Human Tyrosine Receptor Phosphatase J - CS mutant

Target ID:

PTPRJA

Aliases:

CD148DEP1HPTPetaR-PTP-ETASCC1

Deposition Date:

Wednesday 22nd November 2006

Families:

Phosphatases

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2NZ6

Authors:

E.UGOCHUKWU,A.BARR,P.SAVITSKY,A.C.W.PIKE,G.BUNKOCZI,M.SUNDSTROM,J.WEIGELT,C.H.ARROWSMITH,A.EDWARDS,F.VON DELFT,S.KNAPP,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2NZ6

tabs

Structure Details

PTPRJ is a member of the R3 family of protein tyrosine phosphatases. It possesses an extracellular region containing fibronectin type III repeats, a single transmembrane region, and a single intracytoplasmic catalytic domain. PTPRJ is expressed in all hematopoietic lineages in particular in granulocytes and monocytes/macrophages. Weaker expression levels have been described for peripheral blood lymphocytes, including CD4 and CD8-positive T-cell subsets, B cells- in particular memory B cells- platelets, natural killer (NK) cells, dendritic cell types and transformed lymphoid T- and B cell lines. Among non-lymphoid tissues PTPRJ is found on the epithelium of the small and large bowel mucosa, appendix, endometrium, fallopian tubes, mammary glands, prostate glands, pancreatic acini and ducts, glandula parotis, cutaneous sweat glands and proximal and distal tubules of the kidney, fibrocytes, melanocytes and Schwann cells. It is also expressed at high levels in endothelia, including developing renal vascular endothelia. PTPRJ expression is up-regulated in leukocytes present in inflamed tissues as well as after in vitro activation with stimuli such as Con A, PMA or LPS. CSF-1 (macrophage colony-stimulating factor) leads to down-regulation of expression levels.

It is a negative regulator of cellular proliferation and is involved in cell differentiation by inducing cytoskeletal rearrangements, aberrant cell-substratum interactions , reduction of motility and chemotaxis in fibroblasts. It also plays a role in developmental vascular organization, endothelial proliferation and endothelium-pericyte interactions. PTPRJ negatively regulates T cell receptor signaling possibly through interfering with the phosphorylation of phospholipase C gamma 1 (PLCG1) and linker for activation of T cells (LAT).

In eosinophiles activation of PTPRJ leads to degranulation and induction of generation of superoxide anions frequent deletions, loss of heterozygosity (LOH), and missense mutations in the PTPRJ gene is associated with cancer (colon, breast, thyroid, lung). It has been suggested that PTPRJ may be important in the early events leading to colorectal cancer.

PTPRJ knockin mice from which cytoplasmic phosphatase sequences have been removed die at 10.5 days of gestation due to failure of developmental vascularization and associated growth retardation.

PTPRJ has several characteristics of a tumour-suppressor gene: Expression of PTPRJ suppresses the malignant phenotype in transformed rat thyroid cells, human colon, lung and breast cancers frequently present somatic missense mutations, loss of heterozygosity or deletions of the PTPRJ gene dedifferentiation of tumor cells might be associated with a reduced expression of PTPRJ. PTPRJ also plays a potential role in inflammatory diseases, such as allergic and parasitic diseases, associated with eosinophilia. The wild type protein did unfortunately not crystallize and the SGC determined two crystal structures of PTPRJ inactive mutants so far. One mutant in which the WPD loop tryptophane was changed to an alanine and a classical active site Cys/Ser mutant. Both mutants were crystallized in the presence of a substrate peptide. Due to the weak affinity of the peptide no substrate density was observed in the structures. However, the Cys/Ser mutant a peptide containing tryptophan located a C-terminal flanking region bound to its active site suggesting a mechanism of regulation involving residues C-terminal to the PTPRJ catalytic domain.

