Human Protein Receptor Tyrosine Phosphatase E

Target ID:

PTPREA

Aliases:

DKFZp313F1310HPTPEPTPEPTPRER-PTP-EPSILON

Deposition Date:

Monday 31st March 2008

Families:

Phosphatases

Related Diseases:

MetabolismSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2JJD

Authors:

J.M.ELKINS,E.UGOCHUKWU,I.ALFANO,A.BARR,G.BUNKOCZI,O.KING,P.FILIPPAKOPOULOS,P.SAVITSKY,E.SALAH,A.PIKE,C.JOHANSSON,S.DAS,N.BURGESS-BROWN,O.GILEADI,F.VONDELFT,C.H.ARROWSMITH,C.BOUNTRA,A.M.EDWARDS,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2JJD

tabs

Structure Details

PTPRE belongs, together with RPTPA, to the receptor class 4 subfamily of protein-tyrosine phosphatases. Members of this phosphatase family contain a short extracellular domain, a single transmembrane region, and two tandem cytoplasmic catalytic domains (D1 and D2 domain). In addition to the transmembrane form of PTPRE, four cytoplasmic variants (cyt PTPepsilon, p67, p65, cyt-PTPepsilonPD1) lacking the extracellular and membrane spanning domains have been described.

PTPRE is involved in regulation of a variety of signaling pathways including voltage-gated potassium channel activity, negative regulation of insulin-receptor and JAK/STAT signaling. A role of PTPRE in osteoclast and macrophage function, regulation of cerebellar development as well as in suppression of endothelial cell proliferation has also been described.

PTPRE is highly expressed in leukocytes, particularly in monocytes and granulocytes, but moderate expression levels have been detected in most organs. PTPRE mRNA is detected in the neural tube of day 12 postcoitum embryo and in the nervous system including brain and spinal cord, whereas in neonatal mice expression is widely distributed in the central nervous system with strongest expression in the hippocampus, cerebral cortex, and olfactory bulb. In addition, PTPRE expression is significantly elevated in mouse mammary tumours.

PTPRE-/- mice show acute delay in myelination of Schwann cells and enhanced phosphorylation and conductance of voltage-gated potassium channels. Female knockout mice also exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. Transgenic mice overexpressing PTPRE in mammary epithelium develop pronounced and persistent mammary hyperplasia accompanied by residual milk production and solitary mammary tumours are often detected secondary to mammary hyperplasia.

ere we determined the structure of both PTPRE catalytic domains at 3.2 Å resolution. The quality of the refined structure was significantly improved by use of six-fold non-crystallographic symmetry averaging of the electron density maps to aid model building, resulting in a high quality three dimensional model of this interesting enzyme.

Materials and Methods

PTPRE

PDB:2JJD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_006504
Entry Clone Source:Origene
SGC Clone Accession:
Tag:Non-cleavable C-terminal His6 tag.
Host:BL21 (DE3)R3 (Phage resistant strain)

Construct

Prelude:
Sequence:MSGPKKYFPIPVEHLEEEIRIRSADDCKQFREEFNSLPSGHIQGTFELANKEENREKNRYPNILPNDHSRVILSQLDGIPCSDYINASYIDGYKEKNKFIAAQGPKQETVNDFWRMVWEQKSATIVMLTNLKERKEEKCHQYWPDQGCWTYGNIRVCVEDCVVLVDYTIRKFCIQPQLPDGCKAPRLVSQLHFTSWPDFGVPFTPIGMLKFLKKVKTLNPVHAGPIVVHCSAGVGRTGTFIVIDAMMAMMHAEQKVDVFEFVSRIRNQRPQMVQTDMQYTFIYQALLEYYLYGDTELDVSSLEKHLQTMHGTTTHFDKIGLEEEFRKLTNVRIMKENMRTGNLPANMKKARVIQIIPYDFNRVILSMKRGQEYTDYINASFIDGYRQKDYFIATQGPLAHTVEDFWRMIWEWKSHTIVMLTEVQEREQDKCYQYWPTEGSVTHGEITIEIKNDTLSEAISIRDFLVTLNQPQARQ[G]EQVRVVRQFHFHGWPEIGIPAEGKGMIDLIAAVQKQQQQTGNHPITVHCSAGAGRTGTFIALSNILERVKAEGLLDVFQAVKSLRLQRPHMVQTLEQYEFCYKVVQDFIDIFSDYAAHHHHHHHighlighted residue in square brackets corresponds to a mutation Glu581Gly at that position.
Vector: pNIC-CH

