SE, Baca M, Hilton DJ, Alexander WS. (2002) SOCS-6 binds to insulin receptor substrate 4, and mice lacking the SOCS-6 gene exhibit mild growth retardation. Mol Cell Biol. 22, 4567-78.

3. Bayle J, Letard S, Frank R, Dubreuil P, De Sepulveda P. (2004) Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling. J Biol Chem. 279, 12249-59.