Purification and Crystallization of the Catalytic Domain of Human Protein Tyrosine Phosphatase 1B Expressed in Escherichia coli

Barford, David; Keller, James; Flint, Andrew; Tonks, Nicholas K.

doi:10.1006/jmbi.1994.1409

Cited by 50 publications

(38 citation statements)

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“…All subcloned fragments were sequenced completely to confirm that no other mutations had been introduced. Mutants of 37-kDa PTP1B were expressed in E. coli strain BL21 and purified by chromatography on Fast Flow S-Sepharose and Mono Q (22).…”

Section: Methodsmentioning

confidence: 99%

Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases

Flint

Tiganis

Barford

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a ''substrate trap.'' Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substratebinding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligandindependent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.The protein tyrosine phosphatases (PTPs) are a structurally diverse family of receptor-like and nontransmembrane enzymes that have been implicated in the control of numerous physiological processes, including growth and differentiation (1, 2). Approximately 75 PTPs have been identified to date. These enzymes are characterized by the presence of a conserved catalytic domain of Ϸ240 residues which contains the unique signature motif,

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Section: Methodsmentioning

confidence: 99%

Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases

Flint

Tiganis

Barford

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

724

698

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“…Preparation of Crystals-Wild-type protein and the Q262A mutant PTP1B protein and crystals were prepared as described (20). The PTP1B-vanadate complex was prepared by incubating wild-type PTP1B crystals in 100 mM HEPES (pH 7.5), 200 mM magnesium acetate, 18% (w/v) polyethylene glycol 8000, and 1 mM sodium orthovandate for 10 min prior to transfer to the same buffer with 25% (v/v) glycerol and freezing in a nitrogen gas stream at 100 K (22).…”

Section: Methodsmentioning

confidence: 99%

“…By assuming that the rate of substrate diffusion into the crystal exceeds the rate of cysteinyl-phosphate hydrolysis, and with an excess of substrate added to the crystal incubation buffer, a steady state accumulation of intermediate should be obtained, which may be captured by flash-freezing a crystal in a stream of nitrogen gas at 100 K. The cysteinylphosphate intermediate is preserved in the frozen crystals, which are suitable for x-ray data analysis. PTP1B crystals are grown at pH 7.5 and 4°C (20). However, at this pH, the rate of PTP-catalyzed hydrolysis of p-NPP is 10% of its maximum rate observed between pH 5.5 and 6.5 (27).…”

Section: Structure Of a Transition Statementioning

confidence: 99%

Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography

Pannifer

Flint

Tonks

et al. 1998

Journal of Biological Chemistry

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235

214

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The formation of phosphoryl-enzyme intermediates is an essential component of numerous enzymatic mechanisms that involve phosphoryl-transfer reactions, for example the dephosphorylation of Tyr(P) residues catalyzed by protein-tyrosine phosphatases (1). PTPs 1 together with the protein-tyrosine kinases, which catalyze the opposing tyrosine phosphorylation reaction, control the overall levels of cellular tyrosine phosphorylation, and the molecular basis of the regulation and substrate specificity of these enzymes is subject to much investigation (2, 3). Reversible tyrosine phosphorylation is essential to the signal transduction pathways triggered by hormones, mitogens, and oncogenes that regulate such processes as cell growth, differentiation, and proliferation. The PTPs are a diverse family of enzymes that comprise transmembrane receptor-like PTPs (RPTPs) and soluble cytosolic proteins. The catalytic domains of the PTPs are highly conserved, consisting of ϳ250 amino acids and are characterized by an 11-residue PTP signature motif, (I/V)HCXAGXXR(S/T)G, containing the Cys and Arg residues that are essential for catalysis (4, 5). Diversity within the family is generated by the nature of the noncatalytic segments attached to the N and C termini of the PTP domains, which provide regulatory and subcellular targeting functions. Additional diversity is generated within the RPTPs, which frequently possess tandem PTP domains, although for some RPTPs such as CD45, the C-terminal PTP domain lacks catalytic activity (3). The dual specificity phosphatases (DSPs) are related to the PTPs by their possession of the PTP signature motif and related tertiary structure and catalytic mechanism (1, 6).Insights into the catalytic mechanism of PTPs and DSPs have been obtained from structural and kinetic studies of these enzymes (1, 4, 5). A PTP1B-phosphotyrosine peptide complex revealed that the Tyr(P) residue of the peptide is buried within a deep catalytic site cleft present on the protein's molecular surface. The base of the catalytic site is formed by residues of the PTP signature motif with the phosphate group of Tyr(P) being coordinated by main-chain amide groups and the Arg side chain of this motif, such that the phosphorus atom is situated adjacent to the S␥ atom of the catalytic Cys residue (7). Four other loops bearing invariant residues form the sides of the catalytic cleft and contribute to catalysis and substrate recognition. Engagement of phosphopeptides by PTP1B promotes a major conformational change of one of these loops (the WPD loop) consisting of residues 179 -187 that shift by as much as 8 Å to close over the phenyl ring of Tyr(P) and allow the side chain of Asp-181 to act as a general acid in the catalytic reaction. The Arg-221 side chain reorients to optimize salt bridge interactions with the phosphate bound to the catalytic site. This shift is coupled to motion of the WPD loop via a hydrogen bond between NH 2 of Arg-221 and the carbonyl oxygen of Pro-180 and hydrophobic interactions between the aliphatic moiety of ...

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“…Trypsin Digestion-The C-terminally truncated, 37-kDa human PTP1B was purified as described previously (27). PTP1B was treated with H 2 O 2 at 37°C for 10 min or treated with S-nitroso-N-penicillamine (SNAP) (Calbiochem), S-nitrosoglutathione (GSNO) (Alexis Biochemicals), DETANONOate (Sigma), or N-acetylpencillamine (NAP) (Fluka) at 37°C for 20 min.…”

Section: Preparation Of S-nitrosylated Ptp1b For Solution-basedmentioning

confidence: 99%

“…Crystallization and Structural Analysis of PTP1B S-Nitrosylation-Crystallization of the 37-kDa PTP1B was carried out as described previously (27). The crystal was incubated with SNAP (1 mM) at room temperature for 20 min and then mixed with the mother liquid containing 29% glycerol as an antifreezing reagent.…”

Section: Preparation Of S-nitrosylated Ptp1b For Solution-basedmentioning

confidence: 99%

Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation

Chen

Chu

Pan

et al. 2008

Journal of Biological Chemistry

139

120

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Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMPindependent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce sitespecific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 Å resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H 2 O 2 -induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.

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Purification and Crystallization of the Catalytic Domain of Human Protein Tyrosine Phosphatase 1B Expressed in Escherichia coli

Cited by 50 publications

References 0 publications

Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases

Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases

Visualization of the Cysteinyl-phosphate Intermediate of a Protein-tyrosine Phosphatase by X-ray Crystallography

Cysteine S-Nitrosylation Protects Protein-tyrosine Phosphatase 1B against Oxidation-induced Permanent Inactivation

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