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Use of an anaerobic environment to preserve the endogenous activity of protein-tyrosine phosphatases isolated from intact cells¹

Dorrance H. Hamilton Research Laboratories, Division of Endocrinology and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA

³Correspondence: Division of Endocrinology and Metabolic Diseases, Jefferson Medical College, Room 349 Alumni Hall, 1020 Locust St., Philadelphia, PA 19107-6799, USA. E-mail: Barry.Goldstein{at}mail.tju.edu

SPECIFIC AIMS

Protein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is required for enzymatic activity. In this study, we evaluated how a strictly anaerobic working environment with deoxygenated buffers and avoiding exposure to air permits the assessment of endogenous PTPase activity as directly isolated in subcellular fractions. Using similar techniques, we also studied the reactivity of a specific PTPase, PTP1B, by immunoprecipitation. This approach provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu that more closely reflects their endogenous reactivity and their potential effect on signal transduction pathways involving reversible protein-tyrosine phosphorylation.

PRINCIPAL FINDINGS

1. Cellular PTPase activity from 3T3-L1 adipocytes is reversible to a variable extent after oxidative inhibition by exposure to air
The effect of air exposure on PTPases isolated from differentiated murine 3T3-L1 adipocytes was evaluated by comparing the activity of samples obtained by cell lysis on the open bench top to cells that were snap-frozen with liquid nitrogen, introduced into an anaerobic workstation in the frozen state, disrupted into deoxygenated homogenization buffer, and assayed within the anaerobic environment. In the absence of added reducing agents, cell lysis into air resulted in a 27% decrease in PTPase activity (P<0.001) in the cell homogenate compared to handling in the anaerobic chamber, using para-nitrophenylphosphate (pNPP) as substrate. When PTPase activity was measured in the presence of 1 mM dithiothreitol (DTT), there was no significant change in the activity as measured in the chamber. However, the reduced activity in the aerobically isolated cell lysate was restored by treatment with DTT to the level found in the samples maintained in the anaerobic chamber, indicating that the samples isolated aerobically were reversibly oxidized by exposure to air. Cytosol from the 3T3-L1 cells also showed a 33% lower activity in the air-exposed samples vs. the anaerobic samples (P<0.001), with a complete restoration of enzyme activity to the level observed in the anaerobic samples during assay in the presence of DTT. Results with the solubilized particulate fraction, however, showed that the activity of the samples isolated in air, though significantly reduced (P<0.001), was only 16% lower than the anaerobic samples, and there was no significant change after DTT incubation.

Using tyrosine-phosphorylated derivatized lysozyme as substrate, air exposure sharply decreased the PTPase activity of the 3T3-L1 adipocyte cytosol and solubilized particulate fraction to 20% and 54%, respectively, of the activity observed under anaerobic conditions (Fig. 1 ). The presence of DTT during the assay significantly increased the PTPase activity not only in the air-exposed samples by a striking 3.3- and 2.9-fold in the cytosol and particulate fraction, respectively, but also in the anaerobic cytosol and particulate fraction by 71% and 2.2-fold, respectively. These results indicated that PTPases in their endogenous environment are closely regulated by redox changes and variably affected by reduction in vitro, most likely depending on the overall sensitivity of the PTPase homologs present.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of air exposure on PTPase activity in subcellular fractions of 3T3-L1 adipocytes and reversibility with DTT. Differentiated 3T3-L1 adipocytes were snap-frozen with liquid nitrogen, lysed into homogenization buffer, and fractionated into cytosol and a solubilized particulate fraction either within the anaerobic workstation (Forma Scientific, model #901024) or on the bench top under room air. PTPase activity in the presence or absence of 1 mM DTT was measured by the hydrolysis of reduced, carboxamidomethylated, myelated (RCM) lysozyme that was phosphorylated on tyrosine with recombinant insulin receptors. *P = 0.02 and **P = 0.003 vs. anaerobically isolated control samples.