Materials and Methods

Entry Clone Source: Origine

Entry clone accession: gi|18860900

SGC Construct ID: PTPRJA-c012

GenBank GI number: gi|18860900

Vector: pNIC28-Bsa4. Details [PDF]; Sequence [FASTA] or [GenBank]

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence:

The expressed sequence of 2CFV corresponds to the wild type sequence in which the WPD motif has been replaced by APD (highlighted in sequence in red). This mutant is still active using fluorescent based substrates:

mhhhhhhssgvdlgtenlyfq*smKLIRV
ENFEAYFKKQQADSNCGFAEEYEDLKLVG
ISQPKYAAELAENRGKNRYNNVLPYDISR
VKLSVQTHSTDDYINANYMPGYHSKKDFI
ATQGPLPNTLKDFWRMVWEKNVYAIIMLT
KCVEQGRTKCEEYWPSKQAQDYGDITVAM
TSEIVLPEWTIRDFTVKNIQTSESHPLRQ
FHFTSAPDHGVPDTTDLLINFRYLVRDYM
KQSPPESPILVHCSAGVGRTGTFIAIDRL
IYQIENENTVDVYGIVYDLRMHRPLMVQT
EDQYVFLNQCVLDIVRSQKDSKVDLIY

In addition we generated a mutant (2NZ6) in which the active site cysteine (marked in red) was substituted by a serine (C/S mutant):

mhhhhhhssgvdlgtenlyfq*smKLIRV
ENFEAYFKKQQADSNCGFAEEYEDLKLVG
ISQPKYAAELAENRGKNRYNNVLPYDISR
VKLSVQTHSTDDYINANYMPGYHSKKDFI
ATQGPLPNTLKDFWRMVWEKNVYAIIMLT
KCVEQGRTKCEEYWPSKQAQDYGDITVAM
TSEIVLPEWTIRDFTVKNIQTSESHPLRQ
FHFTSWPDHGVPDTTDLLINFRYLVRDYM
KQSPPESPILVHSSAGVGRTGTFIAIDRL
IYQIENENTVDVYGIVYDLRMHRPLMVQT
EDQYVFLNQCVLDIVRSQKDSKVDLIY

Host: BL21 (DE3)

Growth medium, induction protocol: 1ml from a 10 ml LB overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 liter of LB media containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD 600 reached ~0.3. After that the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1mM IPTG at an OD 600 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.

Extraction buffer, extraction method: 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.

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. PTPRJ 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 - 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

Crystallization:
W/A mutant (2CFV): Crystals were obtained using the vapor diffusion method and a protein concentration of 9 mg/ml by mixing 100nl of the concentrated protein with 100nl of a well solution containing 0.01M NiCl₂; 0.1M TRIS pH 8.5 and 1M Li₂SO₄. 1 mM of the peptide IGEHpYVHVNA corresponding to the Met-receptor were added to the protein prior to crystallization.

C/S mutant (2NZ6): Crystals were obtained using similar condition as described for the W/A mutant with a protein concentration of 9 mg/ml

Data Collection: Resolution: 2.5Å (2CFV) and 2.3Å (2NZ6); Crystals were cryo-protected using the well solution and 25% ethylene glycole and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected at the SLS beamline X10 at a single wavelength (0.979 Å).

References

Iuliano R, Le Pera I, Cristofaro C, Baudi F, Arturi F, Pallante P, Martelli ML, Trapasso F, Chiariotti L, Fusco A. (2004). The tyrosine phosphatase PTPRJ/DEP-1 genotype affects thyroid carcinogenesis. Oncogene, 23(52):8432-8438.

Jandt E, Denner K, Kovalenko M, Ostman A, Bohmer FD. (2003). The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell-matrix adhesion.
Oncogene, 22(27):4175-4185.

Takahashi T, Takahashi K, St John PL, Fleming PA, Tomemori T, Watanabe T, Abrahamson DR, Drake CJ, Shirasawa T, Daniel TO. (2003). A mutant receptor tyrosine phosphatase, CD148, causes defects in vascular development. Mol Cell Biol. 2003 Mar, 23(5):1817-1831.

Ruivenkamp CA, van Wezel T, Zanon C, Stassen AP, Vlcek C, Csikos T, Klous AM, Tripodis N, Perrakis A, Boerrigter L, Groot PC, Lindeman J, Mooi WJ, Meijjer GA, Scholten G, Dauwerse H, Paces V, van Zandwijk N, van Ommen GJ, Demant P. (2002). Ptprj is a candidate for the mouse colon-cancer susceptibility locus Scc1 and is frequently deleted in human cancers. Nat Genet 3:295-300.