Human striatal enriched protein tyrosine phosphatase (STEP)

Target ID:

PTPN5A

Aliases:

FLJ14427PTPSTEPSTEP

Deposition Date:

Wednesday 22nd June 2005

Families:

Phosphatases

Related Diseases:

NeurobiologySignalling

Structure type:

apo

Download Coordinates:

2BV5

Authors:

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

Site:

Oxford

ICM Datapack:

ICM Datapack

2BV5

tabs

Structure Details

STEP, a striatal-enriched protein tyrosine phosphatase, is preferentially expressed in neurons of the basal ganglia, hippocampus, cortex and related structures. Alternative splicing produces various STEP family members, and both cytosolic (STEP 46) and membrane-associated (STEP 61) variants exist.

STEP and its non-neuronal homologs like He-PTP have been implicated in the regulation of ERK activity. Both splice products STEP 61 and STEP 46 are phosphorylated in a common kinase-interacting domain (KIM) that binds to ERK2. The kinase PKA phosphorylates STEP 46 at Ser49 (equivalent to Ser221 in STEP 61) which decreases the activity of STEP by reducing its affinity for its substrates. Dephosphorylated STEP binds and inactivates ERK through dephosphorylation of the tyrosine residue in its activation domain and blocks nuclear translocation of the kinase. These findings establish STEP as an important tyrosine phosphatase involved in regulating the duration of ERK signaling in neurons.

Materials and Methods

Entry clone source: Purely Proteins

Entry clone accession: gi|90652860

Final protein sequence:
gplgspgipSRVLQAEELHEKALDPFLL
QAEFFEIPMNFVDPKEYDIPGLVRKNRY
KTILPNPHSRVCLTSPDPDDPLSSYINA
NYIRGYGGEEKVYIATQGPIVSTVADFW
RMVWQEHTPIIVMITNIEEMNEKCTEYW
PEEQVAYDGVEITVQKVIHTEDYRLRLI
SLKSGTEERGLKHYWFTSWPDQKTPDRA
PPLLHLVREVEEAAQQEGPHCAPIIVHC
SAGIGRTGCFIATSICCQQLRQEGVVDI
LKTTCQLRQDRGGMIQTCEQYQFVHHVM
SLYEKQLSHQS

Vector: pGEX-6P2. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence:
mspilgywkikglvqptrllleyleekye
hlyerdegdkwrnkkfelglefpnlpyyi
dgdvkltqsmaiiryiadkhnmlggcpke
raeismlegavldirygvsriayskdfet
lkvdflsklpemlkmfedrlchktylngd
hvthpdfmlydaldvvlymdpmcldafpk
lvcfkkrieaipqidkylksskyiawplq
gwqatfgggdhppksdlevlfq*gplgsp
gip

PreScission? (rhinovirus 3C)- protease cleavable (*) GST tag

Host: BL21 (DE3) phage resistant

Growth medium, induction protocol: Starter cultures from freshly transformed colonies in 10 ml LB, and ampicillin were grown overnight. This was diluted 1:1000 in fresh media (6L) and was grown at 37°C to an OD 600 of 0.3 and than transferred to 18°C. Expression was induced at an OD 600 of 0.8 using 1 mM IPTG. Cells were harvested after 3h by centrifugation, transferred to 50-ml tubes, and frozen in liquid nitrogen.

Extraction buffer, extraction method: Extraction buffer: 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM DTT. The cell pellets (20 g wet wt) were re-suspended in 50 ml extraction buffer, and lysed by a high pressure cell disrupter. Supernatant was centrifuged for 30 minutes at 60,000 rpm.

Column 1: Glutathione Sepharose 4B affinity, 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.

Buffers: Binding buffer: 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM DTT.

Procedure: Supernatant was applied at gravity flow, followed by a wash with 30 ml binding buffer. The GST-fusion was cleaved while bound to the column by addition of PreScission protease. The column was gently rotated overnight a 4°C then protein eluated with 3 bed volumes of binding buffer.

Column 2: SEC

Buffers: 10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM DTT.

Procedure: Akta-Prime

Column 3: HiTrap Q

Protein concentration: Centricons 10 kDa cut off

Mass spec characterisation : LC-ESI-MS TOF confirmed the correct mass expected for this construct (33252 Da).

Crystallization: Crystals were obtained using sitting drop method at 4°C. Drops were prepared using 150 nl of protein (10 mg/ml concentration) and 150 nl of the well solution (25% PEG 3350, 0.2M LiSO 4 , 100 mM Bis Tris Propane pH 5.5).

Data Collection: Resolution: 1.8 Å; X-ray source: Synchrontron BESSY BL1.

References

Pulido, R., Zuniga, A. & Ulrich, A. (1998). PTP-SL and STEP protein tyrosine phosphatases regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by association through a kinase interaction motif. EMBO J. 17 , 7337-7350

Lombroso PJ, Murdoch G, Lerner M. (1991). Molecular characterization of a protein-tyrosine-phosphatase enriched in striatum. Proc Natl Acad Sci 88:7242-7246.

Valjent E, Pascoli V, Svenningsson P, Paul S, Enslen H, Corvol JC, Stipanovich A, Caboche J, Lombroso PJ, Nairn AC, Greengard P, Herve D, Girault JA. (2005). Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum. Proc Natl Acad Sci 102:491-496.