2. PTPase activity from human adipose tissue subcellular fractions is differentially inhibited by air exposure
The effect of air exposure on PTPase activity was also tested in human adipose tissue that was lysed from a frozen state, fractionated, and assayed within the anaerobic chamber compared with using an identical procedure on the bench top under room air. The particulate fraction PTPase activity under aerobic conditions was dramatically reduced to 46% of the activity observed under anaerobic conditions (P<0.001). Inclusion of DTT increased the activity of both the anaerobic and aerobic particulate fraction samples by 15% and 28%, respectively. In contrast to findings with the 3T3-L1 adipocytes, the cytosol PTPase activity did not show a significant difference between aerobic and anaerobic conditions. The cytosol PTPase activity in each of these environments was increased 18–20% by DTT exposure.

3. Air exposure inhibits the specific activity of PTP1B from HepG2 cells more than it inhibits the overall PTPase activity
Isolated subcellular fractions of human HepG2 cells were inhibited by 20%, 18%, and 9.8% by exposure to air in the overall PTPase activity in the cell lysates, the cytosol, and the solubilized particulate fraction, respectively, compared with the activity in samples isolated in the anaerobic chamber. The specific activity of PTP1B was also measured by immunoprecipitation from the HepG2 cell lysates using a monoclonal antibody PTP1B that can adsorb the enzyme in a catalytically active state. Air exposure dramatically reduced the catalytic activity of PTP1B to only 36% of the level observed in the samples maintained under strict anaerobic conditions throughout the experiment (P<0.001).

We then tested how the activity of the isolated PTP1B could be modulated by biochemical oxidation or reduction in vitro (Fig. 2 ). In samples handled under aerobic conditions, the basal activity of PTP1B was again reduced to 45% of the activity observed in the anaerobic chamber (P<0.001). Under either aerobic or anaerobic conditions, treatment of the immunoprecipitated PTP1B with 0.5 mM H₂O₂ for 10 min reduced its PTPase activity to negligible levels, consistent with essentially complete oxidative inactivation of the catalytic thiol. However, treatment of the samples isolated under aerobic conditions with 2 mM DTT for 10 min before PTPase assay increased the enzyme activity to 1.7-fold of the initial level in control samples (P=0.001). Treatment with DTT also caused a smaller (15%) but statistically significant increase in the PTPase activity in the anaerobic samples (P=0.04). These findings suggest that PTP1B isolated under aerobic conditions is reversibly inactivated by oxidation to a large degree. The fraction of activity irreversibly oxidized by air exposure may be estimated from the difference in activity measured under anaerobic conditions after DTT treatment vs. the activity measured after sample processing and assay in the anaerobic chamber. These data indicate that the activity of PTP1B isolated from the endogenous state was preserved by handling in the anaerobic chamber, and remained 46% higher than the aerobic samples even after treatment with DTT.

View larger version (18K): [in this window] [in a new window]   Figure 2. effect of air exposure and in vitro oxidation and reduction reactions on the catalytic activity of PTP1B. A HepG2 cell lysate was prepared within the anaerobic chamber from cells that had been snap-frozen with liquid nitrogen; after a preclearing step, PTP1B was isolated by immunoprecipitation with a monoclonal antibody directed at a carboxyl-terminal epitope that preserves its enzymatic activity (Ab-2; Oncogene Sciences). PTPase activity was measured in washed immunoprecipitates by hydrolysis of para-nitrophenylphosphate (pNPP). Control samples using nonimmune mouse IgG showed minimal background PTPase activity (<5% of the activity with Ab-2). To maintain controlled atmospheric conditions, all steps including and subsequent to cell lysis were performed under the nonreducing, anaerobic gas mixture and compared to results obtained by usual aerobic conditions under room air on the bench top. Where indicated, the immunoprecipitated PTP1B was treated with 0.5 mM H2O2 for 10 min or with 2 mM DTT for 10 min prior to PTPase enzyme assay. *P < 0.001 vs. anaerobically isolated control samples.

