Human protein tyrosine phosphatase, non-receptor type 9

Target ID:

PTPN9A

Aliases:

MEG2PTPMEG2

Deposition Date:

Tuesday 27th March 2007

Families:

Phosphatases

Related Diseases:

MetabolismCancerSignalling

Structure type:

apo

Download Coordinates:

2PA5

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2PA5

tabs

Structure Details

PTPN9 also called MEG2 was originally cloned from a megakaryocytic cell line and is the only member of the NT3 subfamily of PTPs. This tyrosine phosphatase contains a unique 250-amino-acid non-catalytic N-terminus with homology to Sec14p, a yeast protein that has been described to bind to and is activated by polyphosphoinositides PIP2 and PIP3 as well as by phosphatidyl serine. Deletion of PTPN9 in mice results in multiple neurodevelopmental defects and hemorrhages and a more than 90% late embryonic lethality. PTPN9 has been shown to inhibit insulin-induced phosphorylation of the insulin receptor and subsequent studies showed that RNAi-mediated reduction of PTPN9 transcript levels enhances insulin action. In addition, PTPN9 has been identified as a modulator of the insulin-dependent gluconeogenic transcription factor FOXO1. Importantly, adenoviral-mediated depletion of PTPN9 in the liver of diabetic mice results in insulin sensitization and normalization of hyperglycemia suggesting that modulation of PTPN9 activity may be an effective strategy in the treatment of type 2 diabetes.

PTPN9 has also been implicated in the development of Polycythemia vera (PV), a clonal myeloproliferative disorder characterized by trilineage marrow hyperplasia with increased production of red cells, granulocytes, and platelets. Polycythemia has been associated with high mortality rates if untreated as well as with the development of leukemia. A correlation of PTPN9 activity with colony-forming ability of ECFCs ( erythroid colony-forming cells) has been demonstrated suggesting also PTPN9 as a potential target for development of therapeutic drugs to reduce red cell production.

Here we determined the structure of the phosphatase domain at 1.6Å resolution.

Materials and Methods

PTPN9: Human protein tyrosine phosphatase, non-receptor type 9

PDB:2PA5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:IMAGE:3895727
Entry Clone Source:MGC
SGC Clone Accession:
Tag:
Host:Phage-resistant derivative of BL21(DE3)

Construct

Prelude:NON-cleavable C-terminal His 6 tag
Sequence:MSVHVPGPHAMTIQELVDYVNARQKQGIY EEYEDIRRENPVGTFHCSMSPGNLEKNRY GDVPCLDQTRVKLTKRSGHTQTDYINASF MDGYKQKNAYIGTQGPLENTYRDFWLMVW EQKVLVIVMTTRFEEGGRRKCGQYWPLEK DSRIRFGFLTVTNLGVENMNHYKKTTLEI HNTEERQKRQVTHFQFLSWPDYGVPSSAA SLIDFLRVVRNQQSLAVSNMGARSKGQCP EPPIVVHCSAGIGRTGTFCSLDICLAQLE ELGTLNVFQTVSRMRTQRAFSIQTPEQYY FCYKAILEFAEKEGMVSahhhhhh
Vector:pNIC-CH

Growth

Medium:LB
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 37degC until the OD600 reached ~0.3 then the temperature was adjusted to 18degC. 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.

Purification

Procedure

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:LC- ESI -MS TOF indicated a loss of 130 a.m.u. corresponding to the proteolytic cleavage of the initial methionine. Expected mass : 36179; Observed mass: 36049.
Crystallization:Crystals were grown at 20°C in 200 nl sitting drops mixing 100 nl of protein with 100 nl of a solution containing 25% PEG-3350, 0.2 M potassium thiocyanate, 10% ethylene glycol, 0.1 M Bis-Tris propane pH 6.15. The crystals were cryo-protected using 20% ethylene glycol which was added to the drop 30 seconds prior to mounting and flash freezing in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Cho, C. Y., Koo, S. H., Wang, Y., Callaway, S., Hedrick, S., Mak, P. A., Orth, A. P., Peters, E. C., Saez, E., Montminy, M. , et al. (2006). Identification of the tyrosine phosphatase PTP-MEG2 as an antagonist of hepatic insulin signaling. Cell Metab 3 , 367-378.
Huynh, H., Bottini, N., Williams, S., Cherepanov, V., Musumeci, L., Saito, K., Bruckner, S., Vachon, E., Wang, X., Kruger, J., et al. (2004). Control of vesicle fusion by a tyrosine phosphatase. Nat Cell Biol 6 , 831-839.
Wang, Y., Vachon, E., Zhang, J., Cherepanov, V., Kruger, J., Li, J., Saito, K., Shannon, P., Bottini, N., Huynh, H. , et al. (2005). Tyrosine phosphatase MEG2 modulates murine development and platelet and lymphocyte activation through secretory vesicle function. J Exp Med 202 , 1587-1597.
Xu, M. J., Sui, X., Zhao, R., Dai, C., Krantz, S. B., and Zhao, Z. J. (2003). PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells. Blood 102 , 4354-4360.
Zhao, R., Fu, X., Li, Q., Krantz, S. B., and Zhao, Z. J. (2003). Specific interaction of protein tyrosine phosphatase-MEG2 with phosphatidylserine. J Biol Chem 278 , 22609-22614.