Human Tyrosine Receptor Phosphatase kappa

Target ID:

PTPRKA

Aliases:

DKFZp686C2268DKFZp779N1045R-PTP-kappa

Deposition Date:

Monday 28th November 2005

Families:

Phosphatases

Related Diseases:

SignallingCancerNeurobiology

Structure type:

apo

Download Coordinates:

2C7S

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2C7S

tabs

Structure Details

PTPRÃª belongs together with PTPRÃ¬, PTPRÃ± and PTPRÃ« to the R2A/IIb subfamily of receptor protein tyrosine phosphatases. The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPRÃª possesses an extracellular region, a single transmembrane region, and two tandem catalytic domains. The extracellular region contains a meprin-A5 antigen-PTP mu (MAM) domain, an Ig-like domain and four fibronectin type III-like repeats. The purified extracellular domain of PTPRÃª functions as a substrate for adhesion by cells expressing PTPRÃª and induces aggregation of coated synthetic beads.

PTPRÃª is widely expressed with high expression detected in spleen, prostate and ovary brain, lung, skeletal muscle, heart, placenta, liver, kidney, and intestine.
In the brain, expression can be found in cortical, olfactory, hippocampal, hypothalamic, amygdaloid and visual structures.
Expression of PTPRÃª is stimulated by TGF-beta and is induced by high cell density.

PTPRÃª has been shown to stimulate cell motility and neurite outgrowth and is required for both the anti-proliferative and the pro-migratory effects of TGF-beta,
suggesting a role in regulation, maintenance and restitution of cell adhesions.
These functions might be regulated through dephosphorylaion of beta- and gamma-catenin at adherens junctions.

Expression of PTPRÃª is absent or down-regulated in more than 20% of melanoma cell lines and in some un-manipulated melanoma biopsies. Furthermore, the human PTPRÃª gene lies in a region frequently deleted in hematological neoplasms, melanomas, ovary carcinomas, and many other solid tumors and a peptide derived from mutated PTPRÃª has been shown to be immunogenic melanoma-specific .

However, PTPRÃª plays a dual role as tumor suppressor and tumor-promoter in mammary epithelial cells.
RNA interference (RNAi) experiments showed that down-regulation of PTPRÃª results in acceleration of cell cycle progression, enhancement of the cellular response to epidermal growth factor (EGF), and abrogates TGF-beta-mediated antimitogenesis whereas over expression of PTPR k results in inhibition of basal and EGF- induced proliferation and ERbB receptor signalling in cancer cells.

Materials and Methods

PTPRK

PDB:2C7S

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|18860902
Entry Clone Source:Purely Proteins
SGC Clone Accession:
Tag:Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*smPAIRVADLLQHINLMKTSDSYGFKEEYESFFEGQSASWDVAKKDQNRAKNRYGNIIAYDHSRVILQPVEDDPSSDYINANYIDGYQRPSHYIATQGPVHETVYDFWRMIWQEQSACIVMVTNLVEVGRVKCYKYWPDDTEVYGDFKVTCVEMEPLAEYVVRTFTLERRGYNEIREVKQFHFTGWPDHGVPYHATGLLSFIRRVKLSNPPSAGPIVVHCSAGAGRTGCYIVIDIMLDMAEREGVVDIYNCVKALRSRRINMVQTEEQYIFIHDAILEACLCGETAIPVCEF*DSKGGYGSE
Vector:pLIC-SGC

Growth

Medium:
Antibiotics:
Procedure:1mL from a 10 mL LB overnight culture containing 50 Âµg/mL kanamycin was used to inoculate 1 liter of LB media containing 50 Âµg/mL kanamycin. Cultures were grown at 37Â°C until the OD600 reached ~0.3. After that the temperature was adjusted to 18Â°C. Expression was induced for 4 hours using 1mM 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: Ni-affinity chromatography.

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 5% Glycerol. Wash buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol. Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 50 to 250mM Imidazole, 5% Glycerol.

Procedure: 5 mL of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 30 mL binding buffer. The lysate was applied to the column which was subsequently washed with 3 x 10 mL wash buffer. PTPRK was eluted by a step gradient generated by 5 mL portions of elution buffer with increasing concentration of imidazole (concentrations used: 50mM, 100mM, 250mM). The eluted protein fractions were collected and analyzed by SDS Â PAGE . After elution DTT was added to a final concentration of 10mM.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

SEC -Buffers: 10 mM Hepes, pH 7.5, 25 mM NaCl.

Procedure: The fractions eluted of the Ni-affinity chromatography were pooled and concentrated to about 1 mL using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 1 mL/min. Eluted fractions were 95% pure as judged by SDS - PAGE .

Protein concentration: Centricon with a 10kDa cut off in SEC -buffer

Extraction

Procedure
Cell pellets were lysed using a high pressure cell disrupter. The lysate was centrifuged at 50,000 rpm for 40 minutes and the supernatant collected for purification.
Concentration:
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/mL by mixing 100nl of the concentrated protein with 100nl of a well solution containing 0.20 M NaNO₃, 20.0% PEG 3350, 10.0% Ethylene glycole.
NMR Spectroscopy:
Data Collection:Resolution: 1.9 Ã; Crystals were cryo-protected using the well solution and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected at the SLS beamLine X10 at a single wavelength (0.99 nm).
Data Processing:

References

Wang SE, Wu FY , Shin I, Qu S, Arteaga CL (2005). Transforming growth factor {beta} (TGF-{beta})-Smad target gene protein tyrosine phosphatase receptor type kappa is required for TGF-{beta} function. Mol Cell Biol. 25:4703-4715.

Novellino L, Renkvist N, Rini F, Mazzocchi A, Rivoltini L, Greco A, Deho P, Squarcina P, Robbins PF, Parmiani G, Castelli C (2003). Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel, class II HLA-restricted melanoma antigen. J Immunol. 170:6363-6370.

Nakamura M, Kishi M, Sakaki T, Hashimoto H, Nakase H, Shimada K, Ishida E, Konishi N (2003). Novel tumor suppressor loci on 6q22-23 in primary central nervous system lymphomas. Cancer Res. 63:737-741.