Expression cloning of a human dual-specificity phosphatase.

Abstract

Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase.Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phanid prokaryotic expression vector and screened with a monoclonal , was ligated to the EcoRP-Sal I fragment of the expression vector pCEV-lacZ to generate the plasmid pCEV-bek (Fig. 1A). pCEV-lacZ is a pUC-based plasmid expression vector that produces a 3-gal fusion protein under the control of the lac promoter (see below).Construction of ApCEV-IacZ Prokaryotic Expression Vector. The vector designed for prokaryotic cDNA expression cloning was similar to Agtll but incorporated several additional features. cDNAs can be inserted by the automatic directional cloning method (16), allowing construction of unidirectional libraries with high efficiency. Use of unidirectional libraries to screen negative signals excludes antisense cDNA clones, which might inhibit the expression of cDNAs in host cells (like the bek kinase used in these experiments). It also increases the possibility of cDNA expression from the promoter in the vector. Since ApCEV-lacZ is a phagemid vector, the plasmid can be rescued by a simple procedure (17).To construct ApCEV-lacZ, the lacZ structural gene (encoding a-gal) of pMC1871 (Pharmacia) was cloned into pUCSVneo4ARI, a precursor plasmid of pCEV27 (17). Multiple cloning sites similar to those in ApCEV27 were created by inserting oligonucleotides into the EcoRI site of the IacZ gene. The resulting plasmid, pCEV-lacZ, was linearized at the Not I site and, after multiple excision sites were attached (17), cloned into ApCEV27 Not I arms to create ApCEV-lacZ.Library Construction and Screening. An oligo(dT)-primed M426 human fibroblast cDNA library was constructed in ApCEV-lacZ. To screen the library, bacteria (Escherichia coli Y1091; Clontech) containing the pCEV-bek plasmid were infected with phage (1.2 x 104 plaque-forming units per 150-mm plate) and grown for 4 hr at 3TC on agar plates containing ampicillin (50 pug/ml). The plates were overlaid with nitrocellulose filters impregnated with 10 mM IPTG and were incubated for an additional 4 hr at 37cC, as described (18). The filters were removed, washed with phosphatebuffered saline containing 0.05% Tween 20 (PBST), and then blocked in PBST containing 3% dry milk for 1 hr at room temperature. Filters were probed with mouse monoclonal anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY) overnight at room temperature, rinsed four times with PBST, incubated with 1251-labeled protein A for 30 min,

A novel human ERK phosphatase regulates H-ras and v-raf signal transduction

“…An extended HC motif, the highly conserved active site sequence of PTPs, VSVHCXXGXXRSX-XXXXAY(L/I)M (where X is any amino acid), was present in B59 at amino acid position 276 to 296 (Figure 1a). The predicted B59 amino acid sequence encompassing this catalytic site (amino acid residues 238 ± 331) showed signi®cant similarity to several known human dsPTPs, including B23, CL100, PAC1, hVH2 (Guan and Butch, 1995;Misra Press et al, 1995) and VHR (Ishibashi et al, 1992). Among these proteins, the most conserved region lies within a 21-amino acid stretch that includes and (Figure 1b).…”

RK‐682, a potent inhibitor of tyrosine phosphatase, arrested the mammalian cell cycle progression at G1phase

“…GST-VHR fusion protein (VHR) was prepared as described [4]. The plasmid to express GST-cdc25B fusion protein (cdc25B) was constructed by insertion of the AatI fragment (nucleic acids 1394-1844) of the human cdc25B cDNA [14] into the SmaI site of the pGEX1 vector.…”

“…The key step in the activation of MPF is dephosphorylation on Thr-14 and Tyr-15 of cdc2 protein by cdc25, a kind of dual-specificity protein phosphatases [3]. VHR (VHl-related human protein), which was isolated from a human fibroblast has the dual-specificity phosphatase activity towards phosphotyrosine and phosphoserine/threonine like cdc25 [4].…”

Essential role of calcium in the regulation of MAP kinase phosphatase-1 expression

“…Other members of the VH1 family of dual-speci®city protein phosphatases include VHR (Ishibashi et al, 1992), PAC-1 (Rohan et al, 1993), MKP-2/hVH-2/ TYP-1 (Guan and Butch, 1995;Misra-Press et al, 1995;King et al, 1995), B23/hVH-3 (Ishibashi et al, 1994;Kwak et al, 1995), hVH-5 (Martell et al, 1995) and rVH-6/MKP-3 (Mourey et al, 1996;Muda et al, 1996). These gene products have unique but overlapping tissue distribution patterns.…”