PTPN14

Target ID:

PTPN14A

Aliases:

MGC126803PEZPTP36

Deposition Date:

Thursday 18th August 2005

Families:

Phosphatases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2BZL

Authors:

J.E.DEBRECZENI,A.BARR,J.ESWARAN,S.DAS,N.BURGESS,E.LONGMAN,O.FEDOROV,O.GILEADI,F.VON DELFT,M.SUNDSTROM,C.ARROWSMITH,J.WEIGELT,A.EDWARDS,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2BZL

tabs

Structure Details

PTPN14 (Pez) is a member of the cytoplamic FERM-domain containing protein tyrosine phosphatase (PTP) family which are characterized by a FERM (Band 4.1, ezrin, radixin, moesin homology) domains at their N-termini, and PTP (protein tyrosine phosphatase) domains at their C-termini. PTPN14 was first cloned in a screen for PTPs expressed in normal breast tissue. It is also expressed in varying amounts in other tissues including kidney, skeletal muscle, lung and placenta. In addition, PTPN14 is highly expressed in human umbilical vein endothelial cells (HUVEC), suggesting it may be a critical enzyme in regulating endothelial cell function.

PTPN14 belongs to the NT6 subtype of PTPs. Its FERM domain directs PTPN14 to the cytoskeleton/plasma membrane interface in a phosphoinositide-dependent manner. PTPN14 also contains an acidic region and a putative SH3 domain-binding sequence in the region between the ezrin-like and catalytic domain. Conservation of the FERM superfamily PTP in Caenorhabditis elegans but not in yeast suggests a fundamental role in the multi-cellular organism.

Subcellular localization of PTPN14 is regulated in both HeLa and human umbilical vein endothelial cells (HUVEC). In cells grown to confluence PTPN14 is localized in the cytosol, where it is concentrated at intercellular junctions, but it is predominantly nuclear in sparsely plated cells that have not yet formed extensive cell-cell contacts. Other factors also regulate the subcellular localization of PTPN14, including TGFÃ¢ , which inhibits translocation of PTPN14 from the cytosol to the nucleus. Furthermore, PTPN14 has been detected in the nucleus in migrating and proliferative cells at the wound edge. TGFÃ¢, which is known to inhibit cell proliferation but not migration, inhibits translocation of PTPN14 to the nucleus, further strengthening the argument that PTPN14 plays a role in the nucleus during cell proliferation.

Knockout mice of PTPN14 show uterine dilation and the presence of keratin in the uterine lumen. Homozygous mutant females have no or reduced mammary gland tissue and an increased anogenital distance.

Mutation in the PTPN14 gene may also play a role in cancer cell invasion and metastasis since increased phosphorylation of adherens junctions has been shown to increase cell motility and migration. Ã¢-catenin, a central molecule in adherence junction and in the wnt-signaling cascade, has been identified as a PTPN14 substrate. In addition, dominant negative PTPN14 enhances both tyrosine phosphorylation of adherens junctions and cell motility. Because increased phosphorylation of adherens junctions has been shown to increase cell motility and migration, mutational inactivation of these genes may be an important step in cancer cell invasion and metastasis. This notion is supported by a mutational analysis of the tyrosine phosphatase gene superfamily in human cancers which identified somatic mutations in PTPN14, affecting together 26% of colorectal cancers and a smaller fraction of lung, breast, and gastric cancers. Many mutations identified were nonsense, frameshift, or splice site alterations predicted to result in truncated proteins lacking phosphatase activity, suggesting that the mutated tyrosine phosphatases like PTPN14 are tumor suppressor genes, regulating cellular pathways that may be amenable to therapeutic intervention.

Materials and Methods

PDB:2BZL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi:34328899 NP_005392 .
Entry Clone Source:Purely Proteins Ltd.
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)R3 (Phage resistant strain)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsMVDATRVPMDERFRTLKKKLEEGMVFTEYEQIPKKKANGIFSTAALPENAERSRIREVVPYEENRVELIPTKENNTGYINASHIKVVVGGAEWHYIATQGPLPHTCHDFWQMVWEQGVNVIAMVTAEEEGGRTKSHRYWPKLGSKHSSATYGKFKVTTKFRTDSVCYATTGLKVKHLLSGQERTVWHLQYTDWPDHGCPEDVQGFLSYLEEIQSVRRHTNSMLEGTKNRHPPIVVHCSAGVGRTGVLILSELMIYCLEHNEKVEVPMMLRLLREQRMFMIQTIAQYKFVYQVLIQFLQNSRLIDSKGGYGSE
Vector:pLIC-SGC1

Growth

Medium:
Antibiotics:
Procedure:1 mL 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 4 hours 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.

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 and 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 TCEP

Procedure: 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 50 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.

Column 3: Size Exclusion Chromatography

Buffers: 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP

Procedure: TEV-cleaved PTPN14 was directly applied to a S200 16/60 HiLoad gel filtration column equilibrated in 50 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP using either an ÄKTAprime or ÄKTAxpress 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:
Ligand
MassSpec:
Crystallization:Crystals were grown at 20°C in 200nl sitting drops mixing 150 nl of protein with 50 nl of a solution containing 0.1 M Bis-Tris HCl pH 5.0; 0.2 M Li2SO4 and 25% PEG 3350. The crystals were cryo-protected using 20% ethylene glycol which was added to the drop 30 seconds prior to mounting.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Smith, A. L.; Mitchell, P. J.; Shipley, J.; Gusterson, B. A.; Rogers, M. V.; Crompton, M. R. (1995). PEZ: a novel human cDNA encoding protein tyrosine phosphatase- and ezrin-like domains. Biochem. Biophys. Res. Commun. 209 : 959-965.

Wadham,C., Gamble,J.R., Vadas,M.A. and Khew-Goodall,Y. (200). Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation. J. Cell. Sci. 113 , 3117-3123.

Wadham,C., Gamble,J.R., Vadas,M.A. and Khew-Goodall,Y. (2003). The protein tyrosine phosphatase Pez is a major phosphatase of adherens junctions and dephosphorylates beta-catenin. Mol. Biol. Cell 14 , 2520-2529.

Smith,A.L., Mitchell,P.J., Shipley,J., Gusterson,B.A., Rogers ,M.V. Crompton,M.R. (1995). Pez: a novel human cDNA encoding protein tyrosine phosphatase- and ezrin-like domains. Biochem. Biophys. Res. Commun. 209 (3), 959-965.

Wang, Z.; Shen, D.; Parsons, D. W.; Bardelli, A.; Sager, J.; Szabo, S.; Ptak, J.; Silliman, N.; Peters, B. A.; van der Heijden, M. S.; Parmigiani, G.; Yan, H.; Wang, T.-L.; Riggins, G.; Powell, S. M.; Willson, J. K. V.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.; Velculescu, V. E. (2004). Mutational analysis of the tyrosine phosphatome in colorectal cancers. Science 304 : 1164-1166.