Human Tyrosine-protein phosphatase non-receptor type 18

Target ID:

PTPN18A

Aliases:

BDP1

Deposition Date:

Wednesday 20th December 2006

Families:

Phosphatases

Related Diseases:

NeurobiologySignallingCancer

Structure type:

apo

Download Coordinates:

2OC3

Authors:

E.UGOCHUKWU,A.BARR,I.ALFANO,F.GORREC,C.UMEANO,P.SAVITSKY,F.SOBOTT,J.ESWARAN,E.PAPAGRIGORIOU,J.E.DEBRECZENI,A.TURNBULL,G.BUNKOCZI,M.SUNDSTROM,C.H.ARROWSMITH,J.WEIGELT,A.EDWARDS,F.VON DELFT,S.KNAPP,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OC3

tabs

Structure Details

PTPN18 belongs to the non-receptor class 4 subfamily of protein-tyrosine phosphatases which is also called the PEST family of PTPs. Members of this family contain an N-terminal phosphatase domain followed by a proline/serine and threonine rich region. In addition to PTPN18 this family comprises PTP-PEST (PTPN12, PTP-P19, PTPG1) and PEP (PTP, PTPN22, LYP). The precise biological function of PTPN18 is still unclear but this phosphatase seems to be involved regulating a number of cytoskeletal functions and differentiation of neuronal cells. PTPN18 regulates HER2 activity and has therefore been proposed to act as a tumor suppressor and several links to tumor growth have been established. PTPN18 was found to differentially dephosphorylate several auto-phosphorylated tyrosine kinases known to be over-expressed in tumor tissues. PTPN18 has been reported to be coexpressed with the oncogene HER2 in breast cancer cells, and has been shown to regulate HER2 activity. In thyroid cancer, several cancer-associated alternative spliced variants have been reported including isoforms that lack the phosphatase domain.

Materials and Methods

PTPN18 - Human tyrosine-protein phosphatase non-receptor type 18

PDB:2OC3

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|18375655
Entry Clone Source:Purely Proteins
SGC Clone Accession:
Tag: PreScissionÂ (rhinovirus 3C)- protease cleavable (*) GST tag:
mspilgywkikglvqptrllleyleekye hlyerdegdkwrnkkfelglefpnlpyyi dgdvkltqsmaiiryiadkhnmlggcpke raeismlegavldirygvsriayskdfet lkvdflsklpemlkmfedrlchktylngd hvthpdfmlydaldvvlymdpmcldafpk lvcfkkrieaipqidkylksskyiawplq gwqatfgggdhppksdlevlfq*gplgsp gip
Host:BL21(DE3) phage resistant

Construct

Prelude:
Sequence:GPLGSPGIPDSARSFLERLEARGGREGAV LAGEFSDIQACSAAWKADGVCSTVAGSRP ENVRKNRYKDVLPYDQTRVILSLLQEEGH SDYINGNFIRGVDGSLAYIATQGPLPHTL LDFWRLVWEFGVKVILMACREIENGRKRC ERYWAQEQEPLQTGLFCITLIKEKWLNED IMLRTLKVTFQKESRSVYQLQYMSWPDRG VPSSPDHMLAMVEEARRLQGSGPEPLCVH CSAGCGRTGVLCTVDYVRQLLLTQMIPPD FSLFDVVLKMRKQRPAAVQTEEQYRFLYH TVAQMFCSTLQNA
Vector:pGEX-6P2

Growth

Medium:
Antibiotics:
Procedure:Starter cultures from freshly transformed colonies in 10 ml LB, and ampicillin were grown overnight. This was diluted 1:1000 in fresh media (6L) and was grown at 37°C to an OD600 of 0.3 and than transferred to 18°C. Expression was induced at an OD600 of 0.8 using 1 mM IPTG. Cells were harvested after 3h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen.

Purification

Procedure
Column 1: Glutathione Sepharose 4B affinity, 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.
Procedure: Supernatant was applied at gravity flow, followed by a wash with 30 ml binding buffer. The GST-fusion was cleaved while bound to the column by addition of PreScission protease. The column was gently rotated overnight a 4°C then protein eluated with 3 bed volumes of binding buffer.

Column 3: SEC
Procedure: AKTA-prime

Protein concentration: Centricons 10 kDa cut off

Extraction

Procedure
The cell pellets (20 g wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 60,000 rpm.
Concentration:
Ligand
MassSpec:LC-ESI-MStof confirmed the correct mass expected for this construct. Expected mass, 34254; Observed mass, 34255
Crystallization:Crystals were obtained using sitting drop method at 4°C. Drops were prepared using 150 nl of protein (8 mg/ml concentration) and 150 nl of the well solution (0.1M HEPES pH 6.8, 25% PEG-3350 and 6% Jeffamine M-600).
NMR Spectroscopy:
Data Collection:Resolution: 1.5 Å; X-ray source: Synchrontron SLS-X10.
Data Processing:

References

Aoki, N., Ueno, S., Mano, H., Yamasaki, S., Shiota, M., Miyazaki, H., Yamaguchi-Aoki, Y., Matsuda, T., and Ullrich, A. (2004). Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation. J Biol Chem 279 , 10765-10775.
Gensler, M., Buschbeck, M., and Ullrich, A. (2004). Negative regulation of HER2 signaling by the PEST-type protein-tyrosine phosphatase BDP1. J Biol Chem 279 , 12110-12116.
Guimaraes, G. S., Latini, F. R., Camacho, C. P., Maciel, R. M., Dias-Neto, E., and Cerutti, J. M. (2006). Identification of candidates for tumor-specific alternative splicing in the thyroid. Genes Chromosomes Cancer 45 , 540-553.
Wang, B., Lemay, S., Tsai, S., and Veillette, A. (2001). SH2 domain-mediated interaction of inhibitory protein tyrosine kinase Csk with protein tyrosine phosphatase-HSCF. Mol Cell Biol 21 , 1077-1088.