CONCLUSIONS

In signal transduction pathways regulated by reversible tyrosine phosphorylation, PTPases contribute to the steady-state balance of protein-tyrosine phosphorylation by dephosphorylating the cellular substrates of tyrosine kinases. Since PTPases are high turnover number enzymes, physiological suppression of PTPase catalytic activity has been recognized as a key feature of their regulation within the cellular environment. Members of the PTPase superfamily share a characteristic active site sequence motif with a corresponding reaction mechanism that depends on the reduced state of the thiol side chain of the catalytic cysteine residue (cys²¹⁵ in human PTP1B). The spatial interactions of the catalytic thiol hydrogen promotes its ionization and lowers its pKa to more than 3 units below that found in a typical cysteine thiol. Recent studies have shown that alterations in the oxidation state of the catalytic thiol within the cellular environment can occur and have profound effects on the PTPase specific activity. Cellular reactive oxygen species can oxidize the catalytic thiol of PTPases in a stepwise fashion to progressively more inert forms (Fig. 3 ). Initially, this may involve thiol oxidation to the sulfenic (–SOH) form, which is reversible by either cellular enzymatic mechanisms or with reducing agents in vitro. Further sequential steps of oxidation to sulfinic (–SO₂ H) and sulfonic (–SO₃ H) forms can lead to irreversible PTPase inactivation. PTP1B has also been shown to undergo disulfide conjugation to an inactive glutathiolated form, which may also be reversible by cellular reductases. A growing body of evidence has shown that this general scheme constitutes a major regulatory mechanism for PTPases within the cellular environment.

View larger version (17K): [in this window] [in a new window]   Figure 3. Cellular and environmental redox influences on the structure and function of PTPases. Oxidation of the catalytic thiol of PTPases is believed to occur in a stepwise fashion to progressively more inert forms. The catalytic cysteine thiol is first oxidized to the sulfenic (–SOH) form, which is amenable to reduction by cellular enzymatic mechanisms or with reducing agents in vitro, followed by further sequential steps of oxidation, to sulfinic (–SO2 H) and sulfonic (–SO3 H) forms, leading to irreversible PTPase inactivation. The sulfhydryl designation refers to the reactive cysteine side chain in the catalytic site of a given PTPase homologue (e.g., cys215 in human PTP1B). DTT, dithiothreitol; ROS, reactive oxygen species.

Since PTPases isolated from intact cells and tissues are susceptible to oxidation during isolation and assay, these enzymes typically have been assayed in the presence of strong biochemical reducing agents. However, this obviates any assessment of the endogenous reactivity of PTPases as directly isolated from the intact cellular environment. In the present study, we demonstrate how exposure to atmospheric oxygen can differentially affect PTPase activity isolated from a variety of sources, reflecting the known wide array of PTPases present in a given cell type. Our data support the hypothesis that the redox state of the catalytic thiol of cellular PTPases is likely to be proportioned between an active, reduced form and varying degrees of reversible and irreversible oxidation, which inactivates the enzyme. Sample handling under anaerobic conditions not only prevents artifactual oxidation of the catalytic PTPase thiol, but also allows an assessment of the changes in catalytic activity before and after reduction with DTT to indicate the fraction of enzyme present in an oxidized but activatable state in the cell.

Overall, the approach reported here provides a new framework for characterizing the activity of PTPases as isolated from the intracellular milieu that more closely reflects their endogenous reactivity and their potential effect on signal transduction pathways involving reversible protein-tyrosine phosphorylation. The application of these techniques in further studies will help characterize changes in the reactivity of specific cellular PTPases under various physiological conditions.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0795fje ; to cite this article, use FASEB J. (May 18, 2001) 10.1096/fj.00-0795fje

² These two authors contributed equally to this work.

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