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 litre of LB containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD600 reached ~0.3 then the temperature was adjusted to 18°C. Expression was induced for overnight using 1 mM IPTG at an OD600 of 0.8. 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; 0.5 mM TCEP.

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, then washed with 20 ml binding buffer prior to loading the sample.Buffers: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol; 0.5 mM TCEPProcedure: Supernatant was applied by gravity flow, followed by a wash with 100 ml binding buffer. The column flow-through was collected.Column 2: Ni-affinity. Ni-NTA (Qiagen), 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% Glycerol, 0.5 mM TCEP; Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol, 0.5 mM TCEP; Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM Imidazole , 5% Glycerol, 0.5 mM TCEP.Procedure: The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted.Enzymatic treatment : noneColumn 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoadBuffers: 50 mM Tris-HCl, pH 7.5; 250 mM NaCl; 10 mM DTTProcedure: PTPRE was concentrated and applied to a S200 16/60 HiLoad gel filtration column equilibrated in 50 mM Tris-HCl, pH 7.5; 250 mM NaCl; 10 mM DTT using either an ÄKTAprime or ÄKTAexpress system.

Extraction

Procedure
Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification.
Concentration:Protein was concentrated to 10.7 mg/ml using an Amicon 30 kDa cut-off concentrator.
Ligand
MassSpec:LC -ESI -MS TOF gave a measured mass of 69378 for this construct. Predicted mass 69580. Sequencing indicated a base change at position 1427bp from ATG changing A to G (Glu581 to Gly). No human EST has been reported in Genbank suggesting this may be a PCR error. The discrepancy in mass was interpreted as a loss of the N-terminal methionine during expression and the Glu to Gly change.
Crystallization:Crystals were grown at 20°C from a 1:1 ratio of protein to reservoir solution, where the reservoir solution was 1.6 M MgSO4, 0.1 M MES pH 6.5.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using 2.25M Li2SO4. X-ray source: Diffraction data were collected at the SLS beamline SAX10 at a single wavelength.Resolution: 3.2 Å resolution limit.
Data Processing:

References

Sines T, Granot-Attas S, Weisman-Welcher S, Elson A. (2007) Association of tyrosine phosphatase epsilon with microtubules inhibits phosphatase activity and is regulated by the epidermal growth factor receptor. Mol Cell Biol.; 27(20):7102-12.

Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein RM, Goichberg P, Spiegel A, Elson A, Lapidot T. (2006). Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med.; 12(6):657-64.

Granot-Attas S, Elson A. (2004). Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells. Exp Cell Res.; 294(1):236-243.

Chiusaroli R, Knobler H, Luxenburg C, Sanjay A, Granot-Attas S, Tiran Z, Miyazaki T, Harmelin A, Baron R, Elson A. (2004). Tyrosine phosphatase epsilon is a positive regulator of osteoclast function in vitro and in vivo. Mol Biol Cell.; 15(1):234-244.

Toledano-Katchalski H, Tiran Z, Sines T, Shani G, Granot-Attas S, den Hertog J, Elson A. (2003). Dimerization in vivo and inhibition of the nonreceptor form of protein tyrosine phosphatase epsilon. Mol Cell Biol.;23(15):5460-5471.

Tanuma N, Shima H, Nakamura K, Kikuchi K. (2001). Protein tyrosine phosphatase epsilonC selectively inhibits interleukin-6- and interleukin- 10-induced JAK-STAT signaling. Blood.;98(10):3030-3034.