Human Protein-Tyrosine Nonreceptor-type 3 (PTPH1)

Target ID:

PTPN3A

Aliases:

DKFZp686N0569PTPH1

Deposition Date:

Friday 23rd September 2005

Families:

Phosphatases

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2B49

Authors:

E.UGOCHUKWU,C.ARROWSMITH,A.BARR,G.BUNKOCZI,S.DAS,J.DEBRECZENI,A.EDWARDS,J.ESWARAN,S.KNAPP,M.SUNDSTROM,A.TURNBULL,F.VON DELFT,J.WEIGELT,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2B49

tabs

Structure Details

PTPN3 belongs to the non-receptor class of protein tyrosine phosphatses of the NT5 subtype. It contains a C-terminal PTP domain and an N-terminal domain homologous to the band 4.1 superfamily of cytoskeletal-associated proteins (FERM domain, also called ERM - for ezrin, radixin and moesin - domain) as well as a PDZ domain. PTPN3 was found to be mostly associated with membrane structures in cells and this association appears to be mediated by the N-terminal portion of PTPN3. In addition, the PTPN3 N-terminal segment has been shown to exert an inhibitory effect on its enzymatic activity in vitro .

PTPN3 binds to 14-3-3 adaptor proteins in cells, in a manner dependent on phosphorylation of PTPN3 which is regulated by mitogenic signals linking tyrosine and serine/threonine phosphorylation signalling pathways.
So far only a few substrates of PTPN3 have been identified:
Immunoblot analysis showed that wildtype PTPN3 specifically dephosphorylates VCP (valosin-containing protein),
a cell cycle regulator involved in a variety of membrane related functions.

In addition, PTPN3 dephosphorylates the immune tyrosine-based activation motifs of TCR (T cell-receptor) zeta resulting in attenuation of T-cell receptor signalling.
Along these lines, dephosphorylation and of the protein tyrosine kinase lck by PTPN3 results in further reduction of phosphoryaltion of the TCR.

PTPN3 is expressed in lymphoid organs but RT-PCR analysis detected high levels of PTPN3 expression in a wide variety of tumor cell lines.
In addition, a study by Wang et al. showed that PTPN3 is a mutational hotspot in colorectal cancers suggesting that PTPN3 is a tumor suppressor gene regulating cellular pathways that may be amenable to therapeutic intervention.

Materials and Methods

PTPN3

PDB:2B49

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|18104986
Entry Clone Source:Origene
SGC Clone Accession:
Tag:
Host:BL21 (DE3)

Construct

Prelude:
Sequence:MDTLEGSMAQLKKGLESGTVLIQFEQLYRKKPGLAITFAKLPQNLDKNRYKDVLPYDTTRVLLQGNEDYINASYVNMEIPAANLVNKYIATQGPLPHTCAQFWQVVWDQKLSLIVMLTTLTERGRTKCHQYWPDPPDVMNHGGFHIQCQSEDCTIAYVSREMLVTNTQTGEEHTVTHLQYVAWPDHGVPDDSSDFLEFVNYVRSLRVDSEPVLVHCSAGIGRTGVLVTMETAMCLTERNLPIYPLDIVRKMRDQRAMMVQTSSQYKFVCEAILRVYEEGLVQM
Vector:pLIC-SGC1

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 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.

Enzymatic treatment: (His tag cleavage using TEV) Samples containing PTPN3 were pooled and TEV protease added for overnight incubation at 4°C. Cleaved products and TEV protease were removed by binding to Ni-NTA agarose using the following procedure. The buffer was changed to 50 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM TCEP using a 10 kDa cut-off concentrator and the TEV-treated protein sample mixed with Ni-NTA agarose for 30 minutes at 4°C. The resin was centrifuged and the supernatant containing the cleaved protein collected.

Column 3: SEC

Buffers: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM DTT.

Procedure: AKTA-prime

Protein concentration: Centricons 30 kDa cut off

Extraction

Procedure
Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 40 minutes and the supernatant collected for purification.
Concentration:
Ligand
MassSpec:
Crystallization:PTPN3 was crystallized using the vapor diffusion method at 20°C and a protein concentration of 7.5 mg/ml. 150nl of the protein was mixed with 50nl of a solution containing 2 M (NH4)2SO4 0.1 MBIS-TRIS 5.5. Crystals grew within 3 days and were cryo-stabilize using the crystallization solution and 30% sucrose.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Itoh, F.; Ikuta, S.; Hinoda, Y.; Arimura, Y.; Ohe, M.; Adachi, M.; Ariyama, T.; Inazawa, J.; Imai, K.; Yachi, A. (1993). Expression and chromosomal assignment of PTPH1 gene encoding a cytosolic protein tyrosine phosphatase homologous to cytoskeletal-associated proteins. Int. J. Cancer 55: 947-951.

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.

Yang, Q.; Tonks, N. K. (1991). Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin. Proc. Nat. Acad. Sci. 88: 5949-5953.

Zhang, S.-H.; Liu, J.; Kobayashi, R.; Tonks, N. K. (1999). Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1. J. Biol. Chem. 274: 17806-17812.